welcome to the 10 - hashemite university · welcome to the 10 th annual cbp - message from the...
TRANSCRIPT
Welcome to the 10th
Annual CBP - Message from the Chair
Welcome to the 10
th annual Chemical Biophysics Symposium! The CBP brings together researchers at the intersection of
chemistry, biology and physics in an interactive and stimulating environment to foster the exchange of ideas. We are pleased
to have scientists from many disciplines presenting their latest work, and we hope that you find the CBP an appropriate forum
to discuss and debate ideas with your colleagues.
This year, we provide you with many opportunities to learn and discuss with fellow researchers from universities and
industries, including our traditional panel-and-audience discussion on Friday where all attendees are encouraged to participate
in a light-hearted debate and two lively poster sessions on Saturday. We have also planned numerous social events and coffee
breaks throughout the meeting for more informal discussions.
We are constantly thinking to improve the quality of this symposium and have introduced three workshops on Friday morning
aiming to give participants an opportunity to learn about a specific area of research in a greater depth. These areas include
microcalorimetry in biophysical chemistry, hydrophobicity interactions, and the basics of intellectual property, technology
development and commercialization.
We hope that you enjoy the 10th
annual CBP. If you have any questions or concerns, please do not hesitate to speak with any
member of the organizing committee. The next CBP will be in April 2012 – we hope to see you then!
- Soyoung Lee -
Chemical BioPhysics Symposium 2011
University of Toronto, Canada
Contact Information:
Soyoung Lee (Student Chair): [email protected]
Prof. Heiko Heerklotz (External Faculty Advisor): [email protected]
Prof. David McMillen (Faculty Advisor): [email protected]
Prof. Cynthia Goh (Faculty Advisor): [email protected]
CBP 2011 Organizing Committee
Student Chair
Soyoung Lee Co-Chairs
Jordan Ang
Christina Müller
Hiren Patel
Finance
Claire Yu
Food
Nisha Patel
Mary Shan
Henry Yan
Program Planning
Andrew Chan
Richard Cisek
Hannah Morales
Danielle Tokarz
Artistic Design
Jordan Ang
Isaac Li
Adrienne Tanur
Welcome Package
Adrienne Tanur
Venue
Hiren Patel
Sponsorship
Christina Müller
Gaurau Raval
Masood Samim
Kevin Song
Adrienne Tanur
Publicity
Andrew Chan
Rayomond Dinshaw
Grace Li
Isaac Li
Hiren Patel
Sarah Rausher
Zaheen Sadeq
Webmasters
Isaac Li
Baoxu Liu
Speaker Coordination
Calvin Cheng
Grace Li
Nisha Patel
Sarah Rauscher
Faculty Advisors
Prof. Cynthia Goh
Prof. Claudiu Gradinaru
Prof. David McMillen
Prof. R.J. Dwayne Miller
Prof. Anja Nohe
Prof. Gilbert Walker
External Faculty Advisors
Prof. Heiko Heerklotz
i
Workshops
Friday, April 9th
, 9:30 AM -12:00 PM, Leslie L. Dan Pharmacy Building
Workshop 1 Chair: Isaac Li (University of Toronto) PB310
Hydrophobic Interactions: Fundamentals to Applications
Workshop 2 Chair: Hiren Patel (University of Toronto) PB250
Microcalorimetry in Biophysical Chemistry Applications, Protocols, Secrets
Workshop 3 Chair: Christina Müller (University of Toronto) LM128
(Lash Miller Bldg.)
From Lab Results to Societal Benefits – How Knowledge Acquired During Your PhD can Lead to Real-
World Impact Through Commercialization
ii
Conference Program
Friday, April 9th
, Leslie L. Dan Pharmacy Building
12:30-2:00 PM Registration
2:00-2:15 PM Opening Remarks – Soyoung Lee & Prof. Heiko Heerklotz B250
Session I Chair: Soyoung Lee (University of Toronto) B250
2:15-3:00 PM Gary Pielak (The University of North Carolina at Chapel Hill)
“Macromolecular Crowding & Protein Chemistry: Views from Inside & Outside Cells”
3:00-3:20 PM Anton Zilman (University of Toronto)
“Selective Transport Through Nano-Channels: Biology, Physics and Nanotechnology”
iii
Session II Chair: Sarah Rauscher (University of Toronto) B250
3:20-4:05 PM Shekhar Garde (Rensselaer Polytechnic Institute)
“Hydration Phenomena at the Interface of Chemistry and Biology: A New Fluctuations-
based Perspective”
4:05-4:25 PM Grace Li (University of Toronto)
“Molecular Mechanism of Aβ Amyloid Inhibition by Inositol”
4:25-4:45 PM Christopher Ing (University of Waterloo)
“Sampling of Quantum Systems using the Path Integral Langevin Dynamics: Applications to
Weakly Bound Systems and Biological Molecules”
4:45-5:15 PM Coffee Break
Sponsored by MicroCal
5:15-6:15 PM Panel Discussion: "Number of publications, their impact factors, and the value of grants received –
are these the right criteria for the quality of scientific research?"
Chair: David McMillen / Cynthia Goh
With: Gary Pielak, R.J. Dwayne Miller, Rob Macgregor, and Roman Melnyk
6:20-7:20 PM Dinner Main Lobby
iv
Session III Chair: Heiko Heerklotz (University of Toronto) B250
7:20-8:05 PM Guri Giaever (University of Toronto)
“Novel Targets and Chemical Probes Identified by Systematic Chemogenomic Profiling:
A Step Towards an Empirical Definition of the Druggable Genome”
8:05-8:25 PM Kimberley S. Samkoe (Dartmouth Medical School)
“Novel Reference Tracer Model for Quantification of Receptor Binding in vivo with
Fluorescent Molecular Imaging”
8:25-8:45 PM Tiju Thomas (Memorial University of Newfoundland)
“Nanoparticle Synthesis of Luminescent Materials: High Temperature, Mechanical, and
Aqueous Solution Growth Methods”
9:00 PM - Informal Discussions
Pogue Mahone, 777 Bay Street (Corner of Bay and College Sts.)
v
Saturday, April 9th
, Leslie L. Dan Pharmacy Building
9:00-9:30 AM Continental Breakfast
Session IV Chair: Anja Nohe (University of Delaware) B250
9:30-10:15 AM William Shih (Harvard University)
“Self-Assembly of DNA into Nanoscale Three-Dimensional Shapes”
10:15-10:35 AM Shenlin Wang (University of Guelph)
“Structural Changes in Soluble Sensory Rhodopsin Transducer upon the Interaction with
DNA”
10:35-10:55 AM Andrew Rutenberg (Dalhousie University)
“Quantification of Fluorophore Copy Number from Intrinsic Fluctuations During
Fluorescence Photobleaching”
11:00-12:30 PM Poster Session I (Odd Numbered) & Coffee break
Sponsored by SFR
B250 Hall
12:30-1:30 PM Lunch Main Lobby
vi
Session V Chair: Isaac Li (University of Toronto) B250
1:30-2:15 PM Dor Ben-Amotz (Purdue University)
“Hydrophobic Hydration Shell Spectroscopy”
2:15-2:35 PM Cristiano Dias (University of Toronto)
“Exploring the Role of Hydrophobic Interaction in Secondary Structure Formation using a
Simple Atomic Model”
2:35-2:55 PM Mazdak Khajehpour (University of Manitoba)
“Hofmeister Salts, Water Hydrogen Bonding and the Hydrophobic Effect”
2:55-4:25 PM Poster Session II (Even Numbered) & Coffee Break
Sponsored by Gamble Technologies
B250 Hall
vii
Session VI Chair: Nisha Patel (University of Toronto) B250
4:30-5:15 PM Roland Winter (Technical University Dortmund)
“Exploring the Configurational and Free Energy Landscape of Biomolecules under
Extreme Conditions: from Model Biomembranes to Proteins”
5:15-5:35 PM David N. Langelaan (Dalhousie University)
“Structure and Topology of the N-terminal Tail and First Transmembrane Segment of the
Apelin Receptor in Three Different Micelle Types”
5:35-5:55 PM Aron Broom (University of Waterloo)
“Modular Evolution and the Design of a Threefold Symmetric Globular Protein”
5:55-6:15 PM Rayomond Dinshaw (University of Toronto)
“Ultrafast Energy Transfer Dynamics in Light-Harvesting Proteins””
6:15-6:40 PM Break and travel to restaurant
6:40 PM - Symposium Dinner
Forest View Chinese Restaurant, 466-468 Dundas St. W., 2nd
floor
“Do you know your stuff?” Chemical Biophysics game show to follow!
viii
Sunday, April 10th
, Leslie L. Dan Pharmacy Building
8:45-9:30 AM Continental Breakfast Main Lobby
Session VII Chair: Hiren Patel (University of Toronto) B250
9:30-10:15 AM Justin Nodwell (McMaster University)
“Chemical Interrogation of a Bacterial Life Cycle”
10:15-10:35 AM Shun Lu (University of Guelph)
“Changes in the Mechanical Properties of Pseudomonas aeruginosa Bacterial Cells Induced
by Antimicrobial Peptides”
10:35-10:55 AM Coffee Break Main Lobby
Session VIII Chair: Rayomond Dinshaw (University of Toronto) B250
10:55-11:40 AM Régis Pomès (University of Toronto)
“Peptides and Proteins in Detergent and Lipids”
11:40-12:25 PM Villy Sundström (Lund University)
“The Primary Steps of Solar Energy Conversion – from Photosynthesis to Organic Solar
Cells”
12:25-12:35 PM Closing Remarks B250
Abstracts
Oral Presentations
1
Session I, Fri 2:15 pm
Macromolecular Crowding & Protein Chemistry: Views from Inside & Outside Cells
Gary Pielak
Department of Chemistry
The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
I will discuss the successes and challenges of in-cell protein NMR and the quantification of protein folding, stability, and
diffusion under crowded conditions. One challenge is the inability to observe 15
N-1H HSQC spectra from most globular
proteins in cells. To understand this problem we turned to in vitro experiments, using synthetic polymers, globular
proteins, and cell lysates to assess how these crowding agents affect protein diffusion. To examine stability, we used
NMR-detected amide-proton exchange to quantify the opening free energy of test proteins in the presence of crowding
agents. To examine folding in cells, we applied 19
F NMR to an unstable test protein. Our results highlight the differences
between crowding agents and shed light on the biological effects of crowding.
Contact: [email protected]
Session I, Fri 3:00 pm
Selective transport through nano-channels: biology, physics and nanotechnology
Anton Zilman
Department of Physics, University of Toronto, Toronto, ON.
Functioning of living cells requires selective transport of proteins and other molecular signals into and out of the cell, as
well between various cell compartments. Much of such transport is conducted by nano-scale channels and gates, which
function without direct input of metabolic energy and without channel transitions from an ‘open’ to a ‘closed’ state during
transport. Mechanisms of selectivity of such channels provide inspiration for bio-engineering applications, in particular for
design of selective nano-molecular sensors and sieves.
Precise mechanisms of selective transport through such ‘always open’ biological and artificial nano-channels are still
unknown. I will review the biophysics of such channels, with the emphasis on the functioning of the Nuclear Pore Complex
– a biological nano-gate that carries the transport between the cell nucleus and the cytoplasm. I will present a theoretical
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
2
model to explain the mechanisms of selectivity of transport through such nano-channels. The theory provides a simple
physical mechanism for selectivity based on the differences in the kinetics of transport through the channel between
different molecular species. In particular, the theory explains how the channels can remain selective in the presence of
vast amounts of non-specific noise. The theoretical predictions explain previous experimental results and have lead to the
creation of a prototype artificial molecular nano-filter.
Contact: [email protected]
Session II, Fri 3:20 pm
Hydration Phenomena at the Interface of Chemistry and Biology:
A New Fluctuations-based Perspective
Shekhar Garde
Howard P. Isermann Department of Chemical & Biological Engineering
Rensselaer Polytechnic Institute, Troy, New York
Water-mediated interactions (e.g., hydrophobic interactions) govern a host of biological and colloidal self-assembly
phenomena from protein folding, micelle and membrane formation, to molecular recognition. Macroscopically,
hydrophobicity is often characterized by measuring a droplet contact angle on a surface. At the nanoscale, such
measurements are not feasible, e.g., for surfaces of proteins or nanoparticles. Using theory and molecular simulations, we
present a new perspective that connects the behavior of water near nanoscale interfaces to their hydrophobicity/philicity.
Specifically, we show that water density fluctuations (and not the average local density) provide a quantitative
characterization of interfacial hydrophobicity. Density fluctuations are enhanced near hydrophobic interfaces and
suppressed near hydrophilic ones. This new perspective provides a computational tool for characterizing the
hydrophobicity patterns on protein surfaces, which are relevant for binding, recognition, and aggregation. Simulations
also show how the properties of water at interfaces influence binding, folding, and dynamics of flexible molecules in
interfacial environments. Our current understanding of the hydration of ions, osmolytes, and solution additives, when
combined with this new perspective, provides additional insights into the role of water in multicomponent biological
interactions.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
3
Session II, Fri 4:05 pm
Selective Transport Through Nano-Channels: Biology, Physics and Nanotechnology
Grace Li and Régis Pomès
Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, and Department of Biochemistry,
University of Toronto, Toronto, ON.
Alzheimer’s disease (AD) is a severe neurodegenerative disease with no cure. Currently,
one method of targeting the underlying disease is to prevent or reverse the amyloid
formation of Aβ42, a key pathological hallmark of AD. A small-molecule novel drug
candidate, Scyllo-inositol, is a polyol small-molecule that exhibits stereochemistry
dependent inhibition of the formation of Aβ fibrils in vitro. Furthermore, recently
completed phase II clinical trials demonstrated that scyllo-inositol achieved target drug
levels in the cerebral spinal fluid (CSF) of Alzheimer’s patients, a main challenge for AD
drug candidates to to overcome.
Despite its promise as a therapeutic for AD, the mechanism of action of scyllo-inositol at the molecular level is currently
not understood. We perform extensive microsecond timescale atomistic explicit solvent molecular dynamics simulations
of scyllo-inositol and its inactive stereoisomer, chiro-inositol with the Aβ42 protofibril. From our simulations, we predict
binding affinities and characterize the binding modes of inositol and their stereochemistry-dependent effect on the
structure of Aβ42 protofibrils. Our results provide molecular insight for the rational design of small-molecule inhibitors of
Aβ42 and other amyloid-based diseases.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
4
Session II, Fri 4:25 pm
Sampling of Quantum Systems Using the Path Integral Langevin Dynamics: Applications to Weakly Bound Systems and
Biological Molecules
Christopher Ing1, Konrad Hinsen
2, Jing Yang
3 and Pierre-Nicholas Roy
3
1Department of Physics and Astronomy, University of Waterloo, ON, Canada, N2L 3G1 2
2Centre de Biophysique Moleculaire, CNRS, Rue Charles Sadron, 45071 Orleans, France
3Department of Chemistry, University of Waterloo, ON, Canada, N2L 3G1
We present the successful use of path integral molecular dynamics (PIMD) in
conjunction with the path integral Langevin equation thermostat (1) for
sampling systems that exhibit nuclear quantum effects, notably those at low
temperatures or those consisting mainly of hydrogen or helium. To test this
approach, the internal energy of doped helium clusters are compared with
white-noise Langevin thermostatting and high precision path integral monte
carlo (PIMC) simulations. We comment on the structural evolution of these
clusters in the absence of rotation and exchange as a function of cluster size. To
quantify the importance of both rotation and exchange in our PIMD simulation,
we compute band origin shifts for (He)N-CO2 as a function of cluster size and
compare to previously published experimental and theoretical shifts (2). A
convergence study is presented to confirm the systematic error reduction
introduced by increasing path integral beads for our implementation in the Molecular Modelling Toolkit (MMTK) software
package. Applications to sugars are explored at biological temperatures by calculating both equilibrium and dynamic
properties using the methods presented.
(1) M. Ceriotti, M. Parrinello, and D. E. Manolopoulos, J Chem Phys 133, 124104.
(2) H. Li, N. Blinov, P.-N. Roy, and R. J. L. Roy, J Chem Phys 130, 144305.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
5
Session III, Fri 7:20 pm
Novel Targets and Chemical Probes Identified by Systematic Chemogenomic Profiling:
A Step Towards an Empirical Definition of the Druggable Genome
Guri Giaever
Faculty of Pharmacy, Faculty of Molecular Genetics, and McLaughlin Centre for Molecular Medicine
University of Toronto, Toronto, Ontario
Small-molecule chemical probes that selectively inhibit specific cellular proteins are powerful tools for studying biological
processes. While complementary to genetic studies, they have distinct advantages including the ability be applied in a
rapid, tunable and reversible manner. Moreover, chemical probes are often transferable across organisms, increasing their
utility and bypassing the requirement of available genetic tools. The discovery of novel chemical probes is of particular
importance in light of the fact that the number of truly novel drugs emerging each year has remained flat for decades
despite enormous increases in pharmaceutical research and development spending. Using our well-validated chemical
genomic platform in yeast, HaploInsufficiency Profiling and Homozygous Profiling (HIPHOP), our lab has embarked on such
a large-scale discovery effort and screened >2,500 known and novel compounds genome-wide. One of the unique
advantages of our automated HIPHOP platform is the ability to simultaneously identify both the protein target(s) and the
corresponding small molecule inhibitor in vivo, in a single assay. Results from this ongoing endeavor are highlighted.
Contact: [email protected]
Session III, Fri 8:05 pm
Novel Reference Tracer Model for Quantification of Receptor Binding in vivo with Fluorescent Molecular Imaging
Kimberley S. Samkoe1,2
, Kenneth M. Tichauer2, Kristian Sexton
2, Scott C. Davis
2 and Brian W. Pogue
1,2
1Department of Surgery, Dartmouth Medical School, Hanover, NH USA 03755
2Thayer School of Engineering, Dartmouth College, Hanover, NH USA 03755
Over expression of key signalling receptors by cancers increase growth, replication and invasiveness of tumours. As such,
molecular targeted imaging agents provide an abounding resource for non-invasive investigation of tumours in vivo. One
shortcoming of in vivo fluorescent imaging approaches is the inability to accurately quantify targeted agent binding. We
propose a novel method of quantification where a targeted and non-targeted agent are administered and imaged
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
6
simultaneously. Computational analysis of the targeted and non-targeted agent uptake curves provides the binding
potential (BP): a unit-less measure of both receptor density and ligand binding affinity. Here we determine the BP of
epidermal growth factor (EGF) to EGF receptor (EGFR), a receptor overexpressed in many tumours and a target for
antibody therapy. Mice bearing either orthotopic AsPC-1 pancreatic tumours were imaged on a flatbed fluorescence
scanner and simultaneously injected with IRdye800CW conjugated to EGF and IRDye700DX-carboxylate (1:1 molar ratio).
The average BPs in several tissues were determined: 3.29±0.08 (AsPC-1 tumour), 3.37±1.08 (normal pancreas), and
0.10±0.07 (muscle). The muscle has a very low BP as expected and is considered the in vivo negative control. The normal
pancreas was found to have a similar BP to the AsPC-1 tumour; although AsPC-1 tumours overexpress EGFR compared to
normal pancreas, they have significantly lower cellular content (i.e., lower receptor density). For additional comparison,
the BP was determined for sub-cutaneous human U251 glioma tumours, also known to overexpress EGFR. A BP of
2.42±0.44 was determined for the U251 tumour (significant from AsPC-1 BP, p<0.05). The lower BP of the U251 tumour
agrees with the relative expression of EGFR in vitro and cellular content of the tumors. Assessment of the BP in vivo may
have implications for non-invasive stratification of patients for anti-cancer therapies as well as therapeutic monitoring.
Contact: [email protected]
Session III, Fri 8:25 pm
Nanoparticle Synthesis of Luminescent Materials: High Temperature, Mechanical, and Aqueous Solution Growth
Methods
Tiju Thomas1,4
, Kristin Poduska1, Cynthia Goh
2, Francis J. DiSalvo
3 and M. G. Spencer
4
1 Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, NL, Canada
2 Department of Chemistry, University of Toronto, Toronto, ON, Canada
3 Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA
The high chemical stability and low toxicity of gallium nitride (GaN) suggest that
luminescent nanoparticles based on this material could find potential use as
biomarkers. GaN has been widely targeted for optoelectronic applications because
its optical properties can be tuned by using rare earth (RE) dopants. In this work, we
present three low-cost methods for producing luminescent nanoparticles. First, an
ammonothermal route leads to GaN powders through a liquid phase growth
mechanism. A related growth process facilitates the synthesis of red-luminescent
europium (Eu) doped GaN powders. A chemical etching protocol enables 300%
improvement in the 622 nm emission from Eu:GaN powders (as shown in the Figure)
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
7
by removing dark oxide impurities. Second, optically active, nanosized RE:GaN can be made from as-prepared micron-sized
powders using a simple ball-mill process. Third, we describe an eco-friendly aqueous solution based synthesis for inorganic
phosphors based on polymer collapse and cross-linking of poly-acrylic acid. We will discuss the comparative benefits and
drawbacks of these three methods for producing luminescent nanoparticles for optical applications.
Contact: [email protected]
Session IV, Sat 9:30 am
Self-Assembly of DNA into Nanoscale Three-Dimensional Shapes
William Shih
Department of Cancer Biology, Dana-Farber Cancer Institute, Department of Biological Chemistry and Molecular
Pharmacology, and Wyss Institute for Biologically Inspired Engineering
Harvard University, Boston, Massachusetts
I will present a general method for solving a key challenge for nanotechnology: programmable self-assembly of complex,
three-dimensional nanostructures. Previously, scaffolded DNA origami had been used to build arbitrary flat shapes 100 nm
in diameter and almost twice the mass of a ribosome. We have succeeded in building custom three-dimensional structures
that can be conceived as stacks of nearly flat layers of DNA. Successful extension from two-dimensions to three-
dimensions in this way depended critically on calibration of folding conditions. We also have explored how targeted
insertions and deletions of base pairs can cause our DNA bundles to develop twist of either handedness or to curve. The
degree of curvature could be quantitatively controlled, and a radius of curvature as tight as 6 nanometers was achieved.
This general capability for building complex, three-dimensional nanostructures will pave the way for the manufacture of
sophisticated devices bearing features on the nanometer scale.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
8
Session IV, Sat 10:15 am
Structural Changes in Soluble Sensory Rhodopsin Transducer upon the Interaction with DNA
Shenlin Wang1, So Young Kim
2, Kwang-Hwan Jung
2, Vladimir Ladizhansky
1 and Leonid S. Brown
1
1Department of Physics, University of Guelph, ON, Canada
2Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, Korea
Anabaena sensory rhodopsin (ASR) is a recently discovered membrane photosensor with a unique signal transduction
cascade. It interacts with a soluble tetrameric transducer (ASRT), regulating expression of several genes related to the
utilization of light energy. Even though the X-ray crystal structure of ASRT is available, the mechanism of its interaction
with DNA is still unknown. We used solution NMR to characterize structure of ASRT tetramer in solution and to understand
the mechanism of the DNA-binding. Both X-ray crystal structures of ASRT and solution NMR data reveal seven β-strands
forming a rigid scaffold (β-face) and a flexible, partially disordered α-face, comprised by the C-terminal parts and loops.
We found that conformation of the α-face in solution is very different from that in the crystals. While C-termini of
crystalline ASRT are either α-helical or disordered, half of ASRT monomers in solution feature additional C-terminal β-
strand, whereas another half have their C-tails as random coils. Titration of ASRT solution with a 20 bp fragment of the pec
operon promoter showed that this structural heterogeneity may be functionally meaningful, as only monomers with β-
structured C-tails bind the DNA. The DNA binding occurs with submicromolar affinity and 1:1 stoichiometry (DNA:ASRT
tetramer), and results in a significant ordering of the α-face of the interacting monomers, which involves the extension of
the C-terminal β-strand and reorganization of the first loop. Changes in NMR signals of the ASRT sidechains suggest that
specific Arg and Asn/Gln residues are involved in the interaction with DNA.
Contact: [email protected]
Session IV, Sat 10:35 am
Quantification of Fluorophore Copy Number from Intrinsic Fluctuations During Fluorescence Photobleaching
Andrew Rutenberg
Department of Physics, Dalhousie University, Halifax, NS.
I will present a novel technique for quantifying the cellular copy-number of fluorophores that relies on the random nature
of the photobleaching process. Our approach does not require single-molecule sensitivity, and therefore can be used with
commonly used epifluorescence microscopes. Fluctuations arising from photobleaching are used to estimate the
proportionality between fluorescence intensity and copy-number, which can then be used with subsequent intensity
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
9
measurements to estimate copy-number. Significantly, we also calculate and verify the statistical errors of our approach.
Our approach can be applied to immobile fluorophores, does not require cellular division, and has an experimentally
adjustable rather than an intrinsic timescale. I will also present experimental tests of our approach.
Contact: [email protected]
Session V, Sat 1:30 pm
Hydrophobic Hydration Shell Spectroscopy
Dor Ben-Amotz
Department of Chemistry
Purdue University, West Lafayette, Indiana
Vibrational (Raman) spectroscopy and multivariate curve resolution (MCR), are
combined to uncover spectral features arising from the hydration-shells around
dissolved hydrophobic groups. The results reveal a high-frequency OH stretch peak
arising from water dangling OH bonds, as well as lower frequency OH stretch
features arising from H-bonded hydration-shell water molecules. The structure,
spectrum, and dynamics of the water dangling OH bonds are elucidated using
computer simulations in which the OH vibrational frequency is correlated with the
local electric field strength experienced by each OH group in the solution.
Moreover, the hydration-shells of aromatic solutes reveal a peak attributed to a pi-
H-bond between water and the aromatic ring. Temperature dependent
measurements are used to determine the enthalpy difference between the
dangling and pi-H-bonded OH groups bond and an average H-bond in bulk water.
Frequency shifts of the water-water H-bonds in the hydrophobic hydration shells
reveal difference between the strength of hydration shell H-bonds and those in bulk water, as well as how the hydration
shell H-bond distributions change with temperature.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
10
Session V, Sat 2:15 pm
Exploring the role of hydrophobic interaction in secondary structure formation using a simple atomic model
Cristiano Dias, Mikko Karttunen and Hue Sun Chan
Department of Biochemistry and Molecular Genetics, University of Toronto, Toronto, ON.
Hydrophobic interactions play a key role in the life of proteins and are considered a main driving force for folding.
However, their role in the formation of secondary structures is not well understood. Using geometrical arguments and a
simple all-atom implicit-water model for small peptides, we found that the attractive part of hydrophobic interactions
likely favor the formation of beta-sheets. In the context of the model, whether alpha-helices or beta-sheets are favored is
sensitive to the range of the attractive hydrophobic interactions. For example, alpha-helices are favored in poly-alanine
when the the range for attractive interaction in the model is unphysically extended. This trend may account for the
divergent conclusions from different modeling studies. Moreover, we identified a significant role of desolvation barriers in
secondary structure formation. For the cases of poly-alanine, poly-valine, and poly-leucine peptides we have tested,
desolvation barriers penalize alpha helices and thus favor the formation of beta sheets in collapsed conformations of the
peptides. These model observations offer new insights into the physical origins of secondary structure propensities of the
amino acids.
Contact: [email protected]
Session V, Sat 2:35 pm
Hofmeister Salts, Water Hydrogen Bonding and the Hydrophobic Effect
Mazdak Khajehpour
Department of Chemistry, University of Manitoba, Winnipeg, MB.
Hydrophobic interactions are one of the most important interactions maintaining the folded form of proteins. The
theoretical analysis of Dill predicts that the strength of these interactions depend upon the nature of hydrogen bonding
between water molecules. Water molecules participate in hydrogen bonding in two distinct modalities: linear and bent.
According to Dill a preponderance of linear hydrogen bonds strengthens hydrophobic interactions, while an excess of bent
hydrogen bonds increases the solubility of hydrophobic moieties in water. We have tested this hypothesis using
Hofmeister salts in order to perturb the distribution between bent and linear hydrogen bonds in water, and quantified the
effects of these pertubations on hydrophobic interactions such as micelle formation and cyclodextrin intercalation. Our
results demonstrate great correlation between hydrophobic effects and linear hydrogen bonding in water.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
11
Session VI, Sat 4:30 pm
Exploring the Configurational and Free Energy Landscape of Biomolecules under Extreme Conditions:
from Model Biomembranes to Proteins
Roland Winter
Department of Physical Chemistry
Technical University Dortmund, Dortmund, Germany
Lipid bilayers, which provide valuable model systems for biomembranes, display a variety of polymorphic phases,
depending on their molecular structure and environmental conditions, such as pH, ionic strength, temperature and
pressure. By using calorimetric, spectroscopic and diffraction techniques, the temperature and pressure dependent
structure and phase behavior of lipid systems, differing in chain configuration and headgroup structure has been studied
[1-3]. Moreover, neutron small-angle scattering and two-photon excited fluorescence microscopy have been used to study
the lateral organization of phase-separated lipid membranes and raft mixtures as well as the influence of peptide and
protein incorporation on membrane structure and dynamics, also under high pressure conditions [1,4,5]. Furthermore, we
discuss pressure as a kinetic variable. Applying the pressure-jump relaxation technique in combination with time-resolved
synchrotron X-ray diffraction, the kinetics of various lipid phase transformations was investigated, including studies of
membrane fusion processes [1,3,6]. The technique has also been applied for studying protein folding reactions. We
present data on the pressure-induced un/refolding of various proteins using synchrotron X-ray scattering and Fourier-
transform infrared spectroscopy, which monitor changes in the tertiary and secondary structural properties of the proteins
upon pressurization or depressurization [1,2]. A simple thermodynamic approach is used for studying the stability of
proteins as a function of both temperature and pressure and express it as a three-dimensional free energy surface. In this
regard, we take advantage of a series of different techniques in the evaluation of the conformation of the proteins and in
evaluating the changes in the thermodynamic parameters upon unfolding, such as the heat capacity, enthalpy, entropy,
volume, isothermal compressibility and expansivity [1,7,8]. The results demonstrate that combined temperature-pressure
dependent studies can help delineate the free energy landscape of proteins and hence help elucidate which features and
thermodynamic parameters are essential in determining the stability of the native conformational state of proteins.
Finally, recent advances in using pressure for studying misfolding and aggregation (amyloidogenesis) of proteins will be
discussed [1,9-12]. Our approach reveals new insights into the pre-aggregated regime as well as mechanistic details about
concurrent aggregation pathways and the differential stability of the protein aggregates as a function of temperature and
pressure.
[1] R. Winter, D. Lopes, S. Grudzielanek, K. Vogtt, J. Non-Equilib. Thermodyn. 32, 41 (2007)
[2] I. Daniel, P. Oger, R. Winter, Chem. Soc. Rev. 35, 858 (2006)
[3] C. Jeworrek, M. Pühse, R. Winter, Langmuir 24 , 11851 (2008)
[4] C. Nicolini, A. Celli, E. Gratton, R. Winter, Biophys. J. 91, 2936 (2006)
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
12
[5] E. Powalska, S. Janosch, E. Kinne-Saffran, R. Winter, et al., Biochemistry 46, 1672 (2007)
[6] C. Conn, O. Ces, R. Winter, J. M. Seddon, R. H. Templer, et al., Phys. Rev. Lett. 96, 108102 (2006)
[7] L. Mitra, J.-B. Rouget, B. Garcia-Moreno, C. A. Royer, R. Winter, ChemPhysChem 9, 2715 (2008)
[8] L. Mitra, R. Winter, K. Akasaka, C. Royer, et al., J. Am. Chem. Soc. 129, 14108 (2007)
[9] S. Grudzielanek, V. Smirnovas, R. Winter, J. Mol. Biol. 356, 497 (2006)
[10] N. Javid, K. Vogtt, C. Krywka, M. Tolan, R. Winter, Phys. Rev. Lett. 99, 028101 (2007)
[11] R. Mishra, R. Winter, Angew. Chem. Int. Ed. 47, 6518 (2008)
[12] S. Gruzielanek, Y. Zhai, R. Winter, ChemPhysChem 11, 2016 (2010)
Contact: [email protected]
Session VI, Sat 5:15 pm
Structure and Topology of the N-terminal Tail and First Transmembrane Segment of the Apelin Receptor in Three
Different Micelle Types
David N. Langelaan1 and Jan K. Rainey
1,2
1 Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS.
2 Department of Chemistry, Dalhousie University, Halifax, NS.
Membrane mimetics such as micelles, bicelles or bilayers provide an essential tool for studying
membrane proteins and their fragments. In this study, following the “divide and conquer” strategy,
we present the structure of the N-terminal tail and first transmembrane segment of the apelin
receptor (human APJ residues 1-55, APJ55). Using sodium dodecylsuphate (SDS),
dodecylphosphocholine (DPC) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol)
(LPPG) micelles, the structures of APJ55 are compared by both circular dichroism (CD) and nuclear
magnetic resonance (NMR) spectroscopy. Far-UV CD spectroscopy demonstrates that APJ55 adopts a
largely helical structure in each environment. Through 1H-
15N HSQC NMR experiments, APJ55 is
observed to be in similar though distinct conformations in SDS, DPC and LPPG micelles. The high-resolution structures of
APJ55 in the various micelles consist of a helix-kink-helix motif in the micelle-spanning transmembrane region. Overall, this
study provides important insight into the consistency of structures generated through different membrane mimetics.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
13
Session VI, Sat 5:35 pm
Modular Evolution and the Design of a Threefold Symmetric Globular Protein
Aron Broom1, A.C. Doxey
2, Y.D. Lobsanov
3, D.R. Rose
2, P.L. Howell
3, B.J. McConkey
2 and E.M. Meiering
1
1 Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry.
2 Department of Biology, University of Waterloo, Waterloo, ON.
3 Program in Molecular Structure & Function, The Hospital for Sick Children, Toronto, ON.
The high frequency of internal symmetry in common protein folds is presumed to reflect their evolutionary origins from
the repetition and fusion of primordial peptide modules, but very little is known about the primary sequence and physical
determinants of this process. A sequence and structural analysis of symmetric subdomain modules within an abundant
and ancient globular fold, the β-trefoil, reveals that modular evolution is not just a relic of the primordial past, but is an
ongoing and recurring mechanism for rediscovering symmetry within a symmetric fold. In this modular evolution we see
the recurring use of small subdomain modules as building blocks for new symmetric proteins. Based on our analysis, we
designed the first functional and fully threefold symmetric globular protein, ThreeFoil. The design process utilized several
computational design methods such as consensus design, homology modelling, and design via energy functions. ThreeFoil,
which exhibits complete sequence and near structural identity between symmetric modules, is highly soluble, binds
carbohydrates, and has remarkably high thermal and kinetic stability. These findings have far-reaching implications for the
evolution of proteins via subdomain modules, and demonstrate the utility of using such subdomain modules in the design
of proteins with desirable properties (i.e. thermal stability) for therapeutic and biotechnological applications.
Contact: [email protected]
Session VI, Sat 5:55 pm
Ultrafast Energy Transfer Dynamics in Light-Harvesting Proteins
Rayomond Dinshaw and Gregory D. Scholes
Department of Chemistry, Institute for Optical Sciences, and Center for Quantum
Information and Quantum Control, University of Toronto, Toronto, ON.
Photosynthesis is necessary for nearly all life on Earth. Light-harvesting proteins
capture electromagnetic energy and funnel it to reaction centers where it is utilized
in chemical reactions. The electronic energy transfer (EET) happens with near perfect
quantum efficiency on a femtosecond timescale.
Recent experiments suggest that EET in light-harvesting proteins can occur
coherently through a wave-like mechanism even at physiological temperatures. The
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
14
observance of long-lived coherences (~200 femtoseconds) at physiological temperatures is surprising since environmental
effects are expected to dephase the system on a much quicker timescale. These results have far reaching consequences as
they suggest that quantum mechanical phenomena occur in wet, noisy biological systems and maybe relevant to biological
processes. We investigate EET in light-harvesting proteins extracted from cryptophyte algae and phycobilisomes using
two-dimensional photon echo (2D PE) spectroscopy. 2D PE spectroscopy is a nonlinear, ultrafast, optical technique that
allows for femtosecond resolution of dynamics and is particularly sensitive to interchromophoric coupling, electronic
coherences, and coherent energy transfer. By comparing energy transfer dynamics in various light-harvesting proteins we
are able to determine if coherent energy transfer provides an evolutionary advantage for some species or if it is ubiquitous
to all light-harvesting proteins.
Contact: [email protected]
Session VII, Sun 9:30 am
Chemical Interrogation of a Bacterial Life Cycle
Justin Nodwell
Department of Biochemistry and Biomedical Sciences
McMaster University, Hamilton, Ontario
The streptomycetes are an enormous genus of filamentous soil bacteria. They are best known as producers of bioactive
small molecules, many of which are used as antibiotics, immune suppressants, chemotherapeutic agents and other drugs.
These molecules are produced as part of their so-called ‘secondary metabolism’, a poorly understood and dispensable
offshoot of primary metabolism. The streptomycetes are also characterized by a fascinating and unusual life cycle that
involves the generation of differentiated spore-forming and antibiotic-producing cell types. Understanding this life cycle is
important for at least two reasons. First, streptomycetes do not express the majority of their secondary metabolic genes in
the laboratory: finding ways to activate their expression could lead to new drugs. Second, the sporulation process provides
a unique model for understanding bacterial cell division and chromosome segregation – both targets for the possible
develop of new antimicrobial agents. To understand this life cycle in greater detail we screened ~30,000 pure compounds
for inhibitory effects on its various steps. To date we have discovered two classes of molecules that remodel the spectrum
of secondary metabolites produced by the bacterium, and another set that interfere with normal spore-formation.
The two sets of secondary metabolism remodelers are structurally distinct and clearly act on different biological pathways,
we assume via different molecular targets. We note that both classes activate the production of some metabolites and
repress others and that the changes they induce are distinct and reproducible. Importantly, both can be used to trigger the
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
15
production of cryptic secondary metabolites in many different species, including naïve wild isolates. We are elucidating the
mechanism of action of these molecules by transcriptomic analysis of the model species Streptomyces coelicolor.
The six sporulation inhibitors similarly act via several distinct mechanisms and all of them act on the distantly related
bacterium Bacillus subtilis in addition to the streptomycetes – arguing that they bind evolutionarily conserved targets.
Three inhibit cell division, perhaps by direct interaction with the bacterial cytoskeleton. Another three induce a rare cell
foreshortening phenotype. While potency varies, all six of these molecules exhibit antimicrobial activity against Bacillus
subtilis suggesting that they could constitute novel antimicrobial drug leads.
Contact: [email protected]
Session VII, Sun 10:15 am
Changes in the Mechanical Properties of Pseudomonas aeruginosa Bacterial Cells Induced by Antimicrobial Peptides
Shun Lu1, Hanna-Leena Alakomi
2 and John R. Dutcher
1
1 Department of Physics, University of Guelph, Guelph, ON, Canada N1G 2W1.
2 VTT Technical Research Centre of Finland, P.O. Box 1000 (Tietotie 2), FI-02044 VTT.
The cell envelope of gram-negative bacteria plays an important role in maintaining cellular shape, supporting turgor
pressure, selectively transfering molecules in and out of the cell, and adapting the cell to its external environment.
Because of these important structural and functional roles, both the inner membrane and outer membrane of the
bacterial cell can be major targets for antimicrobial peptides, which are a class of antibiotics that rarely induce bacterial
resistance (1). In our research group, we have developed an atomic force microscopy nano-creep technique (2) to study
the mechanical properties of individual Pseudomonas aeruginosa bacterial cells in a liquid environment. In the present
study, we have used this technique to measure changes to the mechanical properties of the cells produced by exposing
the cells to well-studied antimicrobial peptides: polymyxin B (PMB) and its derivative polymyxin B nonapeptide (PMBN).
We found that the creep response of cells under a fixed applied load is very different after exposure of the cells to PMB
and PMBN, which is possibly due to the disruption of its outer membrane. To describe the viscoelastic properties of the
cells exposed to PMB and PMBN, we found that it was necessary to use a four element spring and dashpot model, instead
of the three element standard linear solid model that describes the viscoelastic properties of cells in Millipore water (2).
We also found that PMB and PMBN have qualitatively different effects on the stiffness of the cell membrane. These
measurements provide a first step towards understanding the different mechanisms of action of PMB and PMBN on
bacterial cells.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
16
References:
1. H. Tsubery, I. Ofek, S. Cohen, and M. Fridkin, Biochem., 39, 11827-11844, 2000
2. V. Vadillo-Rodriguez, T. J. Beveridge, and J. R. Dutcher, J. Bacteriol., 190, 4225-4232, 2008.
Contact: [email protected]
Session VIII, Sun 10:55 am
Peptides and Proteins in Detergent and Lipids
Régis Pomès Molecular Structure and Function, Hospital for Sick Children, and Department of Biochemistry
University of Toronto, Toronto, Ontario.
By compartmentalizing cells and organelles, biological membranes provide a structural framework underlying many
essential processes in the chemistry of life. Integral membrane proteins fulfill vital functions involving signalling,
recognition, and the exchange of ions and nutrients. Accordingly, the malfunction of membrane proteins is linked to
numerous diseases such as cystic fibrosis. Membranes are also the arena in which many battles of bacterial infection and
immune response are played out. Furthermore, the interaction of amyloid-forming peptide oligomers with membranes is
thought to play a role in the neurotoxicity of severe degenerative pathologies such as Alzheimer’s and Parkinson’s
diseases.
Despite the importance of these processes to human health and disease, elucidating the molecular mechanisms
underlying the interaction of peptides and proteins with lipid membranes has remained challenging. High-performance
computing and efficient sampling algorithms make it possible to access length- and time-scales relevant to the structure
and function of peptides and proteins in membrane mimetics and lipid bilayers at the atomic level of detail. I will present
molecular simulation studies aimed at characterizing the physical and molecular bases of protein solvation by detergent
and lipids, the action of antimicrobial peptides on lipid bilayers, the self-organization of amyloidogenic peptides at
membrane-water interfaces, and the binding and transport properties of integral membrane proteins.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
17
Session VIII, Sun 11:40 am
The Primary Steps of Solar Energy Conversion – from Photosynthesis to Organic Solar Cells
Villy Sundström
Department of Chemical Physics,
Lund University, Lund, Sweden
Photosynthetic light-harvesting and energy conversion occur in highly sophisticated pigment-protein complexes. The
biological structures and processes are often used as inspiration for the development of artificial systems for solar
electricity or solar fuel production. Various nanostructured organic and hybrid materials are being explored for application
in photovoltaic solar energy conversion. Light harvesting, energy transport, charge photogeneration and recombination,
charge transport are the elementary processes accounting for the conversion of light energy into useful charge carriers.
These processes will be discussed for both photosynthetic and artificial systems. We show how a combination of time
resolved spectroscopy methods, covering the time scales from femtoseconds to milliseconds and spectral range from uv to
far infrared (THz frequencies), are powerful tools to study the light induced processes and provide mechanistic
information valuable for design of novel or optimized materials.
Contact: [email protected]
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
Abstracts
Poster Presentations Sessions: I – April 9
th, 11:00 am – 12:30 pm (Odd Numbered Posters)
II – April 9th
, 2:55 pm – 4:25 pm (Even Numbered Posters)
19
List of Posters
P1 Optical Labeling of Diagnostic Cell Receptors via Immunotargeted Nanoparticles
Ilia Auerbach-Ziogas, Sebastien Besner, David Rioux, Edward Parker, Chen Wang, Michel Meunier, Gilbert Walker.
P2 Characterization of a Novel Aptazyme
Jenna Collier, Dr. Thorsten Dieckmann
P3 Accelerated Molecular Dynamics in the Molecular Modelling Tool Kit using Graphical Processing Units
Stephen Constable, Pierre-Nicholas Roy
P4 The role of entropy and metal ions in the malachite green binding RNA aptamer
Jason Bernard Da Costa, Thorsten Dieckmann
P5 Volumetric Characterization of Sodium-Induced G-Quadruplex Formation
Helen Y. Fan, Yuen Lai Shek, Amir Amiri, David N. Dubins, Heiko Heerklotz, Robert B. Macgregor, Jr., and Tigran V. Chalikian
P6 The Effect of Nanoscale Phase Separated Diblock Copolymer Films on the Settlement of Marine Species
Claudia M. Grozea, John A. Finlay, Maureen E. Callow, James A. Callow and Gilbert C. Walker
P7 Controlling Protein Oligomerization with Surface Curvature on the Nanoscale
M. Kurylowicz, C. Gram, J. Atkinson and J. Dutcher
P8 Signature of Hydrophobic Hydration in Single Polymer
Isaac T. S. Li, Gilbert C. Walker
P9 In vivo photo-controlled gene expression system
Mostafizur Mazumder, Yih Yang Chen, G.Andrew Woolley and David R. McMillen
P10 Annealing effect of temperature on lamellar perforations in bicellar model membranes
Hannah Morales , Mu-Ping Nieh, V. A. Raghunathan, Georg Pabst, Thad Harroun, Kazuomi Nagashima, John Katsaras, Peter
Macdonald
P11 Elastin self-assembly: the contribution of hydrophobicity, proline residues and domain 30
Lisa D. Muiznieks, Megan Miao, Sean E. Reichheld, Eva Sitarz and Fred W. Keeley
P12 Attack Or Retreat: Two Modes Of Membrane Solubilisation By Surfactants
Mozhgan Nazari, Mustafa Kurdi, and Heiko Heerklotz
P13 The influence of perfluorinated surfactants on the performance of model pulmonary lung surfactant mixtures
Matthew F. Paige, Ala’a F. Eftaiha
20
P14 All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713
Hiren Patel, Clemens Tscheka, Katarina Edwards, Goran Karlsson and Heiko Heerklotz
P15 Parsing Partial Molar Volumes of Small Molecules: A Molecular Dynamic Study
Nisha Patel, David N. Dubins, Régis Pomès, and Tigran V. Chalikian
P16 NMR studies of an aggregation prone protein
Michael Piazza, Yay Duangkham, Donald E. Spratt, Thorsten Dieckmann and J. Guy Guillemette
P17 Interaction of Doxorubicin (DOX) with a Negatively Charged, Starch grafted Methacrylic acid (st-g-MAA) Co-polymer: Thermodynamic
and Spectroscopic studies
Gaurav Raval, Alireza Shalviri, Heiko Heerklotz and Xiao Yu Wu
P18 Big fat polymers: Lipids supported on thermosensitive microgels
Qasim Saleem, Neil J. Mackinnon, Baoxu Liu, Claudiu Gradinaru and Peter M. Macdonald
P19 Interactions of dodecyl phosphocholine with lipid membranes
Arpit Shah, Mozhgan Nazari, Hiren Patel, and Heiko Heerklotz
P20 Investigation of Opioid Receptor Internalization Dynamics
Katelyn M. Sheehan, David T. Cramb
P21 Volumetric Characterization of Interactions of Protein Groups with Glycine Betaine
Yuen Lai Shek and Tigran V. Chalikian
P22 Mechanical desorption of single fibronectin type III module from hydrophilic and hydrophobic surfaces
Weiqing Shi, Shan Zou, Gilbert C. Walker
P23 Nanoparticles for Tolerogenesis: Analyzing Key Properties
Amy Tekrony, Usama Al-Atar, Vincent Wright, Anne Cooper, David Cramb, Lori West, Jillian Buriak
P24 Chemically powered molecular machines
Snigdha Thakur, Raymond Kapral
P25 NMR and AFM characterization of recombinant spider silk monomers and fibers
Marie-Laurence Tremblay, Lingling Xu, Paul X.-Q. Liu, Jan K. Rainey
P26 Nanoparticle Interactions in Chicken Embryo Blood Vessels.
Kristin Yaehne and David T. Cramb
P27 Concentration-Dependent Structural Polymorphism of G-Rich DNA Sequences
Rashid Abu-Ghazalah, Steve Rutledge, Lewis Lau, Robert Macgregor, Amr Helmy
21
P28 Role of DNA breathing dynamics in gene expression : Some relevant Brownian functionals
Malay Bandyopadhyay, Shamik Gupta, Dvira Segal
P29 Structural and Functional Insights into Membrane Transporters and Sensors by Solid-State NMR
Lichi Shi, Izuru Kawamura, Shenlin Wang, Meaghan Ward, Ying Fan, Evelyn Lake, Mumdooh Ahmed, Kwang-Hwan Jung, Vladimir
Ladizhansky, Leonid S Brown
P30 Microsecond Pore Dilation Kinetics and Hydrophobic Gating of Magnesium Transport in the CorA System
Chris Neale, N. Chakrabarti, Emil F. Pai, and Régis Pomès
P31 Molecular Mechanism of Proton Uptake in Cytochrome c Oxidase
David Caplan, Rowan Henry, Elisa Fadda, Régis Pomès
P32 The Role of Melatonin in Amyloid Fibril Formation on Lipid Membrane
Youngjik (Vince) Choi, Ravi Gaikwad, Zoya Leonenko
P33 Effect of the lipid environment on interaction of P-glycoprotein with drugs
Adam T. Clay and Frances J. Sharom
P34 Effect of cholesterol and lipid composition on amyloid fibril formation on a model lipid membrane.
Elizabeth Drolle, Francis Hane, Erin Faught, Ravi Gaikwad, Zoya Leonenko
P35 Modelling Pore Formation in Bacterial Membranes by Anti-Microbial Peptides
Chris S. DiLoreto, Robert A. Wickham
P36 Two Novel Rhodopsins of Phaeosphaeria: Representatives of Distinct Subgroups of Fungal Rhodopsins
Y. Fan, R. Oliver, and L.S. Brown
P37 Statistics of amino acid hetero- and homo-multiplets in protein chains. Deviation from random behaviour based on residual polarity
and location in secondary structural elements.
C. Gong and G.A. Arteca
P38 Prediction and Analysis of Novel Interaction Motifs in Yeast
Yuan Gui
P39 Experimental Evidence for Ion Transport in Neuronal Uncoupling Proteins: Implications in Structure – Function relationships.
Tuan Hoang, Matthew D. Smith, and Masoud Jelokhani-Niaraki
P40 Moh1, a new player in repair of double stranded DNA breaks through non-homologous end joining in Saccharomyces cerevisiae
Mohsen Hooshyar, Matthew Jessulat, Ashkan Golshani
P41 Effect of Antimicrobial Agents on MinD Protein Oscillations in E. coli Bacterial Cells
Corey Kelly, Maximiliano Giuliani, Megan Murphy, John Dutcher
22
P42 A General and Efficient All-Atom Simulation Method to Determine the Equilibrium Orientation of Transmembrane Proteins in
Membranes.
Kethika Kulleperuma, John Holyoake and Régis Pomès
P43 Effects of L- vs. D-Isomers of Cationic Antimicrobial Peptides on Alginate Binding and Antimicrobial Activity
Soyoung Lee, Lois M. Yin, and Charles M. Deber
P44 Atomic force spectroscopy to measure lateral compression in lipid membrane.
Victor Malkov, Elizabeth Drolle, Ravi Gaikwad, Gilbert Walker and Zoya Leonenko
P45 Conformation-induced binding of extracellular loops of the apelin receptor with the apelin ligand in lipid environments
Pascaline Ngweniform and Jan K. Rainey
P46 Kinetics of Membrane Fusion Induced by Small Lipophillic Molecules
Trinh T. Nguyen and David T. Cramb
P47 Identification and Characterization of Novel Genes Involved in Non-Homologous End-Joining Pathway in the Yeast Saccharomyces
cerevisiae
Katayoun Omidi, Matthew Jessulat, Mohsen Hooshyar, Bahram Samanfar, Ashkan Golshani
P48 Correlating predicted and observed aggregation for a disease-linked protein.
Heather Primmer
P49 Imaging the Enzymatic Degradation of Cellulose Fibers Using Surface Plasmon Resonance
Adam N. Raegen, Kyle Reiter, Scott G. Allen, John R. Dutcher
P50 Aggregated yet Disordered: A Molecular Simulation Study of the Self-Assembly of Elastin
Sarah Rauscher, Régis Pomès
P51 Functional Genomics of Translation Pathway in the Yeast Saccharomyces cerevisiae
Bahram Samanfar, Le Hoa Tan, Firoozeh Chalabian, Katayoun Omidi, Ashkan Golshani
P52 Transmembrane Domain of the p14 Membrane Fusion Protein: From Structure to Function
Muzaddid Sarker, Jan K. Rainey, and Roy Duncan
P53 The Oligomeric Nature Of The M2 Muscarinic Cholinergic Receptor As Revealed In The Allosteric Effect Of Tacrine
Rabindra V. Shivnaraine, Xi-Ping Huang, John Ellis and James W. Wells.
P54 Dissecting the folding energetics and switching caused by myristoylation of hisactophilin.
Martin TJ Smith, Joseph Meissner, Elizabeth M Meiering
P55 Stability and aggregation studies of fALS-associated mutant Cu-Zn-Superoxide Dismutase
Helen Stubbs
23
P56 Characterization of an Iron-Sulfur Binding Protein in the Tail Tip of Bacteriophage Lambda
William Tam, Alan Davidson
P57 Identification and characterization of novel translation related genes in Saccharomyces cerevisiae
Le Hoa Tan, Bahram Samanfar and Ashkan Golshani
P58 Intrinsic Dynamics of the Exchange Protein Directly Activated By Cyclic AMP (EPAC)
Bryan VanSchouwen, Federico Fogolari, Giuseppe Melacini
P59 Mapping drug interactions in the substrate-binding pocket of the P-glycoprotein multidrug efflux pump
David Ward and Frances J. Sharom
P60 Combining bulk biochemical assays with single-molecule techniques to study the regulation of microtubule dynamics by motor proteins
Michal Wieczorek, Susanne Bechstedt, Gary Brouhard
P61 Morphology and Nanomechanics of Type II collagen
Chuan Xu and Erika F. Merschrod S.
P62 Interaction of α-Synuclein and its A30P Variant with Vesicles that Mimic Mitochondrial Membranes
Yoo Jeong Yang, Imola G. Zigoneanu, Alexander S. Krois, Md. Emdadul Haque, Gary J. Pielak
P63 Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in their interactions with bacterial membranes and
biofilm polysaccharides
Lois M. Yin, Michelle Edwards, Jessica Li, Christopher M. Yip, and Charles M. Deber
P64 Influences of Native Energetic Heterogeneity and Many-body Interaction in ΦΦΦΦ Value Analysis of Protein Folding
Zhuqing Zhang, Hue Sun Chan
P65 Peroxisome proliferator-activated receptor δ limits pathogenic Th cell responses in EAE
Fei Zhao, Roopa Bhat, Ajay Chawla, Lawrence Steinman, Shannon Dunn
P66 In-Solution Single-Molecule Studies of a Protease Conformational Dynamics
Abdullah Bahram, Amir Mazouchi, Walid Houry and Claudiu C. Gradinaru
P67 Refining the Electronic Hamiltonian of the Photosynthetic Protein, Phycocyanin 645, by Quantitative Fit of Linear Spectra
Chi-Han Chang, Rayomond Dinshaw, Gregory D. Scholes
P68 Theory and experiment of the first hyperpolarizability tensor of biological spherocrystals for second harmonic generation microscopy
Richard Cisek, Adam Tuer and Virginijus Barzda.
P69 The molecular love triangle – developing triple color FCCS for detecting complex biomolecular interactions
Holly Wobma, Megan Hartwick, Katya Grekova, Dr. David Cramb
24
P70 Drift-oscillatory steering with the forward-reverse method for calculating the potential of mean force
Bryan W. Holland, Mostafa NategholEslam, Bruno Tomberli, C. G. Gray
P71 Implementing the Photon Counting Histogram for Protein Studies
Yuchong Li and Claudiu Gradinaru
P72 An Improved Method for Studying Single Proteins Trapped in Lipid Vesicles
Baoxu Liu, Amir Mazouchi and Claudiu Gradinaru
P73 The Average Size of the Unfolded State of SH3 Domain of the Drosophila Adapter Protein Drk
Amir Mazouchi, Abdullah Bahram, Baoxu Liu, Sarah Rauscher, Régis Pomès, Julie Forman-Kay, Claudiu Gradinaru
P74 Using Opto-acoustics to Track Magnetite Nanoparticles for the Detection of Sentinel Lymph Nodes in Breast Cancer
GA Noble, MC Kolios, CJ Kumaradas
P75 Updates from the construction of the fast nonlinear microscope
Masood Samim and Virginijus Barzda
P76 Numerical analysis of third harmonic generation in nonlinear microscopy
Daaf Sandkuijl, Virginijus Barzda
P77 Unit Sphere Representation of the First Hyperpolarizability Tensor
Adam Tuer, Serguei Krouglov, Richard Cisek, Danielle Tokarz, Virginijus Barzda
P78 Innovative Nanostructures as Harmonophores for Third Harmonic Generation Microscopy
Danielle Tokarz, Richard Cisek, Adam Tuer, Virginijus Barzda, and Ulrich Fekl
25
P1
Optical Labeling of Diagnostic Cell Receptors via Immunotargeted Nanoparticles
Ilia Auerbach-Ziogas, Sebastien Besner, David Rioux, Edward Parker, Chen Wang, Michel Meunier, Gilbert Walker.
Department of Chemistry, University of Toronto.
The stage of development at the time of diagnosis is an essential indicator in the prognosis of cancer; the earlier that
diagnosis is accomplished, the better the patient outcome. In order to facilitate the pathology of blood cancers, we seek
the ability to characterize the cells that are diagnostically-relevant on a single-cell basis, considering their signature of
surface receptors in combination with their cellular morphology. Whereas current fluorescence techniques can only
accomplish the simultaneous detection of 3 or 4 receptor types in population screening, the research that I'll present
attempts to meet a higher standard of multimodal imaging where receptor labeling overlays morphology. Gold-silver
alloy nanoparticles (ANPs), synthesized with an arbitrary composition, are functionalized with targeting antibodies and
used as receptor labels. The ANPs exhibit a plasmon peak that is commensurate with their constitution, and so scatter
preferentially at wavelengths corresponding to this peak. Under dark-field microscopy, this scattering-bias is seen as
colour, making particles of different compositions visually differentiable, and thus suitable as labels for the multiplexed
recognition of surface cell receptors. A library of compositionally-varied alloys, each programmed to target a single
diagnostic receptor, could represent a simple and affordable advance for clinical hematopathology.
P2
Characterization of a Novel Aptazyme
Jenna Collier, Dr. Thorsten Dieckmann
Department of Chemistry, University of Waterloo
The recent discovery of catalytically active RNAs, ribozymes, has expanded the role of
RNA in biology. Unlike protein enzymes, RNA catalysts are composed of only four
different monomer units. Relative to protein enzymes, this greatly limits the amount
of dissimilar functional groups they may have. In spite of these limitations, RNA
catalysts boast impressive acceleration of catalyst mediated reaction rates. The
details of these mechanisms have not yet been confirmed. However, results from our
laboratory suggest that internal electrostatic fields present in the active sites of
ribozymes are a significant factor in their catalytic potential. To further confirm this theory, an in vitro selection project
was conducted to identify RNAs that can catalyze the ester bond hydrolysis of Fluorescein-diacetate (FDA) to Fluorescein.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
26
This was conducted via repeated rounds of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This was
done using a pull-down assay involving biotinylated FDA and magnetic streptavidin beads (see figure 1). To test the
relative binding of each round of selected RNAs to Fluorescein-diacetate and Fluorescein, the Ribogreen® RNA
quantification protocol was employed. The kinetics and thermodynamics of the hydrolysis reaction both inside and
outside the RNA binding pocket was studied using isothermal calorimetry. The results of this project aim to examine the
characteristics of the ribozyme active sites to which the binding of small molecule ligands occurs.
P3
Accelerated Molecular Dynamics in the Molecular Modelling Tool Kit using Graphical Processing Units
Stephen Constable, Pierre-Nicholas Roy
Department of Chemistry, University of Waterloo.
Molecular Dynamics (MD) is a computational tool that allows for microscopic
investigation of chemical systems, including the prediction of free energy changes and
reaction rate constants. Recently, it has been found that using Graphical Processing
Units (GPUs) for MD offers orders of magnitude increases in computational power
versus traditional Central Processing Units (CPUs). This allows for more accurate
simulations yielding more useful results, especially for the large system size inherent
in the life sciences. The Molecular Modelling Tool Kit (MMTK) software is used to
perform MD simulations, and can be augmented to take advantage of a GPU MD
code. This will provide optimal computational efficiency to software users.
Programming of the GPU will be achieved by utilization of the Open Molecular
Mechanics (OpenMM) library. Applications of the software to the prediction of the
kinetic and thermodynamic properties of pentose sugars are discussed, in particular the conformational analysis of beta-
arabinose will be considered.
Figure 1: The SELEX Pull-down Assay
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
27
P4
The role of entropy and metal ions in the malachite green binding RNA aptamer
Jason Bernard Da Costa, Thorsten Dieckmann
Department of Chemistry, University of Waterloo.
The binding of small molecule ligands to nucleic acid aptamers provides an
excellent model for the study of RNA drug interactions. The malalchite green
aptamer allows the detailed study of the importance of stacking and electrostatic
interactions, since ligand recognition does not involve hydrogen bonding
between ligand and RNA. Our studies have shown, that the ligand undergoes
significant changes in its electronic structure and charge distribution when bound
inside the binding pocket. In the studies presented here, the thermodynamics of
binding of the aptamer to its original ligand (Malachite Green) and three
derivatives (Cyrstal Violet, Tetramethylrosamine, PyroninY) were studied by
isothermal titration calorimetry. These studies revealed that entropy plays a large role in binding affinity and specificity.
This data combined with previous structural information reveals the role of the A30 nucleotide and a magnesium ion in
the adaptive nature of the binding. The presence of high salt causes a switch in the ligand preference, the affinity for TMR
at high salt or magnesium is higher than MG, while at low salt the affinity for MG is higher than for TMR. The ability of an
RNA aptamer that was selected for binding affinity to a specific target to change its ligand preference depending on the
solution conditions is intriguing and suggestive of a progression from simple binding to more complex functions.
P5
Volumetric Characterization of Sodium-Induced G-Quadruplex Formation
Helen Y. Fan, Yuen Lai Shek, Amir Amiri, David N. Dubins, Heiko Heerklotz, Robert B. Macgregor, Jr., and Tigran V.
Chalikian
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto
Oligodeoxyribonucleotides (ODN) with repeats of the human telomeric sequence can adopt different tetrahelical
conformations that exhibit similar energetic parameters. We studied the volumetric properties of the folded and
unfolded states of an ODN with four repeats of the human telomeric sequence, d[A(GGGTTA)3GGG], by combining
pressure perturbation calorimetry (PPC), vibrating tube densimetry, ultrasonic velocimetry, and UV melting under high
pressure. The volumetric measurements were carried out in aqueous buffers at pH 7 containing 20, 50, and 100 mM
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
28
NaCl. All of the methods employed yielded volumetric parameters that were in excellent agreement. The molar volume
changes, ∆V, of the conformational transition leading to formation of the folded state are large and positive. At 50mM
NaCl, the average transition volume, ∆Vtr, obtained from all the methods is 56.4 ± 3.5 cm3mol
-1 at the transition
temperature of 47˚C, with ∆Vtr decreasing with an increase in temperature. We carried out a molecular dynamics
simulation of the change in the intrinsic geometric parameters of the ODN accompanying quadruplex formation. On the
basis of the experimental and computational results, the folding transition of the ODN is accompanied by a loss of ~18%
of the net hydration of the coil conformation.
P6
The Effect of Nanoscale Phase Separated Diblock Copolymer Films on the Settlement of Marine Species
Claudia M. Grozea,* John A. Finlay,+ Maureen E. Callow,
+ James A. Callow
+ and Gilbert C. Walker*
*Department of Chemistry, University of Toronto, +School of Biosciences, University of Birmingham
Marine biofouling is the undesired accumulation of living organisms such as algae on artificial surfaces immersed in
seawater. The most effective coatings have been based on toxic metal compounds such as tin and are currently banned
or highly regulated. Novel materials must be developed free of biocides, with antifouling and/or foul release properties.
We investigated the settlement response of zoospores of the alga Ulva and the diatom
Navicula on diblock copolymer surfaces that have both nanoscale topographic features
and variation in local chemistry. In confined geometries, block copolymers can self-
assemble in a variety of ordered structures such as cylinders or lamella. Self-assembled
cylindrical nanopatterned surfaces with hydrophobic and hydrophilic domains were
produced using the diblock copolymer polystyrene-block-poly(2-vinyl pyridine) (PS-b-
P2VP) and polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA). Thin films were
obtained by spin-coating the polymers from toluene solutions onto nylon substrates.
The films were annealed using appropriate solvent gradients. In particular, the PS-b-
PMMA films were annealed by a new low temperature solvent annealing method.
Patterns with cylinders varying in size from 30 nm to 80 nm were obtained. Additional
patterns such as lamellar and spherical were also studied. Atomic force microscopy was used to characterize the films
throughout the fabrication process. The settlement response of zoospores and diatoms was evaluated using
fluorescence microscopy.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
29
P7
Controlling Protein Oligomerization with Surface Curvature on the Nanoscale
M. Kurylowicz, C. Gram, J. Atkinson and J. Dutcher
Physics Department, University of Guelph.
The influence of surface geometry on adsorbed proteins promises new possibilities in protein biophysics such as
molecular recognition of geometric cues, reporting on subcellular nanostructures, and modulation of ligand binding or
fluorophore quenching. We investigate the effect of surface curvature on the structure of beta-lactoglobulin (βLG) using
Single Molecule Force Spectroscopy. βLG is a model interfacial protein which stabilizes oil droplets in milk and is known
to undergo structural rearrangement when adsorbed onto a surface. We reliably control nanoscale surface curvature by
creating a series of close-packed monolayers of monodisperse polystyrene (PS) nanoparticles with diameters of 20, 60,
100, 140 and 180 nm, which are stable in aqueous buffer. By adsorbing βLG onto these hydrophobic surfaces and
collecting force-extension curves in the fluid phase we can compare the conformation of βLG on 5 different surface
curvatures with its conformation on a flat PS film. We demonstrate a transition from oligomeric to monomeric βLG as
the surface curvature is increased. Histograms of contour length from fits to peaks in the force-extension curves show a
single maximum near 60 nm for βLG adsorbed onto nanoparticles with diameters less than ~100 nm. For larger
nanoparticles this histogram approaches the profile which is observed for βLG adsorbed onto a flat PS film, with 2 and 3
maxima indicative of βLG monomers, dimers and trimers.
P8
Signature of Hydrophobic Hydration in Single Polymer
Isaac T. S. Li, Gilbert C. Walker
Department of Chemistry, University of Toronto.
Hydrophobicity underlies self-assembly in many natural and synthetic
molecular and nanoscale systems. A signature of hydrophobicity is its
temperature dependence. We report the first experimental evaluation
of the temperature and size dependence of hydration free energy in a
single hydrophobic polymer that tests key assumptions in models of
hydrophobic interactions in protein folding. Here, the hydration free
energy to extend three hydrophobic polymers with differently sized
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
30
aromatic side-chains was directly measured by single molecule force spectroscopy. Our results are three-fold: first, the
hydration free energy per monomer is a strong function of temperature and does not follow interfacial thermodynamics;
second, the temperature-dependence profiles are distinctively different among the three hydrophobic polymers as a
result of a hydrophobic size effect at the sub-nanometer scale; and lastly, the results indicate that free monomer
hydration free energy cannot be directly applied to monomers on a chain of a macromolecular systems without carefully
correcting for intra-chain hydrophobic interactions.
P9
In vivo photo-controlled gene expression system
Mostafizur Mazumder, Yih Yang Chen, G.Andrew Woolley and David R. McMillen
Department of Chemical physical Sciences, University of Toronto Mississauga. Department of Chemistry, University of
Toronto St.George.
Regulatable gene network systems providing easily controlled, conditional
induction or repression of expression are valuable tools in biomedical,
agricultural, biotechnology and synthetic biology research. Most of these
networks (including bistable switches, oscillators, intercell signalling devices,
and logic gates) rely on the administration of a limited set of exogenous
chemicals. Despite the general success of many of these systems, the potential
problems, such as unintended (pleiotropic) effects of the inducing chemical or
treatment, can impose limitations on their use. Moreover, it is experimentally
challenging to get intracellular responses using chemical as a time varying
stimulus. Synthetic biology to date has worked with a limited set of promoters
to design its networks. Placing gene expression under optical control is thus an
attractive prospect: it provides a simple method of altering the internal behaviour of cells without the need for time-
varying extracellular inducers; and it offers a new set of control mechanisms to be used in complex synthetic network
designs. The Woolley group has engineered a fused protein system that can be switched between two states using light
(schematically represented in the figure): a conformational change upon exposure to a specific wavelength causes the
protein to switch to a light-adapted state in which it binds to a specific DNA sequence. By incorporating the target
sequence into a modified promoter, this light-induced conformational change can be used to modify the expression level
of any desired gene. Here, we have designed such a promoter system along with regulatable light induced protein
expression to control the expression of tdtomato fluorescent protein using blue light. The system was introduced into
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
31
Escherichia coli to observe the light dependent tdtomato expression, which was validated with fluorimetry and SDS-PAGE
analysis. The results indicate that we are in fact able to control gene expression using an optical signal.
P10
Annealing effect of temperature on lamellar perforations in bicellar model membranes
Hannah Morales 1, Mu-Ping Nieh
2, V. A. Raghunathan
3, Georg Pabst
4, Thad Harroun
5, Kazuomi Nagashima
1, John
Katsaras 6,7
, Peter Macdonald 1*
1 Department of Chemistry, University of Toronto and Department of Chemical and Physical Sciences, University of
Toronto Mississauga 2 Chemical, Materials and Biomolecular Engineering Department, University of Connecticut
3 Raman Research Institute, Bangalore, India
4 Institute of Biophysics and X-ray Structure Research, Austrian Academy of
Sciences 5 Department of Physics, Brock University,
6 Oak Ridge National Laboratory, Neutron Sciences Directorate
7 National Research Council, Canadian Neutron Beam Centre
The thermal evolution of bicelles, composed of 1,2-dimyristoyl phosphatidylcholine (DMPC) and 1,2-dihexanoyl
phosphatidylcholine (DHPC), with a DMPC / DHPC molar ratio of 5, and doped with the negatively charged lipid, 1,2-
dimyristoyl phosphatidylglycerol (DMPG) at DMPG / DMPC molar ratios of 0.02 or 0.1, was studied using a combination
of 31
P NMR, 1H Pulsed Field Gradient (PFG) diffusion NMR, and Small Angle
Neutron Scattering (SANS). At temperatures above the gel-to-liquid crystalline
phase transition, the DHPC/DMPC/DMPG bicelles exhibit a morphology
consisting of DMPC/DMPG-rich lamellae perforated by DHPC-lined toroidal
holes. SANS results showed that the interlamellar spacing increased with
increasing temperatures. 31
P NMR demonstrated the migration of DHPC from
regions of high-curvature into planar regions as the temperature was
heightened. In addition, PFG diffusion NMR experiments show a decrease of
transbilayer water diffusion in positively aligned bicelles with increasing
temperatures. Thus, both NMR techniques indicate that the fractional surface
area of the pore defects is lessened as the temperature is increased. We
propose that the water previously occupying the lamellar perforations is
forced out as DHPC migrates to the planar regions at higher temperatures,
which accounts for the expansion of interlamellar spacing observed in SANS
measurements.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
32
P11
Elastin self-assembly: the contribution of hydrophobicity, proline residues and domain 30
Lisa D. Muiznieksa, Megan Miao
a, Sean E. Reichheld
a, Eva Sitarz
a and Fred W. Keeley
a,b
aMolecular Structure and Function, The Hospital For Sick Children, Toronto
bDepartment of Biochemistry, The University
of Toronto
Elastin is a hydrophobic, self-assembling protein of the extracellular matrix that provides vertebrate tissue with
properties of repetitive extension and recoil. Self-assembly is characterised by a temperature dependent phase
separation, called coacervation, where elastin monomers self-associate through hydrophobic sequences. Although
hydrophobic domains are typically rich in valine, alanine, glycine and proline, they display little sequence alignment.
While a high (40%) combined proline and glycine residue content bestows structural disorder and flexibility to most
hydrophobic elastin domains, the native sequence of hydrophobic domain 30 is uncharacteristically proline-poor across
many species. Recent studies show that reducing the number of proline residues within short elastin-like polypeptides
disrupts the reversibility of coacervation and results in the formation of aggregates, rich in b-structure, while removal of
proline residues promotes hydrophobic collapse into amyloid-like fibrils. Moreover, isolated domain 30 forms amyloid-
like fibrils from solution. Thus, we postulate that domain 30 may play a unique role in elastin assembly, potentially by
mediating hydrophobic interactions through b-motifs.
Here we investigated the effect of proline-poor elastin domains, including domain 30, on the assembly of elastin-like
polypeptides. Preliminary results suggest the addition of native proline-poor sequences, either individual domains in the
presence of control polypeptides, or incorporated into an elastin-like sequence, increased the stability of the coacervate.
Furthermore, mechanical testing of sheets cast from polypeptides containing domain 30 revealed the formation of stiffer
materials compared to control sequences. These data remain consistent with an increased number of interactions
promoted by proline-poor sequences.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
33
P12
Attack Or Retreat: Two Modes Of Membrane Solubilisation By Surfactants
Mozhgan Nazari, Mustafa Kurdi, and Heiko Heerklotz
Department of Pharmaceutical Sciences, University of Toronto
Solubilization of a lipid membrane by a micelle-forming surfactant starts with the appearance of mixed micelles
coexisting with mixed membranes and aqueous phase. This occurs at a characteristic membrane composition (often
given as the mole ratio Resat) and a characteristic aqueous concentration of surfactant that is typically somewhat below
the CMC of the pure surfactant. Thermodynamically, both criteria are fully equivalent because they linked by the
partition coefficient. However, there are two scenarios when it comes to the mechanism of solubilization. On one hand,
the surfactant may destroy the membrane by inducing a critical curvature strain superseding the mechanical stability of
the bilayer. On the other hand, the surfactant may de-mix from the membrane and associate in the aqueous phase to
form micelles that extract lipid from the membrane. Time-resolved fluorescence anisotropy data of DPH derivatives
agree with other parameters reflecting membrane order in that there is a characteristic, minimum order of a given
membrane (at a certain temperature) and most detergents have to disorder the membrane to this critical state before
micelles appear and solubilization proceeds. However few, mainly bio- and bioanalogous surfactants were found to
solubilize by demixing without critical disordering of the membrane. This may account for the superior performance of
the latter in solubilizing membrane proteins in their active state.
P13
The influence of perfluorinated surfactants on the performance of model pulmonary lung surfactant mixtures
Matthew F. Paige, Ala’a F. Eftaiha
Department of Chemistry, University of Saskatchewan.
Pulmonary lung surfactants play a crucial physiological role in reducing alveolar surface tension and thereby decreasing
the mechanical force (pressure) needed for normal respiration. A variety of medical conditions (e.g. Acute Respiratory
Distress Syndrome, traumatic lung injury) can inhibit production and performance of lung surfactant, and a viable
treatment for these conditions is introduction of artificial pulmonary lung surfactant mixtures into the lungs. While there
are numerous commercial surfactant mixtures available and approved for use, all current formulations suffer from
various shortcomings, and there is significant potential for improvements in performance.
In our group, we are investigating the physical chemistry of surfactant films prepared from mixtures of phospholipids
(DPPC and DPPG, the primary phospholipid components of native lung surfactant) and perfluorinated fatty acids, with a
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
34
view towards using these as artificial lung surfactant mixtures. We have characterized mixed monolayer films using a
combination of thermodynamic (compression isotherms and affiliated thermodynamic functions), kinetic (spreading and
recovery, rate of domain formation) as well as structural (film morphology) measurements. Our observations indicate
that addition of a perfluorinated component to pulmonary lung surfactant mixtures can impart a number of favorable
properties to the films, and that the use of this class of molecules for biomedical applications may be highly beneficial.
P14
All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713
Hiren Patel1, Clemens Tscheka
2, Katarina Edwards
3, Goran Karlsson
3 and Heiko Heerklotz
1
1Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, ON, M5S3M2 Canada.
2Dept. of
Pharmacy, University of the Saarland, Campus, Geb. A4.1, D-66123 Saarbrücken, Germany. 3Dept. of Physical and
Analytical Chemistry, Uppsala University, Box 579, 751 23 Uppsala, Sweden.
The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe
protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the
fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual
FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux
measurements and electron microscopy show that these FEs show a unique scenario of membrane permeabilization.
Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only ≈ 1µM free FE
and in 15% of the vesicles at 10 µM. We explain why this limited, all-or-none leakage could nevertheless account for the
killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then,
crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological
activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid
lipid resembles the behavior of detergent resistance.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
35
P15
Parsing Partial Molar Volumes of Small Molecules: A Molecular Dynamic Study
Nisha Patel1, David N. Dubins
1, Régis Pomès
2, and Tigran V. Chalikian
1*
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto
We used molecular dynamics simulations in conjunction with the Kirkwood-Buff theory to compute the partial molar
volumes for a number of small solutes of various chemical natures. We repeated our computations using modified pair
potentials; firstly, in the absence of the Coulombic term and, secondly, in the absence of the Coulombic and the
attractive Lennard-Jones terms. Comparison of our results with experimental data and the volumetric results of Monte
Carlo simulation with hard sphere potentials and scaled particle theory-based computations led us to conclude that, for
small solutes, the partial molar volume computed with the Lennard-Jones potential in the absence of the Coulombic term
nearly coincides with the cavity volume, VC. On the other hand, MD simulations carried out with the pair interaction
potentials containing only the repulsive Lennard-Jones term produce unrealistically large partial molar volumes of solutes
which are close to their excluded volumes. Our theoretical results are in good agreement with the reported empirical
schemes for parsing partial molar volume data on small solutes. In particular, our determined interaction volumes, VI,
and the thickness, δ, of the thermal volume, VT, for individual compounds are in good agreement with empirical
estimates. This work is the first theoretical study that supports and lends credence to the empirical algorithms of parsing
partial molar volume data that are currently in use for molecular interpretations of volumetric data.
P16
NMR studies of an aggregation prone protein
Michael Piazza, Yay Duangkham, Donald E. Spratt, Thorsten Dieckmann and J. Guy Guillemette
Department of Chemistry, University of Waterloo
Nuclear magnetic resonance (NMR) spectroscopy is an efficient method for studying the dynamics and structures of
protein-protein and protein-peptide complexes. Calmodulin (CaM), a ubiquitous Ca2+
-sensing protein, is able to bind and
regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. The investigation of CaM bound
to the CaM binding region of the inducible nitric oxide synthase (iNOS) isoform of NOS proved to be difficult due to the
propensity of the iNOS CaM binding domain to aggregate when not bound to CaM. In the present study an isotopically
labeled peptide of the CaM binding region of iNOS has successfully been expressed and purified. The peptide identity was
verified by both SDS-PAGE and mass spectroscopy (MS) and further characterized by NMR spectroscopy. These results
demonstrate an efficient approach for the expression and purification of individually stable isotope labeled protein
complexes for NMR analysis, even if one partner is prone to aggregation or has very low solubility. This technique can
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
36
also be used to label a specific type of amino acid or radiolabel all of the amino acids in one of the components
participating in a protein-protein or protein-peptide complex.
P17
Interaction of Doxorubicin (DOX) with a Negatively Charged, Starch grafted Methacrylic acid (st-g-MAA) Co-polymer:
Thermodynamic and Spectroscopic studies
Gaurav Raval, Alireza Shalviri, Heiko Heerklotz and Xiao Yu Wu
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto
Purpose: The aim of this study was to characterize the interactions of the anti-cancer drug doxorubicin with negatively
charged, starch grafted methacrylic acid co-polymer (St-g-PMAA) and develop an applicable thermodynamic model to
describe the binding data of the DOX/St-g-PMAA system. Methods: Using Isothermal Titration Calorimetry (ITC),the
mechanism of interaction between doxorubicin and St-g-MAA was determined by studying effect of pH and by
performing dilution experiments by lowering concentration of both drug and St-g-MAA (in parallel). Zeta potential and Z-
average size of DOX/St-g-PMAA complexes were monitored in DLS titration experiments. Fluorescence intensities were
monitored as a function of increasing concentration of DOX and also by performing titration of St-g-PMAA into DOX
solutions. Results: ITC results showed that there is a strong interaction between DOX and St-g-MAA co-polymer. In
Dilution experiments, the stochiometric ratios of [DOX]/[COOH] decreases by decreasing the concentration of both drug
and polymer(in parallel). These changes in the apparent stochoimetric ratios of [DOX]/[COO-] give some information
about how the drug binds to accessible binding sites COO-(present on St-g-PMAA NP’s) and if DOX molecules forms
dimers or trimers. DOX/St-g-PMAA are predominantly governed by electrostatics as suggested by ITC experiments at
various pH. DLS titration experiments suggest that DOX molecules have a tendency to form dimers and give detailed
insight into the mechanism of DOX dimer formation. In fluorescence experiments, we found that with increasing DOX
concentrations, the fluorescence intensities decrease and get quenched completely at certain DOX concentration. The
effect of adding St-g-PMAA to DOX solution on fluorescence intensities are variable and depends on which initial
concentration of DOX solution is chosen for titration experiment. Conclusions: We have shown that the interaction
between DOX and St-g-MAA are governed by electrostatics and depends on the aggregation behaviour of DOX. An
applicable model for self aggregation of DOX enables us to assess quantitatively the possible effects of drug aggregation
on the interpretation of drug-polymer binding data.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
37
P18
Big fat polymers: Lipids supported on thermosensitive microgels
Qasim Saleem*, Neil J. Mackinnon$, Baoxu Liu*, Claudiu Gradinaru* and Peter M. Macdonald*
*Department of Chemical and Physical Sciences, University of Toronto at Mississauga $Department of Biophysics,
University of Michigan
Lipid supported on polymer spheres has a variety of applications as model cell structures, drug delivery vehicles, and
biosensors. A subset of these systems employ "smart" polymers, which undergo an abrupt conformational change as a
result of a small stimulus. An example of this is poly(N-isopropylacrylamide) (pNIPAM) based microgels which undergo a
large volume decrease upon heating above the so-called volume phase transition (VPT) temperature. This property of
pNIPAM microgels have been employed to create drug delivery vehicles in which temperature acts as the trigger to
release a variety of loaded up molecules into the environment. However, these drug delivery vehicles generally suffer
from premature diffusion of contents and biocompatibility issues. To counter these issues, enveloping the microgels with
lipids (akin to biological membranes on cells) would be the logical step forward.
Phospholipid bilayer membranes are an essential feature in all living cells and hence, used as the coating for
macromolecules intended for biological use. To encase microgels with such membranes, we have employed two main
methodologies. One involves binding small lipid structures called liposomes to modified microgels. Liposome bound
microgels can be envisioned as drug delivery vehicles in their own right with the liposome, rather than the microgel,
acting as the drug carrier. The second method involves mixing the lipid with hydrophobically modified microgels followed
by multiple freeze-thaw cycles. The nature of the polymer-lipid structures that are formed in both cases can be
interrogated by fluorescence spectroscopy and microscopy, and solid-state NMR techniques.
Some of our major findings are that (1) both methods result in a lipid covering of the microgel albeit of different
structural morphology, (2) the microgel morphology can be viewed clearly using Total Internal Reflection Fluorescence
Microscopy, and (3) VPT disrupts the stability of bound liposomes but not an encompassing bilayer’s.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
38
P19
Interactions of dodecyl phosphocholine with lipid membranes
Arpit Shah, Mozhgan Nazari, Hiren Patel, and Heiko Heerklotz
Leslie Dan Faculty of Pharmacy, University of Toronto.
As a bio-analogous surfactant, n-dodecylphopsphocholine (abbreviated DPC, LPC, or FOS-choline-12) has been proven
useful for membrane protein studies. We present a detailed characterization of its temperature-dependent self-
association (CMC, ΔCp) and its interaction with liposomes. We describe membrane partitioning, membrane permeation,
membrane permeabilization (i.e., leakage to aqueous solutes), membrane lysis/solubilization to micelles and structural
aspects by means of ITC (demicellization, uptake-and-release, solubilization-and-
reconstitution assays), static and dynamic light scattering (NIBS), the lifetime-based dye
leakage assay (for details on the method see Soft Matter 2009, 5:2849), and the time-
resolved fluorescence anisotropy of membrane probes. The results give rise to the
hypothesis that DPC does virtually not flip across the membrane. As a consequence, it may
start to selectively solubilize the outer lipid leaflet at only half the overall detergent
concentration required for the onset of solubilization in an equilibrated system.
These results are important for the optimization of membrane protein solubilization and
reconstitution into proteoliposomes.
P20
Investigation of Opioid Receptor Internalization Dynamics
Katelyn M. Sheehan, David T. Cramb
Department of Chemistry, University of Calgary
Opioid agonists are the gold standard in the clinical treatment of chronic pain. Unfortunately, prolonged treatment with
certain opioids, specificially morphine, results in the development of tolerance whereby the analgesic efficacy of the drug
is diminished over time. Evidence has suggested that when triggered by morphine, opioid receptors behave differently
than when triggered by other drugs. Specifically, morphine does not efficiently promote receptor internalization and
recycling after binding. The dynamics of ligand-receptor binding and internalization may prove critical in understanding
the agonist-dependent response of opioid receptors, and thereby, the development of tolerance. This study investigates
the internalization kinetics for opioid peptides labelled with quantum dots in model cells expressing human µ-opioid
receptors.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
39
P21
Volumetric Characterization of Interactions of Protein Groups with Glycine Betaine
Yuen Lai Shek and Tigran V. Chalikian
Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences, University of Toronto
We report the volumetric properties of N-acetyl amino acid amides with non-ionizable side chains and oligoglycines in
binary solutions of water and glycine betaine, a protective cosolvent. We analyze these data within the framework of a
statistical thermodynamic formalism to determine the association constants for the reaction in which glycine betaine
binds to the peptide backbone and amino acid side chains replacing six water molecules. The association constants are
linked to the free energy of transfer of functional groups from water to a glycine betaine solution, ΔGtr. The transfer free
energy, ΔGtr is the sum of a change in the free energy of cavity formation, ΔΔGC, and the differential free energy of
solute-solvent interactions, ΔΔGI, in a concentrated glycine betaine solution and water. We compare our results with
similar data on volumetric measurements in water-urea binary mixtures. We find qualitative similarities in ΔΔGI for
protein functional groups in urea and glycine betaine solutions. Comparative analysis reveals that solute-cosolvent
interactions are favourable for the glycyl unit and the majority of amino acid side chains in both glycine betaine and urea
solutions. These results are consistent with the picture in which the water-to-betaine transfer free energy for a particular
atomic group is determined by a fine balance between an unfavourable change in the free energy of cavity formation,
ΔΔGC, and a generally favourable change in the free energy of solute-solvent interactions, ΔΔGI.
P22
Mechanical desorption of single fibronectin type III module from hydrophilic and hydrophobic surfaces
Weiqing Shi1, Shan Zou
2, Gilbert C. Walker
1*
1Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada
2 Steacie Institute for Molecular Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6,
Canada
A central dogma in biology is that protein structure dictates function; hence structural changes upon adsorption to a
surface should change protein function. This report tests the hypothesis that a fibronectin domain (FNIII10) is denatured
by a hydrophobic surface. A single molecule mechanical unfolding method was developed wherein protein was tethered
to a hydrophobic or hydrophilic surface at either its C- or its N-terminus and pulled from the other end by a soft
cantilever probe. Force transitions that occur at the position corresponding to the end of beta-strands are identified with
the secondary structure. Because sheering beta-strands gives the greatest resistance to pulling. The transitions may be
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
40
associated at least half of the time with 2 characteristic protein conformations at the surface. Identifying protein
orientations and denaturation at surfaces should be useful in identifying the loss and retention of protein activity when
surface associated, such as in biomolecular diagnostic tests, tissue engineering, as well as natural surface mediated
processes, such as amyloid formation.
P23
Nanoparticles for Tolerogenesis: Analyzing Key Properties
Amy Tekrony, Usama Al-Atar, Vincent Wright, Anne Cooper, David Cramb, Lori West, Jillian Buriak
Department of Chemistry, University of Calgary.
Up until a certain age, infants do not produce the same immunological response to foreign ABO blood group antigens as
older individuals who have a fully developed immune system. Therefore, with appropriate precautions, organs from
donors of incompatible blood types can be transplanted into infants safely, without rejection. ABO-incompatible infant
heart transplantation has been shown to result in development of immune tolerance to donor ABO antigens, which is
likely associated with persistence of donor antigens. Therefore, we hypothesized that intentional introduction of
synthesized ABO antigens to infants would also induce tolerance. Inducing tolerance to foreign ABO antigens in this
manner would extend the window of time during which safe transplantation from ABO-incompatible donors could be
performed, thus expanding the potential donor pool for infants and children who have passed the age at which their
immune system matures. A proposed method to induce tolerance is by conjugating antigens to synthesized silica
nanoparticles for introduction to immature individuals, which will allow maximum exposure of circulating lymphocytes to
the antigens. Here, characterization of the newly synthesized nanoparticles is shown. The nanoparticles are first
characterized in solution using fluorescence correlation spectroscopy (FCS) to determine brightness and tendency to
aggregate. Next, bright, non-aggregating nanoparticles are injected into the chorioallantoic membrane of chicken
embryos to determine detection efficiency, aggregation, and uptake characteristics using FCS. It has been found that the
PEG-coated silica nanoparticles with a core-shell synthesis can be easily detected and be made monodisperse with few
aggregation tendencies in the 100-200 nm range. Uptake into blood vessel walls has not been observed for these
nanoparticles over 100nm, indicating that the particles can circulate for a prolonged period of time in the blood stream,
thus enhancing the likelihood of successful tolerance induction.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
41
P24
Chemically powered molecular machines
Snigdha Thakur, Raymond Kapral
Chemical Physics Theory Group, Department of Chemistry, University of Toronto
Self-propulsion of micron and nanoscale objects in the low Reynolds number regime is commonly observed in biological
systems. Examples include bacteria that swim by executing nonreciprocal motions and molecular motors that undergo
conformational changes that lead to directed motion on filaments to carry out active transport tasks in the cell. The
directed biological molecular motor dynamics has inspired the design of synthetic nanomotors, with the ultimate goal of
utilizing their autonomous unidirectional motion in drug delivery, pick up and delivery of cargo, motion-based biosensing,
nano and micro fluidics etc.
Self-propelled nanodimers fueled by a chemical reaction taking place under nonequilibrium steady state conditions have
been designed. Particle-based simulations and analytical estimates of the velocity provide insight into the nature of
nanomotor directed motion. In addition to the individual motions of these self-propelled motors, ensembles of such
active objects exhibit distinctive collective motions.
P25
NMR and AFM characterization of recombinant spider silk monomers and fibers
Marie-Laurence Tremblay1, Lingling Xu
1,2, Paul X.-Q. Liu
1, Jan K. Rainey
1,3
1Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, Canada.
2 Department of
Biotechnology, Donghua University, Shanghai, China 3 Department of Chemistry, Dalhousie University, Halifax, NS,
Canada.
Spider silks are among the strongest and toughest naturally produced biomaterials. Despite these appealing properties,
very little is known about the mechanisms by which these fibers form or about how fiber structure relates to mechanical
properties. Using solution-state nuclear magnetic resonance (NMR) spectroscopy methods, chemical shift assignment
and analysis of a variety of heteronuclear NMR experiments was performed for a recombinant 13
C and 15
N labeled
aciniform spidroin spider silk protein 199 amino acid repeat. This protein is a key constituent of egg case sacs, providing
both flexibility and strength. When this protein is expressed as more the one repeat, it forms fibers that can be manually
drawn from solution and characterized by atomic force microscopy (AFM) and fluorescence spectroscopy. CD
spectroscopy and an initial low-resolution NMR structure both indicate that monomeric aciniform spidroin protein is
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
42
predominantly α-helical in solution but β-sheet in the fiber, as indicated by thioflavin T fluorescence co-localized with
fibers. Our ongoing work is correlating structure to mechanical properties to elucidate the mechanism by which spider
silk fibers form.
P26
Nanoparticle Interactions in Chicken Embryo Blood Vessels.
Kristin Yaehne and David T. Cramb
Department of Chemistry, University of Calgary
The propensity of a substance to be absorbed by an organism upon exposure greatly affects its toxic impact. For
nanoparticles, this retention and distribution behaviour, as well as its inherent toxicity, may be different from that of the
bulk material. As such, nanotoxicology is an increasingly popular and important area of study in adult organisms.
However, the behaviour of different nanoparticles in embryos is largely unknown and requires more investigation. In this
study, fluorescent polystyrene nanoparticles, with varying sizes and surface chemistries, were injected into chicken
embryos. Chicken embryos are used as models for developing organisms because the blood vessels of the chorioallantoic
membrane (CAM) can be easily accessed but are not yet fully developed and are leaky, or angiogenic. This property of
developing organisms puts them at greater risk of nanoparticle deposition and consequent uptake into developing
tissues. Two-photon excitation fluorescence correlation spectroscopy (TPE-FCS) was used to measure the concentration
of fluorescent particles in the blood stream of the chicken embryos and to monitor the subsequent uptake dynamics.
Previous studies by our group using TPE-FCS have shown that nanoparticles (quantum dots) smaller than 100nm are
absorbed by embryonic endothelial vasculature rapidly post-injection, and that nanoparticles of different surface
chemistries have different uptake characteristics. This study investigates the differential uptake of positively and
negatively charged, polystyrene fluospheres with diameters between 500 and 20 nm in chicken embryos.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
43
P27
Concentration-Dependent Structural Polymorphism of G-Rich DNA Sequences
Rashid Abu-Ghazalah§, Steve Rutledge
§, Lewis Lau
†, Robert Macgregor
†, Amr Helmy
§
§ The Edward S. Rogers Sr. Department of Electrical and Computer Engineering, Faculty of Applied Science and
Engineering, University of Toronto and † The Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy,
University of Toronto.
G-quadruplexes are four-stranded nucleic acids structures that arise from DNA (or RNA) sequences that have a large (>
40%) fraction of guanine residues. Such sequences are predominantly found in 5’-untranslated promoter regions (UTRs),
gene promoter regions, and at telomeric ends of chromosomes. In the presence of cations, these sequences either fold
to form monomeric or dimeric quadruplexes; or form tetramolecular complexes composed of four separate DNA (or
RNA) strands. The structures are stabilized via Hoogsteen hydrogen bonding networks that are formed between the
guanine residues and from the O6 carbonyl-cation coordination complexes.
G-quadruplexes are very polymorphic structures. Factors such as the type of cation in solution; the number of
consecutive guanine residues; the number of guanine-tract repeats; and the sequence that constitutes the non-guanine
portion, are all critical in determining the final topology of the G-quadruplex structure. Changing any of the parameters
can lead to a change in the polarity of the interacting strands (i.e. antiparallel vs. parallel) and/or the stoichiometry of the
four-stranded complex.
In this work, using circular dichroism (CD) and photonic crystal fibre (PCF)-enhanced Raman spectroscopy we show that
variants of the human telomeric sequence d(TTAGGG)n undergo previously uncharacterized structural transitions as a
function of oligonucleotide concentration. In the micromolar range, all sequences conform to previously published data;
however, in the millimolar range all variants display a time-dependent structural transition towards an all-parallel
complex. In addition, Raman modes of the guanine glycosidic conformations show a consistent trend moving from a
mixed anti and syn conformations to structures with predominantly anti modes with increasing cation concentration.
Finally, kinetic data reveal that the structural transitions stem from the conversion of monomolecular G-quadruplexes to
tetramolecular complexes. These results could have important implications for the interpretation of structural data
obtained by NMR and crystallography reported by others.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
44
P28
Role of DNA breathing dynamics in gene expression : Some relevant Brownian functionals
Malay Bandyopadhyay, Shamik Gupta, Dvira Segal
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6.
Experimental studies have recently revealed the active role of DNA breathing dynamics as a determinant of promoter
strength and transcription start site location. These studies have also invoked the fact that DNA bubble lifetime,
probability, and average amplitude are distinct dynamical properties which should be examined separately. In this
context, we introduce and analytically analyze the properties of several probability distribution functions characterizing
DNA bubble lifetime, reactivity and maximum size. We also study the joint probability distribution of the maximum
bubble size at a particular time. Understanding the scaling of these quantities with temperature and DNA energetics (e.g.,
base-pair dissociation energy) may become useful for determining the role of DNA breathing dynamics as an active
participant in gene expression.
P29
Structural and Functional Insights into Membrane Transporters and Sensors by Solid-State NMR
Lichi Shi, Izuru Kawamura, Shenlin Wang, Meaghan Ward, Ying Fan, Evelyn Lake, Mumdooh Ahmed, Kwang-Hwan Jung,
Vladimir Ladizhansky, Leonid S Brown
Department of Physics, University of Guelph, ON N1G 2W1, Canada
Membrane proteins are among the most attractive goals for structural studies, due to their biochemical and medical
importance. However, they are often not amenable to studies by the traditional structural methods, such as
crystallography and solution NMR, because of the difficulties with functional expression, solubilization, and
crystallization. Solid-state NMR is an attractive emerging alternative, which can be applied to these important proteins
after reconstitution into native-like membrane-mimicking environment.
Recently, we have demonstrated that solid-state NMR can yield valuable information on the secondary structure,
dynamics, and environment of functionally important sidechains for a 7TM helical membrane protein, proton-pumping
proteorhodopsin (1,2), which resists crystallization. Next, we could refine the X-ray model for another 7TM protein,
Anabaena sensory rhodopsin (3), in the lipid environment, using torsional and distance constraints obtained by solid-
state NMR. We revealed the structure of fragments disordered or distorted in crystals, and, using site-specific monitoring
of H/D exchange, obtained unique information on the protein dynamics and its location in the membrane. Finally, we
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
45
recently showed that such methodology can be extended to eukaryotic membrane proteins of similar size and
architecture, using expression of Leptosphaeria rhodopsin in methylotrophic yeast Pichia pastoris as an example (4).
REFERENCES:
1. Shi L. et al. (2009) Three-dimensional solid-state NMR study of a seven-helical integral membrane proton pump –
structural insights. J. Mol. Biol., 386: 1078.
2. Shi L. et al. (2009) Solid-state NMR study of proteorhodopsin in the lipid environment: secondary structure and
dynamics. Biochim. Biophys. Acta, 1788: 2563.
3. Shi L. et al. (2011) Conformation of a seven-helical transmembrane photosensor in the lipid environment. Angew.
Chem. Int. Ed. Engl., 50: 1302.
4. Fan Y. et al. (2011) Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables
high-resolution solid-state NMR studies in the lipid environment. J. Biomol. NMR, 49: 151.
P30
Microsecond Pore Dilation Kinetics and Hydrophobic Gating of Magnesium Transport in the CorA System
Chris Neale, N. Chakrabarti, Emil F. Pai, and Régis Pomès
Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto.
Department of Biochemistry, University of Toronto, Toronto, Canada
Although magnesium homeostasis is essential for life, the mechanism by which cells select Mg2+
among more prevalent
biological ions like Na+ and K
+ is unknown. In our earlier molecular dynamics (MD) simulation study [Chakrabarti et al., An
Iris-like Mechanism of Pore Dilation in the CorA Magnesium Transport System, Biophys. J. (2010) 98: 784-792], we
identified a 1.5-nm-long hydrophobic constriction in the CorA Mg2+
transport system which is dehydrated in the
crystallographic state of the channel. In our simulations, an iris-like dilation of the pore led to a rapid wetting transition of
the hydrophobic bottleneck, but only after regulatory magnesium ions were removed from their intracellular binding
sites. Here we report large-scale atomistic simulations of CorA in a hydrated bilayer successively with and without
regulatory ions. The results of hundreds of simulations confirm the allosteric mechanism linking the ionic occupancy of
the regulatory sites to hydrophobic gating of the pore and provide quantitative insight into the early kinetics of pore
dilation and hydration. Free energy simulations for magnesium permeation through a relatively dilated conformation of
the channel are presented, and the interplay of hydration, ionic occupancy of the lumen, and structural fluctuations of
the channel are analyzed. Together, these results provide insight into the molecular mechanisms governing the
regulation, transport, and selectivity of magnesium permeation in the unusually long channel lumen of CorA.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
46
P31
Molecular Mechanism of Proton Uptake in Cytochrome c Oxidase
David Caplan1,2
, Rowan Henry1,2
, Elisa Fadda3, Régis Pomès
1,2
1 Department of Biochemistry, University of Toronto
2 Molecular Structure and Function, Hospital for Sick Children
3 Department of Chemistry, University of Galway, Ireland
Cytochrome c oxidase (CcO), the fourth enzyme complex in the respiratory chain, is a proton pump powered by the
reduction of dioxygen into water. The resulting proton-motive force drives ATP synthesis. Although the mechanism
coupling proton pumping to dioxygen reduction remains to be elucidated, mutagenesis studies have identified several
single point mutations in the D-channel (the principal proton uptake pathway within CcO) which lead to decoupled
phenotypes, whereby redox chemistry occurs but proton pumping activity is compromised.
Using molecular dynamics and free energy calculation techniques, we perform comparative simulation studies of fully
unrestrained wild-type and mutant enzyme structures (R. sphaeroides) embedded in a lipid bilayer. The detailed analysis
of the interplay between structural fluctuations, functional hydration, and the energetic bias of ionic movement in the
protein interior provides insight into the molecular mechanism of proton pumping.
P32
The Role of Melatonin in Amyloid Fibril Formation on Lipid Membrane
Youngjik (Vince) Choi1, Ravi Gaikwad
2, Zoya Leonenko
1,2
1 Department of Biology
2 Department of Physics and Astronomy, University of Waterloo, Ontario, Canada
Alzheimer’s disease is a progressive neurodegenerative disease associated with amyloid fibril formation in the brain. The
exact molecular mechanism of amyloid fibril formation and toxicity is not well understood, which delays development of
novel and effective approaches to prevent and cure the disease. It has been suggested by animal studies that hormone
“melatonin”, which regulates and maintains the body's circadian rhythm, may counteract the effects of amyloid toxicity.
We hypothesize that melatonin affects physical and structural properties of the cell membrane lipid to prevent amyloid
toxic effect. We used advanced Atomic Force Microscopy (AFM) imaging and AFM-based force spectroscopy, in order to
elucidate the effects of melatonin on the physical property of model lipid membrane composed of 2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC). We monitored changes in adhesion, Young’s modulus, and breakthrough forces in
DPPC lipid bilayer with and without melatonin, and also how these properties affect amyloid beta binding and fibril
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
47
formation on the surface of DPPC membrane. Our findings may help understand the molecular mechanism of protective
role of melatonin in relation to Alzheimer’s disease.
P33
Effect of the lipid environment on interaction of P-glycoprotein with drugs
Adam T. Clay and Frances J. Sharom
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON.
P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily, is a ~170 kDa integral plasma membrane
protein. Pgp is known to be over-expressed in ~50 % of human cancers, and it contributes to multidrug resistance (MDR).
Its ability to confer MDR arises because the protein restricts the entry of hundreds of different amphipathic compounds
into the cell, by actively pumping them out of the membrane, using the energy of ATP hydrolysis. Previous studies
suggested that membrane lipid phase, composition and fluidity are important factors in Pgp’s ability to bind and
transport drugs. However, the complex interplay of these factors as a result of drug-membrane, Pgp-membrane and
drug-Pgp interactions is not well understood. To further explore the relationship between drug binding and membrane
phase state, Pgp was purified and reconstituted into bilayers composed of either 1-palmitoyl-2-
dimyristoylphosphatidylcholine or dimyristoylphosphatidylcholine. The gel-to-liquid crystalline phase
transitiontemperature, Tm, of the proteoliposomes was verified using differential scanning calorimetry. Intrinsic Trp
fluorescence quenching was used to determine the binding affinity of Pgp for various drugs at several temperatures
spanning the phase transition. Pgp was found to have a higher binding affinity for all drugs in the gel phase state.
Furthermore, the enthalpies of binding to Pgp for some drugs were substantially lower in the gel phase compared to the
liquid crystalline phase. Enthalpies of drug binding to Pgp do not appear to be dependent on phospholipid acyl chain
length, but rather on membrane phase state. Drug partitioning into the membrane of synthetic liposomes was
investigated using a dialysis method. These studies showed that drug partitioning was also affected by membrane phase
state, which inherently affects drug interaction with Pgp. These data support earlier work which showed that Pgp
transports drugs at a higher rate in the rigid membrane environment of the gel phase.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
48
P34
Effect of cholesterol and lipid composition on amyloid fibril formation on a model lipid membrane.
Elizabeth Drolle1, Francis Hane
1, Erin Faught
1, Ravi Gaikwad
2, Zoya Leonenko
1,2
1Department of Biology,
2Department of Physics, University of Waterloo
Amyloid-beta 1-42 is known to form amyloid fibrils which are involved in Alzheimer’s disease for which no cure is
currently available. Normally an alpha helical structure, amyloid-beta 1-42 can misfold into beta sheets forming
neurotoxic oligomers and amyloid fibrils. Although fibril plaque formation is associated with biological membranes in
vivo, the role of membrane heterogeneity and effect of cholesterol in the process of amyloid fibril formation and
toxicity are not well understood, and therefore research in this area is of great interest and necessity. We used atomic
force microscopy (AFM) to study amyloid-beta fibril formation on the surface of lipid membrane. We investigated effect
of charge and lipid phase on amyloid binding and fibril formation using DPPC, DOPC zwitterionic bilayers in gel and fluid
phase, as well as negatively charged DOPG and positively charged DOTAP supported bilayers. The surface of the lipid
membrane becomes more disrupted by Amyloid-beta in the case of fluid phase bilayers DOTAP and DOPC, compared to
DPPC and DOPG. Effect of cholesterol was also investigated on DOPC supported bilayer. We found that cholesterol
induces domain formation in model DOPC membrane, which serve as a template for amyloid binding and fibril formation.
P35
Modelling Pore Formation in Bacterial Membranes by Anti-Microbial Peptides
Chris S. DiLoreto, Robert A. Wickham
Department of Physics, University of Guelph.
A common strategy employed to destroy harmful bacteria is to disrupt the bacterial membrane through the action of
pore-forming anti-microbial peptides. The manner in which the peptides arrange themselves spatially to form a pore in
the membrane, which is important for understanding both the mechanism of pore formation and pore function, is a topic
of current debate. We present a simple model where the peptides are taken to be rigid cylinders and the membrane
lipids are flexible polymeric star-block copolymers, with a hydrophilic head block and two hydrophobic tail blocks. The
cylinder surface has hydrophobic and hydrophilic regions, designed so as to model a typical antimicrobial peptide, such as
magainin. We then use self-consistent field theory to determine the equilibrium state of the lipid membrane, subject to a
fixed peptide configuration.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
49
Through this approach, we can compute the system free-energy as a function of peptide configuration, and determine
the most stable peptide-membrane configuration. For a six peptides arranged on a circle, we find that a pore will form in
the membrane below a certain circle radius. Although our model is quite simple, it will allow us to perform a systematic
and comprehensive study of the parameter space for this system. This should reveal interesting trends and behaviours
which can then be examined using more detailed models.
P36
Two Novel Rhodopsins of Phaeosphaeria: Representatives of Distinct Subgroups of Fungal Rhodopsins
Y. Fan [1], R. Oliver [2], and L.S. Brown [1]
[1] University of Guelph (Canada), [2] Murdoch University (Australia)
Two rhodopsin-like genes, ops1 and ops2, were discovered in the genome of the wheat pathogen Stagonospora nodorum
(syn. Phaeosphaeria). The gene ops1 has a high degree of similarity to the gene of Leptosphaeria rhodopsin (LR), the first
proton-pumping rhodopsin from a eukaryotic organism. On the other hand, the gene ops2 is not close either to the gene
of LR or to the gene of Neurospora rhodopsin (jNR), which has a slow photocycle and was suggested to work as a sensory
rhodopsin.
The proteins, expressed in Pichia pastoris, are membrane-associated, bind all-trans retinal, and have λmax at 545 nm
(PhR1) and 538 nm (PhR2). The close λmax suggests these two rhodopsins are unlikely to be sensors for different colors of
the visible spectrum. The photochemical reaction cycle and the structural changes following the retinal
photoisomerization of PhR1 were quite similar to that of LR. Those data suggested PhR1 may have proton-pumping
ability, consistent with prediction based on high sequence similarity. In contrast, PhR2 possesses a unique photocycle in
accord with its unusual amino acid sequence, suggesting its photosensory role. However, that photocycle is fast, which is
the typical characteristics of a proton pump. Despite the different photocycle kinetics, FTIR showed high similarity of the
vibrational spectra of the late intermediates of the two proteins. The data from the mutant PhR2 D126N demonstrated
that D126 is a primary proton donor in PhR2, similar to other retinal proteins. The FTIR measurements indicated the
mutation E146D on the unique glutamate in PhR2, which supposedly faces the lipids or other proteins, perturbed the
photocyle and the protein structure. Thus, our initial results indicate that both PhR1 and PhR2 are proton pumps,
although proton pumping by PhR2 may be rudimentary and no observed when it interacts with other proteins.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
50
P37
Statistics of amino acid hetero- and homo-multiplets in protein chains. Deviation from random behaviour based on
residual polarity and location in secondary structural elements.
C. Gong and G.A. Arteca
Département de Chimie et Biochimie, Laurentian University, Sudbury, Ontario, Canada P3E 2C6.
Amino-acid multiplets, also known as “tandem oligopeptide repeats”, are short repeats of either single amino acids or
distinct amino-acid multiplets (at least doublets). Single amino acid repeats follow the form (X)m, m>2, and are often
referred to as “homopolymeric tracts.” These homorepeats have been associated with genetic disorders (when m>4),
protein misfolding or aggregation, and the possible promotion of evolutionary changes. Heteromultiplets (e.g., AB-
doublets for two different A and B amino acids) are often associated with enzymatic function and protein-ligand
recognition. Here, we contrast the distributions of homodoublets (AA) and heterodoublets (AB) for single protein chains.
To this end, we use a parsed ensemble of over 8,000 non-redunctant structures deposited in the Protein DataBank (PDB).
We discuss first how the distributions compare in terms of amino acid frequency and relative polarity in heterodoublets
(e.g., considering whether A or B are polar, nonpolar or charged residues). In addition, we study the placement of homo-
and heterodoublets in a-helical and b-strand regions, and coil regions, as well as the helix/coil and strand/coil frontiers.
We discuss how the results deviate from a purely random distribution based on the experimental frequency for each
amino acid. Our findings provide insight into what constitutes a typical "mean natural primary sequence," as opposed to
a random heteropolymer, as well as contraints that can be used to understand protein function and sequence evolution.
P38
Prediction and Analysis of Novel Interaction Motifs in Yeast
Yuan Gui
Department of Biology, Carleton University
Domains are short fragments of a protein’s sequence or structure that convey an evolutionarily conserved function.
Proteins carry out biological function in the cell not as singular units, but by forming interactions, either stable or
transient, with other proteins. These physical interactions are mediated by a number of interacting domains found
within the binding sites of protein. Changes in amino acid sequences, by mutation to the corresponding genes, can cause
an alteration to these domains. Such alterations may lead to disruption of the protein interactions and onset of disease.
A number of recent bioinformatics tools have been developed to predict the likelihood of interaction between two
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
51
proteins by screening for the presence of these domains and comparing them to existing databases of known protein-
protein interactions. PIPE, or Protein-Protein Interaction Prediction Engine, is a computational tool that we have recently
developed to predict protein interactions and locate the interacting sites by screening for short polypeptide sequences,
and searching for homologous sequences in databases of known interactions. Using PIPE we have identified 272 novel
interacting motif pairs of 30 amino acids long in Saccharomyces cerevisiae. The majority of these domains appear to be
highly conserved in human proteome suggesting that these new motifs have been conserved during the course of
evolution. We are currently confirming the activity of some of these motif pairs using yeast two-hybrid analysis.
P39
Experimental Evidence for Ion Transport in Neuronal Uncoupling Proteins: Implications in Structure – Function
relationships.
Tuan Hoang1, Matthew D. Smith
2, and Masoud Jelokhani-Niaraki
1
Department of 1Chemistry and
2Biology, Wilfrid Laurier University
Located in the inner mitochondrial membrane of brown fat adipose tissue, uncoupling protein -1 (UCP-1) dissipates the
proton electrochemical gradient causing reduction in the rate of ATP synthesis and generates heat by non-shivering
thermogenesis. Three other UCP homologues (UCP2, UCP4 and UCP5), expressed in neurons, are suggested to have
potential roles in the function and protection of the central nervous system (CNS). Extensive biochemical studies on UCP2
have provided adequate evidence for its participation in proton and anion transport. So far, no functional studies in
proteoliposome systems have been performed on UCP4 and UCP5. Due to the lack of high resolution structures,
structural information for UCPs remains limited. The goals of this study are to gain further information on the
conformations and functional properties of neuronal UCPs reconstituted in liposomes. The emphasis is on UCP4 and
UCP5 and their comparison to UCP2. Recombinant versions of all five UCPs have been successfully expressed, purified
and reconstituted in liposomes. All UCPs formed dominantly helical conformations in negatively charged phospholipid
vesicles as detected by CD spectroscopy [Ivanova et al (2010) Biochemistry 49(3): 512-21]. CD and fluorescence spectra
showed common structural and binding properties of neuronal UCPs and the well-studied UCP-1, implying a common
physiological role in addition to their specific roles in the CNS. Ion transport assays (proton and chloride) for
reconstituted neuronal UCPs have been developed using anion-sensitive fluorescent probes. In-vitro proton transport has
been confirmed for all neuronal UCPs. Purine nucleotide inhibitions of these proteins resulted in decrease in the proton
transport rate. Preliminary results have also shown the ability of transporting chloride anions in UCP2 and UCP4. Overall,
the outcome of this study will clarify several aspects of the structure-function relationships of the least studied UCPs 4
and 5 and their possible physiological roles in the CNS.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
52
P40
Moh1, a new player in repair of double stranded DNA breaks through non-homologous end joining in Saccharomyces
cerevisiae
Mohsen Hooshyar, Matthew Jessulat, Ashkan Golshani
Department of Biology, Carleton University, Ottawa, Ontario
DNA double stranded breaks (DSBs) are the most severe forms of DNA breaks and they can be induced exogenously or
endogenously. Unrepaired DSBs could cause genomic instability, cell lethality and may lead to production of cancer cells.
There are two main pathways to repair DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ).
NHEJ is achieved through direct ligation of broken ends of DNA without using homologues template and it is the
preferred repair pathway in mammalian cells. Several main proteins involved in NHEJ in Saccharomyces cerevisiae have
functional homologues in human. Therefore lower organism such as S. cerevisiae can serve as good model organism to
study NHEJ. In this study, novel proteins involved in NHEJ in S. cerevisiae are investigated. Potential protein candidates
were screened using a computational method called protein-protein interaction prediction engine (PIPE). Moh1 was one
of the candidate proteins that formed novel interaction with a number of known NHEJ proteins. We therefore
hypothesize that it is a novel NHEJ protein and we subjected it to further molecular and biochemical assays. Using
plasmid repair assay it was shown that Moh1 mutant strain reduces the repair efficiency of DSBs by 60% compared to wt.
Moh1 mutant are also sensitive to DNA damaging drugs such as hydroxyurea and methyl methanesulfonate. To further
investigate involvement of Moh1 in NHEJ a chromosomal break assay was carried out and Moh1 mutants showed
sensitivity to this assay. To better understand the role of Moh1 in NHEJ and DSBs repair synthetic genetic array was
carried out and Moh1 genetically interacted with several genes which have known functions in DNA repair. Based on the
results obtained so far it is evident that Moh1 is involved in NHEJ however details of its activity in NHEJ pathways require
further investigations.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
53
P41
Effect of Antimicrobial Agents on MinD Protein Oscillations in E. coli Bacterial Cells
Corey Kelly, Maximiliano Giuliani, Megan Murphy, John Dutcher
Department of Physics, University of Guelph.
The pole-to-pole oscillation of the MinD proteins in E. coli determines the
location of the division septum, and is integral to healthy cell division. It has
been shown previously that the MinD oscillation period is approximately 40 s
for healthy cells [1] but is strongly dependant on environmental factors such
as temperature, which may place stress on the cell [2,3]. We use a strain of E.
coli in which the MinD proteins are tagged with green fluorescent protein
(GFP), allowing fluorescence visualization of the MinD oscillation. We use high
resolution total internal reflection fluorescence (TIRF) microscopy to observe
the effect of exposure to antimicrobial agents on the MinD oscillation period and, more generally, to analyze the time
variation of the spatial distribution of the MinD proteins within the cells. These measurements provide insight into the
mechanism of antimicrobial action.
[1] Raskin, D.M.; de Boer, P. (1999) Proc Natl. Acad. Sci. 96: 4971-4976.
[2] Colville, K.; Tompkins, N.; Rutenberg, A. D.; Jericho, M. H. (2010) Langmuir 2010:26.
[3] Downing, B.; Rutenberg, A.; Touhami, A.; Jericho, M. (2009) PLoS ONE 4: e7285.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
54
P42
A General and Efficient All-Atom Simulation Method to Determine the Equilibrium Orientation of Transmembrane
Proteins in Membranes.
Kethika Kulleperuma, John Holyoake and Régis Pomès
Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto. Department of Biochemistry,
University of Toronto, Toronto, Canada
Knowledge of the spatial orientation of Transmembrane (TM) proteins is broadly useful in both in-vitro and in-silico
approaches that aim to elucidate the dynamic and functional properties of TM proteins. Such approaches include site-
directed spin labeling, cys-scanning mutagenesis, and atomistic molecular dynamics (MD) simulations using lipid bilayers.
Because three-dimensional structures of TM proteins are generally obtained in a detergent solvent, these structures do
not reveal the native orientation of the protein in the lipid membrane. For this reason, we aimed to develop a general
and computationally-efficient MD approach to predict the most favorable orientation of TM proteins in a lipid
membrane. In order to avoid the long relaxation time scales characterizing protein and lipid bilayer dynamics, our
method treats the TM protein as a rigid body in a membrane-mimetic hydrated octane slab, allowing the protein to reach
a stable orientation within 10 ns. The method was systematically tested on alpha-helical and beta-barrel TM proteins,
each with different starting orientations in the hydrated octane slab. Each protein attained a consistent orientation
irrespective of its starting orientation. Furthermore, the converged orientations are in good agreement with the known
orientations of these test proteins in lipid bilayers. These results indicate that this method is reliable as a general protocol
that can be used to determine the orientation of TM proteins of known structure.
P43
Effects of L- vs. D-Isomers of Cationic Antimicrobial Peptides on Alginate Binding and Antimicrobial Activity
Soyoung Lee, Lois M. Yin, and Charles M. Deber
Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8;
and Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada
Bacteria colonized into biofilms cause significant environmental and disease-related problems, including dental and
medical device-related infections, infective endocarditis, and cystic fibrosis pneumonia. Biofilm formation is
characterized by the overproduction of exopolysaccharides (EPS), which act to prevent the diffusion of conventional
antibiotics to the bacterial cell membrane. There is thus an urgent need for new approaches to treat biofilm microbial
infections. Cationic antimicrobial peptides (CAPs) have become increasingly recognized in current research as templates
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
55
for prospective antibiotic agents. The bacterium Pseudomonas aeruginosa, an opportunistic pathogen colonized in the
lungs of cystic fibrosis patients, forms biofilms by secreting alginate, a high molecular weight anionic polymer of L-
guluronate and D-mannuronate. We have designed novel CAPs of prototypical sequence KKKKKKAAXAAXAAXAANH2,
where X = Phe/Trp – that display high antibacterial activity to various bacteria at micromolar concentrations, but exhibit
little or no hemolytic activity towards mammalian cells. In comparisons of the antimicrobial activities of L- vs. D-isomers
of these CAPs against P. aeruginosa, minimum inhibitory concentration (MIC) levels of D-CAPs were found to be
significantly lower (i.e., higher antimicrobial activity) than corresponding L-CAPs. When interactions of L- and D-isomers
with alginate were studied by circular dichroism spectroscopy, we found that alginate induces partial alpha-helical
conformations in L-CAPs, suggesting that the alginate polymer behaves as an “auxiliary membrane” for the bacteria; in
contrast, D-CAPs do not exhibit similar helical induction under the same conditions. We discuss possible mechanisms as
to the relationship of CAP chirality to alginate-forming bacteria and antibacterial activity.
P44
Atomic force spectroscopy to measure lateral compression in lipid membrane.
Victor Malkov1, Elizabeth Drolle
2, Ravi Gaikwad
1, Gilbert Walker
3 and Zoya Leonenko
1,2
1 Department of Physics and Astronomy, University of Waterloo
2 Department of Biology, University of Waterloo and
3
Department of Chemistry, University of Toronto
Biological membrane is an essential part of the cell and plays a crucial role in cell functioning. Lateral compression of
biological membrane is an important physical parameter that changes during various cell processes. The measurement
and control of the membrane lateral compression is not easily achieved at the nanoscale, but could be very helpful in
studying the functions of biological membranes or model membranes. Solid supported lipid membranes (SLM) are widely
used as model membranes, and a precise determination of the lateral compression is crucial for understanding their
structure and function. Vesicle fusion is a popular technique used for the formation of SLMs or bilayers, but it does not
provide the desired control of the surface tension. We propose a new method for determining a membrane lateral
compression by measuring the breakthrough forces, obtained with atomic force microscopy. Lipid bilayers of 1,2-
dioleoyl-sn-glycero-3-phosphocholine (DOPC) at varying surface pressures were produced by Langmuir-
Blodgett/Langmuir-Schaefer deposition onto mica, which permits the control of the surface compression of the resulting
lipid bilayer. The formulation of a mathematical model that relates the breakthrough forces and lateral surface
compression will also be discussed. The proposed method would help to analyze the lateral compression of a lipid
membrane at the nanoscale in a non-destructive manner.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
56
P45
Conformation-induced binding of extracellular loops of the apelin receptor with the apelin ligand in lipid environments
Pascaline Ngweniforma and Jan K. Rainey
a,b
a Department of Biochemistry & Molecular Biology and
b Department of Chemistry, Dalhousie University, Halifax, NS B3H
1X5
The apelin receptor is a G protein-coupled receptor highly expressed in the central nervous system (CNS), cardiovascular
system, the adipoinsular axis and mammary glands, among other tissues and organ systems. The apelinergic system
(receptor and its cognate peptide ligand apelin) plays important biological functions in the regulation of blood pressure,
blood glucose, drinking behavior and food intake. The action of this system is also implicated in tumour angiogenesis,
diabetes and cardiovascular diseases. Despite these roles, the mechanism of activation of apelin receptor by its cognate
ligand has not been studied at the molecular level. We present the biophysical characterization of the interaction of two
extracellular loops of the apelin receptor (extracellular loops 1, EL1 and 3, EL3) with a fluorescently-tagged apelin
analogue in a variety of lipid environments. Peptides were synthesized by solid phase peptide synthesis and purified by
high-performance liquid chromatography. Characterization both of the peptides in isolation and of the binding between
apelin and EL peptides was carried out using circular dichroism spectroscopy, fluorescence resonance energy transfer and
nuclear magnetic resonance spectroscopy. Structural changes in the presence of lipids, vs. buffer, are apparent for the
EL1 and EL3 peptides. Förster resonance energy transfer (FRET) between pyrene-apelin and EL1 demonstrates binding,
with the requirement for the lipid-induced EL conformation change. These results provide insight into understanding of
the apelinergic system at the molecular level and provide the first structural information on the apelin receptor for the
development of therapeutics targeting this system.
P46
Kinetics of Membrane Fusion Induced by Small Lipophillic Molecules
Trinh T. Nguyen and David T. Cramb
Department of Chemistry, University of Calgary.
Lipid vesicles are attractive drug and gene delivery vehicles in the field of nanomedicine, due to their small size and non-toxic
behavior. However, the mechanism for drug release/uptake once the liposome contacts the cell surface remains speculative.
Liposomes can be taken up by endocytosis, phagocytosis, or fusion could occur between the liposome and the plasma
membrane, releasing the vesicle contents (drug) into the cell.
The mechanism of membrane fusion has been under debate for many years, it is now generally accepted that both proteins
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
57
and lipids play an important role in the process of fusion. There have been studies conducted using pure lipids membranes
that have also illustrated fusion, which indicates that proteins are not necessarily required for fusion to occur. It has been
previously shown that a small lipophilic molecule (halothane) can be used as a fusion agent in unilamellar vesicles of
dioleoylphosphatidylcholine (DOPC). Fluorescence based assays were utilized to monitor fusion events and determine
kinetics of lipid mixing. This study investigates the ability of small lipophilic molecules to induce membrane fusion in a
lipid membrane model.
P47
Identification and Characterization of Novel Genes Involved in Non-Homologous End-Joining Pathway in the Yeast
Saccharomyces cerevisiae
Katayoun Omidi, Matthew Jessulat, Mohsen Hooshyar, Bahram Samanfar, Ashkan Golshani
Department of Biology, Carleton University, Ottawa, Canada.
Successful organization and stable maintenance of cell genomes are essential for survival. One of the main mechanisms
for repairing DNA double-stranded breaks (DSBs), which are the most severe forms of DNA breaks, is the non-
homologous end-joining (NHEJ) pathway. In this pathway, the two broken strands of DNA are directly re-ligated without a
homologous template. If unrepaired, DSB can cause chromosomal rearrangement, loss of genetic information, cell death
or cancer. Using a plasmid repair assay screen we have previously shown that deletion of two genes linked with the
spindle assembly checkpoint, NEK1 and BEC3, reduced the NHEJ efficiency in Saccharomyces cerevisiae. We hypothesized
these represent novel genes that affect NHEJ and subjected them to follow-up analysis. As expected our synthetic genetic
analysis supports a role for NEK1 and BEC3 genes in cell cycle. These analyses also identified a number of genetic
interactions between the two genes and several previously reported NHEJ-related genes such as XRS2 and RAD50,
supporting a role for these genes in NHEJ pathway. Overexpression of NEK1 was further found to compensate for the
sensitivity for deletion of key NHEJ genes LIF1 and MRE11 against different DNA-damaging agents such as hydroxyurea
and bleomycin. NEK1 overexpression also compensated the sensitivity of deletion RAD50, another key NHEJ gene, to
bleomycin. Therefore we conclude that NEK1 and BEC3 are two novel genes that can affect the efficiency of NHEJ. Details
of their activities remain unknown. One possibility is that they may bridge a communication between cell cycle and NHEJ.
This possibility merits further investigations.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
58
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
No
rma
lize
d P
red
icte
d
Ag
gre
ga
tio
n P
rop
en
sit
y
Prediction Algorithms
Short Disease Duration mutants (<2 years)
A4V
A4T
V148I
H43R
Figure 1. A comparison of the predicted aggregation
results for mutations linked to short ALS disease
durations.
P48
Correlating predicted and observed aggregation for a disease-linked protein.
Heather Primmer
Department of Chemistry, University of Waterloo
Protein aggregation is a hallmark of a number of neurodegenerative
disorders including Alzheimer’s Disease, Huntington’s Disease, and
Amyotrophic Lateral Sclerosis. Despite the common occurrence of
protein aggregation in disease, the fundamental mechanisms
controlling the propensity of a protein to aggregate are not well
understood. Recently, various prediction algorithms have been
developed to attempt to predict the likelihood of a protein to form
aggregates. In many cases the algorithms were designed based on a set
of known amyloid-forming proteins or peptides. In order to test the
ability of these algorithms to predict aggregation propensity for a
known disease-linked protein, human copper/zinc superoxide
dismutase (SOD1) was used as a model system. Currently, over 140
mutations of SOD1 have been linked to the familial form of
amyotrophic lateral sclerosis (ALS). Nine different prediction
algorithms were used to predict the aggregation propensity of twenty-four of the most common SOD1 mutations. The
results were correlated to experimental aggregation data for twelve of the mutants and to the well documented disease
durations found to be specific to each mutant for all twenty-four mutations. Interestingly, there were large differences in
the predicted aggregation propensity for a given mutant using the nine different algorithms (Figure 1). Additionally, the
algorithms demonstrate poor correlations between predicted aggregation and the experimentally observed aggregation1.
Furthermore, the predicted propensities for the mutants do not show a convincing relationship with the duration of the
disease in ALS patients1. There are many factors that could be contributing to the poor correlations observed here
including a general lack of understanding of the factors contributing to protein aggregation and the lack of amyloid
formation by mutant SOD1. The implications of this work for future studies in protein aggregation and the aggregation of
disease-linked proteins will be discussed.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
59
P49
Imaging the Enzymatic Degradation of Cellulose Fibers Using Surface Plasmon Resonance
Adam N. Raegen, Kyle Reiter, Scott G. Allen, John R. Dutcher
Department of Physics, University of Guelph
We present the first study of the interaction of a cellulase enzyme mixture with bundles of cellulose fibers using surface
plasmon resonance (SPR) imaging. The cellulose fibers, obtained from the bacterium Acetobacter xylinum, were
heterogeneously distributed on a thin layer of thioglucose deposited onto a gold film. SPR images collected as a function
of time allowed us to observe the adsorption of the enzymes onto both the cellulose fibers and the bare surface, and the
subsequent degradation of the cellulose fibers in real time. In particular, we were able to characterize the decrease in the
thickness and variations in thickness of the cellulose fiber-coated regions with time, and to define a characteristic time
for the removal of cellulose from the surface. These results demonstrate the usefulness of the SPR imaging technique for
measuring the effectiveness of enzymes on cellulose fibers and provide useful metrics of enzyme activity that are of
relevance to the cellulosic ethanol industry.
P50
Aggregated yet Disordered: A Molecular Simulation Study of the Self-Assembly of Elastin
Sarah Rauscher, Régis Pomès
Molecular Structure and Function, Hospital for Sick Children. Department of Biochemistry, University of Toronto
Elastomeric proteins are essential to the function of biological machinery as
diverse as the human aorta, the capture spiral of spider webs, and the jumping
mechanism of fleas. In humans, elastin is responsible for the elastic recoil of skin,
arteries, and lungs. Elastin exhibits remarkable resilience and durability, making it
ideal for biomaterials applications. In addition, elastin undergoes phase separation
followed by self-assembly into a fibrillar structure upon heating. However, the
precise molecular details underlying this temperature-induced self-organization
are not well understood. Elastin is an intrinsically disordered protein; it is
challenging to study experimentally due to its structural heterogeneity and
tendency to aggregate. Computer simulations, which are not impeded by these properties, have recently emerged as a
useful tool to characterize the conformational ensembles of intrinsically disordered proteins.1,2
We have performed
extensive atomistic molecular dynamics simulations totalling more than 0.4 ms of monomers and aggregates of elastin-
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
60
like peptides using simulated tempering distributed replica sampling.3 In this approach, a random walk in temperature is
used to overcome energetic barriers, thereby enhancing conformational sampling. The analysis of ensemble-averaged
structural properties of several elastin-like peptides provide insight into the molecular basis of elastin self-assembly.
Image: A large aggregate of elastin-like peptides (snapshot taken from an MD simulation).
1. Rauscher S, Pomès R. Biochemistry and Cell Biology. 2010; 88(2): 269-290.
2. Rauscher S, Baud S, Miao M, Keeley FW, Pomès R. Structure. 2006; 14(11): 1667-1676.
3. Rauscher S, Neale C, Pomès R. J. Chem. Theory Comput. 2009; 5(10): 2640-2662.
P51
Functional Genomics of Translation Pathway in the Yeast Saccharomyces cerevisiae
Bahram Samanfar1, Le Hoa Tan
1, Firoozeh Chalabian
1, 2, Katayoun Omidi
1, Ashkan Golshani
1
1 Department of Biology, Carleton University, Ottawa, Canada.
2Department of Biology, Islamic Azad University, Tehran
North Campus, Tehran, Iran
Gene function prediction is a major goal of systems molecular biology in the post genome sequencing era. In this context
the yeast, Saccharomyces cerevisiae, has emerged as the eukaryotic model organism of choice for large-scale functional
genomic investigations. Proteins are responsible for the majority of the biological processes in the cell. Generally, they
achieve this task in association with other protein partners. Therefore one way of studying novel protein functions is
through the interactions they make with other proteins with known functions. Since protein synthesis or translation is an
essential process within a cell with significant implication in human diseases like cancer, having a better understanding of
translation process and characterizing novel genes involved in this pathway is the focus of a number of current
investigations. Based on human all to all protein interaction analysis using PIPE, a novel protein-protein interaction
prediction engine that we developed based on the re-occurring short polypeptide sequence of known interacting protein
pairs, we identified yeast homolog of 3 genes (rge58∆, ysl22∆, aph7∆) to form novel interactions with a number of
proteins involved in translation pathway. We therefore hypothesized that these represent novel translation genes.
Plasmid based β-galactisidase stop codon read through assay (premature stop codon in the middle of LacZ expression
cassette), has indicated that aph7∆, ysl22∆ and rge58∆ have higher stop codon bypass compare to the wild type. Note
that qRT-PCR analysis have shown that there is no significant differences in mRNA content between these deletions and
control. Serial dilution analysis using translation inhibitory drugs has shown higher sensitivity of these mutants to
translation drugs streptomycin and cycloheximide in comparison with wild type. Therefore we have concluded that
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
61
RGE58, YSL22 and APH7are three novel genes that affect the process of translation in yeast. Details of their molecular
activities require further investigations.
P52
Transmembrane Domain of the p14 Membrane Fusion Protein: From Structure to Function
Muzaddid Sarker1,2
, Jan K. Rainey2,3
, and Roy Duncan1,2
1Department of Microbiology & Immunology,
2Department of Biochemistry & Molecular Biology, and
3Department of
Chemistry, Dalhousie University, Halifax, NS, Canada.
The fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins
encoded by the fusogenic reoviruses. Rather than inducing virus-cell fusion, the FAST proteins have evolved to specifically
induce fusion between virus-infected host cells and neighbouring uninfected cells. The FAST proteins share no significant
sequence similarity and each has its own unique repertoire of structural motifs. However, all of the FAST proteins assume
a bitopic topology in the plasma membrane, with a single transmembrane domain (TMD) flanked by small N-terminal
exoplasmic and larger C-terminal endoplasmic domains. Recent studies indicate the FAST protein TMDs play a direct role
in the membrane fusion reaction. Furthermore, the FAST protein TMDs are functionally interchangeable, implying the
presence of conserved, family-specific structural features essential for fusion activity. To better understand the
mechanism of action of the FAST protein TMDs at the molecular and sub-molecular levels, we utilized solution nuclear
magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to determine the high-resolution 3D structure of a
32-residue peptide representing the p14 FAST protein TMD in a membrane mimetic micelle environment. Results
indicate that interesting structural features of the TMD α-helix are likely to perturb the lamellar structure of membranes,
thereby contributing to cell-cell membrane fusion.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
62
P53
The Oligomeric Nature Of The M2 Muscarinic Cholinergic Receptor As Revealed In The Allosteric Effect Of Tacrine
Rabindra V. Shivnaraine1, Xi-Ping Huang
2, 3, John Ellis
2 and James W. Wells
1.
1Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S
3M2. 2Departments of Psychiatry and Pharmacology, Hershey Medical Center, Penn State University College of Medicine,
Hershey, PA 17033. 3Current address: Department of Pharmacology, UNC at Chapel Hill, Chapel Hill, NC 27599
The M2 muscarinic cholinergic receptor has been shown to exist as
a tetramer. Each constituent protomer possesses two
topographically distinct but functionally linked binding sites: an
orthosteric site recognised by antagonists such as N-
[3H]methylscopolamine (NMS), and an allosteric site recognised
by modulators such as tacrine. Upon binding to the allosteric site,
such modulators change the kinetics of binding at the orthosteric
site. The allosteric effects of tacrine have been examined for
properties that relate to the oligomeric status of the M2 receptor
in membranes and solubilized extracts from porcine atria, CHO
cells and Sf9 cells. The rate of dissociation of [3H]NMS was
decreased by tacrine with a Hill coefficient significantly greater
than 1, indicative of co-operativity between at least two allosteric sites. The binding of [3H]NMS at
thermodynamic equilibrium was decreased by tacrine in a biphasic manner (log K1 ≈ −5.5, log K2 ≈ −3.1), each
component of which was characterised by a Hill coefficient greater than 1; accordingly, there appear to be at
least four allosteric sites overall, consistent with the notion of a tetrameric receptor. The attainment of
equilibrium was confirmed by varying the time and temperature of incubation and by varying the order in which
tacrine and [3H]NMS were mixed with the receptor. The noncompetitive nature of the inhibition at equilibrium
was confirmed by the lack of change in the potency of tacrine (K1, K2) when the concentration of [3H]NMS was
varied around its equilibrium dissociation constant (i.e., 0.1KD—10KD). As the level of occupancy by [3H]NMS was
increased, however, there was a corresponding increase in the fraction of labelled sites exhibiting low affinity for
the allosteric ligand. Tacrine therefore appears to identify an asymmetry that is induced among the constituent
protomers of the tetramer upon the binding of [3H]NMS. (Supported by the CIHR and the Heart and Stroke
Foundation of Ontario)
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
63
P54
Dissecting the folding energetics and switching caused by myristoylation of hisactophilin.
Martin TJ Smith, Joseph Meissner, Elizabeth M Meiering
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Waterloo.
Myristoylation, the covalent linkage of a saturated, C14 fatty acyl chain to the N-
terminal glycine in a protein, plays a vital role in reversible membrane binding
and signaling by the modified proteins. Currently, little is known about the
effects of myristoylation on protein folding and stability, or about the
energetics and molecular mechanisms of switching involving states with
sequestered versus accessible myristoyl group. Analysis of results obtained by
NMR, protein folding kinetics and equilibrium denaturation techniques
illustrate the effects of myristoylation on hisactophilin, a histidine-rich protein
that binds cell membranes and actin in a pH-dependent manner. They
demonstrate that myristoylation significantly increases hisactophilin stability,
while also markedly increasing global protein folding and unfolding rates. The switching between sequestered and
accessible states is pH-dependent, with an apparent free energy change of 2.0 kcal/mol. This pH dependence of
switching appears to be the physical basis of the sensitive, pH-dependent regulation of membrane binding
observed in vivo. We conclude that an increase in protein stability upon modification and burial of the attached
group is likely to occur in numerous proteins modified with fatty acyl or other hydrophobic groups, and that the
biophysical effects of such modification are likely to play an important role in their functional switches. In
addition, the increased global dynamics caused by myristoylation of hisactophilin reveals a general mechanism
whereby hydrophobic moieties can make nonnative interactions or relieve strain in transition states, thereby
increasing the rates of interconversion between different states.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
64
P55
Stability and aggregation studies of fALS-associated mutant Cu-Zn-Superoxide Dismutase
Helen Stubbs
Department of Chemistry, University of Waterloo.
Familial Amyotrophic Lateral Sclerosis (fALS) is a neurological disorder characterized by rapid degeneration of motor
neurons, progressive paralysis and inevitably death. Mutations in the metalloenzyme, Cu,Zn-SOD are associated with
~20% of all fALS cases, and a prominent disease hypothesis is that aggregation of mutant SOD is neurotoxic. However,
the mechanism by which mutant SOD aggregates is unknown. Furthermore, because SOD undergoes various post-
translational modifications (metal binding, disulfide bond formation, and dimerization), the form of SOD that is relevant
to ALS toxicity is unknown. This work focuses on two forms of SOD: the most immature form, disulfide-reduced
unmetallated SOD (apo SOD2SH
) and the most mature form, disulfide-oxidized, metallated SOD (Cu,Zn-SODS-S
). Previous
studies suggest a relationship between decreased stability and increased aggregation propensity and recent attention has
focused on apo SOD2SH
, for fALS-associated mutations appear to have the most profound effect on the stability of this
form of the protein. Here we have focused on characterizing the aggregation propensity of fALS-associated SOD mutants
in the apo SOD2SH
state under physiologically relevant conditions. Our results suggest that mutants may aggregate via
different mechanisms, forming aggregates that differ in size.1 However,
fALS disease characteristics cannot be fully explained by mutant apo
SOD2SH
aggregation and it is important to investigate the effects of fALS-
mutations on other forms of SOD. Therefore, the stability and folding
behaviour of Cu,Zn-SODS-S
has been investigated. These experiments
show that mutant SODs generally have decreased thermodynamic
stability and that unfolding deviates from two-state behaviour, which
may be related to Cu,Zn-SODS-S
aggregation. In studying the relationship
between stability and aggregation propensity of mutant SOD, we propose that many forms of SOD are relevant to ALS
toxicity. Aggregation of mutant SOD may involve different mechanisms and pathways, which may be associated with
different disease characteristics, such as age of onset and duration.
1. Vassall, K. A.; Stubbs, H. R.; Primmer, H. A.; Tong, M. S.; Sullivan, S. M.; Sobering, R.; Srinivasan, S.; Briere, L. A.; Dunn,
S. D.; Colon, W.; Meiering, E. M., Decreased stability and increased formation of soluble aggregates by immature
superoxide dismutase do not account for disease severity in ALS. Proc Natl Acad Sci U S A 2011, 108, (6), 2210-5.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
65
P56
Characterization of an Iron-Sulfur Binding Protein in the Tail Tip of Bacteriophage Lambda
William Tam, Alan Davidson
Department of Biochemistry, University of Toronto.
The assembly of λ bacteriophage tails requires the action of 11 gene products which must interact in an organized
fashion to assemble infectious tail particles. One of these gene products, gpL, is essential for the assembly of the initiator
complex in λ tails. The initiator complex, which is an ensemble of tail proteins, provides a platform for polymerization of
the tail tube. Therefore the assembly of the initiator complex requires gpL to interact with a number of tail proteins. GpL
and its homologs contain two domains: the N-terminal domain and the C-terminal domain. The C-terminal domain
coordinates an oxygen-sensitive [4Fe-4S] cluster using 4 highly conserved cysteines. Through two individual cysteine
mutants, C184A and C228A, it was found that these mutant proteins coordinate a [2Fe-2S] cluster also using 4 cysteines;
the fourth cysteine being non-conserved. Mutating all four cysteines individually resulted in a 10-100 fold decrease in the
number of assembled phage particles. We propose that the coordination of a [4Fe-4S] cluster with the four conserved
cysteines keeps gpL in a conformation that can optimally interact with other tail proteins for efficient tail assembly.
However, mutations to the conserved cysteines likely results in an altered conformation of gpL, one which coordinates a
[2Fe-2S] cluster. This altered conformation of gpL may not interact as strongly with other tail proteins resulting in lower
efficiency of tail assembly. This is the first report of a morphogenetic phage protein that coordinates an iron-sulfur
cluster.
P57
Identification and characterization of novel translation related genes in Saccharomyces cerevisiae
Le Hoa Tan, Bahram Samanfar and Ashkan Golshani
Department of Biology, Carleton University.
Translation in eukaryotic organisms is a complex process that requires numerous factors including ribosomes to read the
genetic material from the mRNA transcripts and synthesize polypeptides. It is essential that the translation process be
highly accurate from initiation to elongation and termination to generate functional proteins that mediate cell function
and survival. Dysfunctional translation machinery has been linked to a number of human conditions such as cancer and
aging. In addition, the majority of antibiotics exert their activities by targeting translation. Therefore, it is essential to
understand translation and the factors that can affect this process. To identify novel genes that can affect the accuracy of
translation, we have subjected a collection of yeast gene knockouts to an in vivo stop codon read-through assay. This
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
66
assay is based on a β-galactosidase system which contains different premature stop codons in the middle of the β-gal
gene. Using a large scale analysis, we have identified four open reading frames called Tef1, Tef2, Tef3 and Tef4 that
when deleted cause stop codon read-throughs. We have used a small scale analysis to confirm our observations. Since
the target gene deletions appear to cause defects in translation fidelity (stop codon read-through) we investigated their
effect on translation efficiency using an inducible β-galactosidase assay. Our preliminary results indicate that when Tef1
and Tef2 are deleted the efficiency of translation seems to be increased. In contrast when Tef3 and Tef4 are deleted the
efficiency of translation seems to be reduced. Our results clearly indicate that Tef1, Tef2, Tef3 and Tef4 are novel genes
that can affect the process of translation. To confirm that our targeted genes work independent of the transcription
machinery, we are currently evaluating their activity on the content of mRNA using qRT-PCR.
P58
Intrinsic Dynamics of the Exchange Protein Directly Activated By Cyclic AMP (EPAC)
Bryan VanSchouwen1, Federico Fogolari
2, Giuseppe Melacini
1
1Department of Chemistry and Chemical Biology, McMaster University, Canada.
2Facolta di Medicina e Chirurgia,
Università degli Studi di Udine, Italy.
The exchange protein directly activated by cyclic AMP (EPAC) is a critical component of the intracellular G-protein-
mediated signalling network of eukaryotic organisms. EPAC plays a regulatory role in such processes as cell adhesion,
cell-cell junction formation, insulin secretion by pancreatic β-cells, and cleavage of amyloid precursor protein (APP) by α-
secretase in the central nervous system. When cAMP binds to EPAC, the protein undergoes a conformational change
that exposes the catalytic site, thus permitting the binding and activation of the
downstream signalling proteins Rap1 and Rap2. Previous experimental studies
have shed light on key structural attributes of EPAC allostery, and have begun to
explore key dynamical attributes. However, several other pivotal dynamic
attributes of EPAC allostery are still not fully understood. Specifically, the
following questions remain unanswered:
1.) What is the dynamic profile of full-length EPAC?
2.) What are the distinct contributions of cAMP binding and of the
coupled conformational changes to the variations in dynamics between
the apo-inactive and the holo-active states?
Experimental difficulties have so far precluded investigation of these questions. Therefore, the questions were
addressed with molecular dynamics (MD) simulations.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
67
P59
Mapping drug interactions in the substrate-binding pocket of the P-glycoprotein multidrug efflux pump
David Ward and Frances J. Sharom
Department of Molecular and Cellular Biology, University of Guelph
P-Glycoprotein (Pgp, ABCB1 in humans), is a 170 kDa polyspecific multidrug transporter which interacts with hundreds of
structurally and functionally unrelated compounds. Pgp has been implicated in multidrug resistance (MDR) in many
human cancers, where it transports chemotherapeutic drugs out of tumour cells, limiting their cytotoxicity. The Pgp
drug-binding pocket appears to be large and flexible, with several overlapping sub-sites, and both the X-ray crystal
structure and biochemical studies indicate that two drug molecules can bind simultaneously. Bound drug molecules
interact with each other in a complex way to either inhibit or stimulate each other’s transport. Our long term aim is to
map functional interactions inside the Pgp binding pocket by covalently linking fluorescent substrates to two previously
identified interacting transport sites, the R-site and the H-site. The R-site drug azido-tetramethylrosamine (AzTMR) was
crosslinked to Pgp with a drug:protein stoichiometry of 1, and the binding affinity of other substrates to the Pgp-drug
adduct was estimated by quenching of the intrinsic Trp fluorescence. Results have revealed that a second drug molecule
can bind with lower affinity when compared to native Pgp, suggesting a partial overlap of the sub-site where the drug
binds with that of TMR binding. In contrast, some drugs, and large (>750 Da) drug dimers joined by an aliphatic linker,
bind to Pgp-AzTMR with higher affinity, providing evidence of binding cooperativity. It also appears that both native and
Pgp-AzTMR prefer to bind drug dimers with a specific separation between the monomers. The local environment around
AzTMR appears to have a polarity similar to that of methylene chloride. This is consistent with the location of the lower
sub-site in the X-ray crystal structure of Pgp. Understanding the biochemical basis of drug binding to Pgp will facilitate
the design of selective inhibitors to reverse MDR in clinical oncology.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
68
P60
Combining bulk biochemical assays with single-molecule techniques to study the regulation of microtubule dynamics
by motor proteins
Michal Wieczorek, Susanne Bechstedt, Gary Brouhard
Department of Biology, McGill University.
Kinesin protein superfamily members are microtubule (MT)-interacting enzymes involved in vesicle & biomolecule
transport, meiosis/mitosis and axon guidance. These proteins convert the chemical energy released from ATP hydrolysis
into mechanical work such as the directional transport of cargo along MTs. Recently, however, kinesins have surfaced as
regulators of the dynamic nature of MT polymers. We have recombinantly expressed and purified two candidate kinesins
implicated in modulating MT growth rates and have examined their effect on MT dynamics using a combination of bulk
biochemistry and single-molecule fluorescence microscopy. A total internal reflection fluorescence (TIRF) assay has been
employed to observe polymerization of single MTs off of nucleating seeds immobilized on silane-functionalized glass. By
comparing to controls with no added motor protein, and using a simple model for dynamic instability of MTs, we have
identified the effect of these motors on the kinetics of MT polymerization. In addition, several bulk assays have been
used to complement these data. For example, the increase in net polymer mass can be followed by observing changes in
turbidity, offering a crude MT polymerization assay as a control for the single molecule experiments. Lastly, a
modification of a colorimetric ATPase assay will be developed to track changes in kinesin ATP hydrolysis kinetics in the
presence of dynamic MTs. These results will eventually be used to support in vivo observations of MTs and these motors
inside of single cells and in model organisms. The confirmation and characterization of motor proteins that modulate MT
growth rates will significantly advance the motor protein field of research and will provide an important piece to the
complex puzzle that is the eukaryotic cytoskeleton.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
69
P61
Morphology and Nanomechanics of Type II collagen
Chuan Xu and Erika F. Merschrod S.
Department of Chemistry, Memorial University of Newfoundland.
Cartilage is a hyperelastic tissue that can be found in the bone joint, the ear, the nose, the knee, the elbow, etc. Water,
type II collagen fibers and proteoglycans are the major components of cartilage. Type II collagen molecules form
patterned fibers dispersed throughout a proteoglycan/water gel to reinforce the cartilage. Changes in type II collagen
molecule structure, fiber pattern, or fiber orientation are associated with diseases like osteoarthritis, epiphysitis,
semilunar cartilage disorders, osteochondritis, etc. Therefore, understanding the morphology and mechanical properties
of type II collagen fibers and collagen-proteoglycan composites is important for type II collagen related illness treatment.
Our study focuses on the morphology and mechanic properties of type II collagen fibers. Atomic force microscopy is a
powerful tool to manipulate and characterize nanometer size objects like collagen fibrils. It is extensively used in this
study for both morphology and mechanical measurements.
P62
Interaction of α-Synuclein and its A30P Variant with Vesicles that Mimic Mitochondrial Membranes
Yoo Jeong Yang1, Imola G. Zigoneanu
1, Alexander S. Krois
1, Md. Emdadul Haque
1, Gary J. Pielak
1,2,3
1Department of Chemistry,
2Department of Biochemistry and Biophysics,
3Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
α-Synuclein, an intrinsically-disordered protein associated with Parkinson’s disease, interacts with mitochondria, but the
details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles
by using fluorescence anisotropy and 19
F nuclear magnetic resonance spectroscopy. Both proteins interact strongly with
large unilamellar vesicles of composition similar to that of the inner mitochondrial membrane, but no affinity for vesicles
mimicking the outer mitochondrial membrane. Decreasing the amount of cardiolipin reduces protein affinity, with the
variant showing a larger decrease. The degree of cardiolipin acyl side chain also affects binding. One double bond
increases the proteins’ affinity. The presence of two double bonds decreases the affinity. The 19
F data show that the
interaction involves α-synuclein N-terminal region. Increasing the temperature increases binding of wild-type α-
synuclein, but not the A30P variant. The results are discussed in terms of the types of interactions between the proteins
and the membrane, lipid packing efficiency, and the effects of the mutation on α-synuclein structure.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
70
P63
Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in their interactions with bacterial
membranes and biofilm polysaccharides
Lois M. Yin1,2
, Michelle Edwards2, Jessica Li
2, Christopher M. Yip
2, and Charles M. Deber
1,2
1Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Toronto M5G 1X8, Ontario;
and 2Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada
Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in a wide range of living organisms
ranging from plants to insects to mammals – including humans. These CAPs offer a viable alternative to conventional
antibiotics as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death.
Despite the highly favourable properties of CAPs, once bacteria – such as Pseudomonas aeruginosa – become colonized
in body tissues, they often form “biofilms”. Biofilm formation is characterized by the overproduction of bacterial
exopolysaccharides, and of which alginate is a major component. Successful antimicrobial agents must evade the
extracellular polysaccharide ‘matrix’ of the biofilm to reach the bacterial membrane. Our laboratory has developed a
series of CAPs, such as KKKKKKAAFAAWAAFAA-NH2 and analogs with up to four Leu residues replacing Ala at various
positions (6K series), that display significant antimicrobial activity against bacteria with little or no toxicity to mammalian
cells. Using a variety of biophysical and biochemical approaches, including atomic force microscopy (AFM) measurements
performed in tandem with attenuated total reflectance – Fourier transform infrared spectroscopy (ATR-FTIR), we have
investigated how peptide hydrophobicity and charge distribution correlate with the membrane disruptive powers of
CAPs. Results indicate the propensity of CAPs such as the 6K series to convert from alpha-helical to beta-aggregated
structures as a function of time when embedded in bacterial membrane mixtures, while CAPs with a segregated N- and C-
terminal 3K-3K charge distribution reduce beta-aggregation in membranes and improve antimicrobial activities. Varying
hydrophobicity levels and residue compositions of CAPs also affect their alginate-binding and alginate-diffusion abilities.
These findings suggest routes to an optimal balance of hydrophobicity and charge distribution in CAP design to allow
both biofilm penetration and bacterial membrane destruction.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
71
P64
Influences of Native Energetic Heterogeneity and Many-body Interaction in ΦΦΦΦ Value Analysis of Protein Folding
Zhuqing Zhang, Hue Sun Chan
Department of Biochemistry, University of Toronto
Single-point mutant perturbations have been dominantly used to probe protein folding mechanism experimentally,
which turned φ-value into a critical parameter to infer the transition state ensemble. Recently, Munoz group’s large scale
analysis of mutational effects showed a near single universal φ-value with normally distributed error for 24 proteins, and
at zero stability conditions, the intrinsic variabilityφ0 is around 0.36. It provides some new understanding of protein
folding based on φ-value probe. To detect which kind of interaction mechanism behind this, in the present study, we use
native centric, explicit chain coarse-grained model, to systematically detect the perturbation of single-point mutations for
four single domain two-state model proteins. The results indicate that the uniform, additive contact interactions could
not conduce to the experimental Brønsted plot. Those significantly deviated from the main set points usually happen to
terminal residues, and mostly are hydrophobic and formed contact with the other terminal fragment in native structure.
By adjusting the native contact energetic heterogeneity and considering many-body interaction in the model, we found
that these two elements might be important for more accurate understanding of protein folding.
P65
Peroxisome proliferator-activated receptor δ limits pathogenic Th cell responses in EAE
Fei Zhao1, Roopa Bhat
3, Ajay Chawla
3, Lawrence Steinman
3, Shannon Dunn
1,2
1Department of Immunology, University of Toronto, University Health Network, Toronto, Canada,
2WCRI, Toronto
Ontario, and 3Stanford University, Stanford, CA
Multiple sclerosis (MS) is a neurological disorder currently affecting more than a million people world-wide. It is
characterized by recurrent episodes of immune-mediated attack on myelin in the brain, leading to neuron damage and
progressive disability. Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune disease
model of MS. Peroxisome proliferator-activated receptor δ (PPAR-δ) is a member of the steroid hormone nuclear
receptor superfamily that regulates inflammation as well as lipid metabolism. We previously reported that mice deficient
in PPAR-δ (PPAR-δ-/-) exhibit a more severe course of EAE compared to wild type (WT) mice. Part of the defect associates
with the expansion of T helper type 1 (Th1) and Th17 cells. We and others have shown that PPAR-δ-/- T cells proliferate
more and produce more cytokines compared to WT T cells. PPAR-δ-/- T cells are also more prone to activation as
evidence by higher CD25 and CD69 expression. In addition, PPAR-δ agonists have been shown to inhibit the pathogenic
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
72
Th cell responses in EAE. In this study, we are interested in elucidating the underlying mechanism of PPAR-δ functioning
in T cells. We conducted microarray studies to identify genes that are differentially expressed in the WT and PPAR-δ-/- T
cells. We have identified a gene profile that may explain the enhanced expansion and cytokine production by pathogenic
PPAR-δ-/- Th cells. Additional studies are now underway to investigate the roles of these genes in the signaling pathways
of T cell activation and proliferation in the WT and PPAR-δ-/- mice. These studies will shine light on the molecular
mechanism of inhibition of Th1 and Th17 inflammatory cells by PPAR-δ.
P66
In-Solution Single-Molecule Studies of a Protease Conformational Dynamics
Abdullah Bahram1, Amir Mazouchi
1, Walid Houry
2 and Claudiu C. Gradinaru
1
1 Department of Chemical and Physical Sciences, University of Toronto Mississauga
Caseinolytic protease from E. coli (eClpP), a known target of antibacterial drugs like
acyldepsipeptides (ADEPs), is a classical representative of serine proteases. eClpP is a
proteolic chamber that consists of two stacked heptameric rings. The rings are brought
together by the interaction between handle domains of opposite monomers. Several
biochemical studies reveal the plasticity of the handle regions and it is suggested that the
truncated peptides exit from the temporarily made side pores (1).
The goal of this study is to characterize the intrinsic, the substrate- and chaperone-induced dynamics of eClpP scaffold,
especially in the contact "handle" domain. We use a rigidity sensitive fluorophore (TMR) as an indicator of local
fluctuations in the protein structure. We performed in-solution single molecule measurements such as Fluorescence
Correlation spectroscopy (FCS) and Burst Analysis Spectroscopy (BAS). Experiments were performed on a custom-built
multiparameter photon-counting microscope (2).
In the pM concentration limit, lifetime and anisotropy analysis of photon bursts
revealed that the molecular chaperone eClpX induces a conformational change in the
handle domain of the proteolitic chamber. This suggests that the function of
chaperons is not only to unfold the substrates and feed it to the proteolic chamber
but also to induce a long range conformational change in the host ClpP protease. FCS
measurements were also performed to characterize the kinetics of this
conformational motions. These results give insight into the mechanism of release of
products from the ClpP proteolic chamber, hypothesized to involve transient pores in the handle domain (1).
-0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0 ClpP
ClpP+ peptide+ClpX
Rh6G
no
rma
lize
d f
req
ue
nc
y
Anisotropy
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
73
1. Yu, A. Y. H., and W. A. Houry. 2007. ClpP: A distinctive family of cylindrical energy-dependent serine proteases. FEBS
Letters 581:3749-3757.
2. Mazouchi, A., B. Liu, A. Bahram, and C. C. Gradinaru. 2011. On the performance of bioanalytical fluorescence
correlation spectroscopy measurements in a multiparameter photon-counting microscope. Analytica Chimica Acta
688:61-69.
P67
Refining the Electronic Hamiltonian of the Photosynthetic Protein, Phycocyanin 645, by Quantitative Fit of Linear
Spectra
Chi-Han Chang, Rayomond Dinshaw, Gregory D. Scholes
Department of Chemistry, and Center for Quantum Information and Quantum Control, University of Toronto
Photosynthesis is a complex biophysical and biochemical process that converts solar energy into chemical energy stored
in biomass. Central to the process are the light-harvesting proteins which absorb photons and transfer the resulting
excitation energy to reaction centers with greater than 95% quantum efficiency. To study such energy transfer with
quantum chemical approaches, an accurate Hamiltonian is needed.
By simulating linear spectra, we are able to refine the electronic Hamiltonian of the light-harvesting protein Phycocyanin
645 (PC645), isolated from the cryptophyte algae species Chroomonas CCMP270. Fluorescence, absorption, and circular
dichroism spectra of PC645 at physiological temperature and 77K are simulated. We use the Frenkel exciton model to
describe the electronic structure and two Brownian oscillators and eight discrete high-frequency modes to model the
protein backbone. Quantitative fit of the simulated spectra to experimental data enables us to decompose the spectra
into eight excitonic contributions and, above all, refine the site
(chromophore) energies in the electronic Hamiltonian of PC645.
An accurate Hamiltonian is crucial for further simulation of nonlinear
spectra such as two dimensional photon echo spectroscopy and for
elucidating the dynamics of the electronic energy transfer (EET) in light-
harvesting proteins. This complex biological system can be well described
using a quantum model; it is noteworthy that the results and methods may
be applicable both in advancing EET theories and in designing efficient solar
cells.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
74
Key words And Topics: Photosynthesis. Quantum Mechanics. Biophysics. Simulation of Linear Spectra. Electronic
Hamiltonian. Excitation Energy Transfer (EET). Dynamics. PC645.
P68
Theory and experiment of the first hyperpolarizability tensor of biological spherocrystals for second harmonic
generation microscopy
Richard Cisek, Adam Tuer and Virginijus Barzda.
Department of Chemical and Physical Sciences, Department of Physics and Institute for Optical Sciences, University of
Toronto.
Direct visualization of the tensor elements of the first hyperpolarizability can be achieved in radially arranged crystals
using second harmonic generation (SHG) microscopy. Two representative biological spherocrystals, starch and otoconia,
have been investigated. Both structures are very important for biological function, former providing storage of the
chemical energy in form of starch granules in plants, and later sensing a linear acceleration and gravity in animals.
Despite a very different physiological function, the radial structure of both biological spherocrystals carries organizational
similarities. We used nonlinear differential multicontrast microscopy to study polarization dependent SHG imaging of
freshly dissected otoconia from mouse ear. The imaged features were compared with experimental examination of
starch granules, and principles of growing radial spherocrystals were investigated. Imaging SHG of otoconia and starch
granules has been performed with a home built multicontrast laser scanning microscope coupled to a femtosecond
Yb:KGW laser radiating at 1030 nm with a pulse repetition rate of 15 MHz. We could directly visualize the
hyperpolarizability tensor elements by imaging with linearly polarized light and appropriately arranged analyzer in front
of the detector. Information about the susceptibility tensor elements was obtained with single scan using differential
microscopy, a technique where two beams with alternating polarizations are simultaneously deployed to image the same
structure. SHG microscopy is sensitive to small variations in the structure revealing growth rings in SHG granules, and
heterogeneities in otoconia. Therefore it is highly beneficial for numerous applications such as starch quality control and
medical diagnosis of otoconia related disorders.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
75
P69
The molecular love triangle – developing triple color FCCS for detecting complex biomolecular interactions
Holly Wobma, Megan Hartwick, Katya Grekova, Dr. David Cramb
Department of Chemistry, University of Calgary.
Currently, there is great interest in developing techniques to study biomolecular interactions. Applications of such
techniques include determining critical binding events in signal transduction pathways, assessing the affinity of potential
therapeutic agents for a receptor, and studying the rates of biologically relevant chemical reactions. One promising
method to detect binding events at even the picomolar (10-12
) level is Two-Color Fluorescence Cross-Correlation
Spectroscopy (2C-FCCS). This technique works by tracking whether the diffusion of the two different fluorophores in
solution is correlated. By fluorescently labeling different biological molecules (e.g. proteins, lipids, DNA), it is possible to
detect whether the biomolecules themselves interact. To date, FCCS has been limited to studying associations between
only two different fluorescently labeled species. Many biological interactions are quite complex, however, often involving
more than two players. Expanding FCCS to detect more than two simultaneous interactions would thus make it a more
powerful technique. In this study, a FCCS apparatus was developed to track the fluctuating fluorescence intensities in
three color channels, with the goal to cross-correlate these data. The validity of this system will be assessed by collecting
fluorescence data from known tri-colored nanocrystal species connected by oligonucleotides. Once optimized, this
technique will be the first example of direct Three-Color FCCS, and it can then be used to address specific biological
questions.
P70
Drift-oscillatory steering with the forward-reverse method for calculating the potential of mean force
Bryan W. Holland*, Mostafa NategholEslam*, Bruno Tomberli†, C. G. Gray*
*Department of Physics, Biophysics Interdepartmental Group, University of Guelph †Department of Physics and
Astronomy, Brandon University
We present a method that enables the use of the nonequilibrium forward-reverse (FR) method of Kosztin et al. on a
broader range of problems in soft matter physics. Our method, which we call the oscillating forward-reverse (OFR)
method, adds an oscillatory steering potential to the constant velocity steering potential of the FR method. This enables
the calculation of the potential of mean force (PMF) in a single unidirectional oscillatory drift, rather than multiple drifts
in both directions as required by the FR method. By following small forward perturbations with small reverse
perturbations, the OFR method is able to generate a piecewise reverse path that follows the piecewise forward path
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
76
much more closely than any practical set of paths used in the FR method. We calculate the PMF for four different
systems: a dragged Brownian oscillator, a pair of atoms in a Lennard-Jones liquid, a Na+ -Cl− ion pair in an aqueous
solution, and a deca-alanine molecule being stretched in an implicit solvent. In all cases, the PMF results are in good
agreement with those published previously using various other methods, and, to our knowledge, we give for the first
time PMFs calculated by nonequilibrium methods for the Lennard-Jones and Na+ -Cl− systems.
P71
Implementing the Photon Counting Histogram for Protein Studies
Yuchong Li and Claudiu Gradinaru
Department of Chemical and Physical Sciences, University of Toronto Mississauga, Institute for Optical Sciences and
Department of Physics, University of Toronto
Photon Counting Histogram (PCH) is a very versatile biophysical technique based on the
analysis of fluorescence intensity fluctuations. PCH is primarily used to measure the
brightness and the local concentration of a heterogeneous mixture of diffusive
fluorescent species based on the photon counts per molecule and the number of
molecules present in the detection volume. The histogram is generated by binning the
individual photon arrival times in a confocal setup and then fit the curve obtained to
super-Poissonian models in order to recover the molecular parameters of interest.
We have implemented the PCH analysis on a custom-built fluorescence microscope by
developing LabView software code for photon binning and curve fitting. Our approach
was tested using simulation data and measurements on dilute, homogeneous solutions
of standard fluorescent dyes. Initial biophysical applications include the quantization of
protein labels, e.g. Alexa555-streptavidin, and the investigation of the excited state photophysics of the DsRed
fluorescent protein. These results open up interesting possibilities for research of protein aggregation involved in amyloid
formation and for the study of single biomolecules trapped in lipid vesicles.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
77
P72
An Improved Method for Studying Single Proteins Trapped in Lipid Vesicles
Baoxu Liu, Amir Mazouchi and Claudiu Gradinaru
Department of Chemical and Physical Sciences, University of Toronto Mississauga
Institute for Optical Sciences and Department of Physics, University of Toronto
We report on an improved method to encapsulate proteins inside surface-tethered liposomes in order to reduce or
eliminate environmental interference for single-molecule investigations. The liposomes are large enough for the
molecule to experience free diffusion, but sufficiently small so that the molecule appears effectively immobile for
fluorescence imaging. Various single-molecule fluorescence experiments were performed to fully characterize this
anchoring method relative to direct immobilization via biotin-streptavidin linkers. Multidimensional histograms of
intensity, polarization and lifetime reveal that molecules trapped in liposomes display a narrow distribution around a
single peak, while the molecules directly immobilized on surface show highly dispersed values for all parameters1. For
instance, when TMR-labelled molecules were immobilized directly on surface, we recorded large intensity fluctuations
(6.30 ± 4.91 emission states per molecule), whereas the fluctuations were much smaller for the vesicle-trapped
molecules (1.37 ± 0.71 emission states per molecule).
During sample preparation, by hydrating the lipid film at low volumes, high encapsulation efficiencies can be achieved
with ~10 times less biological material than previous protocols. By measuring directly the vesicle size distribution, we
found no significant advantage for using freeze-thaw cycles during vesicle preparation. On the contrary, the temperature
jump can induce irreversible damage of fluorophores and it reduces significantly the functionality of proteins, as
demonstrated on single-molecule binding experiments involving a peptidic inhibitor for the oncogenic protein STAT3. Our
improved and biologically gentle molecule encapsulation protocol has a great potential for widespread applications in
single-molecule fluorescence spectroscopy.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
78
P73
The Average Size of the Unfolded State of SH3 Domain of the Drosophila Adapter Protein Drk
Amir Mazouchi1, Abdullah Bahram
1, Baoxu Liu
1, Sarah Rauscher
2, Régis Pomès
2, Julie Forman-Kay
3, Claudiu Gradinaru
1
1 Department of Chemical and Physical Sciences, University of Toronto Mississauga
2,3 Hospital for Sick Children and
Department of Biochemistry, University of Toronto
The SH3 fold is commonly found in nature and is often involved in mediating protein-protein interactions. drkN SH3
domain (6.8 kDa) has a highly populated unfolded state under non-denaturing conditions, with a folding and unfolding
rate of around 1 s-1
(1). These characteristics make it a useful model system for studying the physical properties of the
unfolded state, which is important to understanding protein folding in general.
Molecular dynamics (MD) simulations and NMR measurements lead to estimates
of the ratio of hydrodynamic radii (Rh) of the unfolded vs. folded drkN SH3 states
(2). Since drkN SH3 is the one of the few model proteins used for studying the
unfolded state, it is important to obtain an accurate measure of the average
hydrodynamic radius of the ensemble. The goal of our work is to obtain an
independent estimate of the hydrodynamic radius of the unfolded state of drkN
SH3.
Fluorescence correlation spectroscopy (FCS) (3) was used to measure the Rh ratio
and the fraction of unfolded/folded protein at 20°C. Meseth et. al. (4) showed
that the discrimination between two diffusive components in a mixture with a Rh
ratio of less than 1.6 is impossible by standard one-color FCS. Here we used ssDNA and dsDNA to demonstrate the
overcoming of this limitation if the diffusion properties of at least one of the pure components are known. The same idea
was applied to determine the ratio of unfolded vs. folded Rh of the SH3 domain, by making use of a mutant (T22G) which
is fully folded at room temperature.
1. M Tollinger et al., J. Am. Chem. Soc.;123(46):11341-11352 (2001)
2. WY Choy et al., J. Mol. Biol.;316(1):101-112 (2002)
3. A Mazouchi et al., Anal. Chim. Acta 688 (1), 61-69 (2011)
4. U Meseth et al., Biophys. J., 76, 1619–1631(1999)
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
79
P74
Using Opto-acoustics to Track Magnetite Nanoparticles for the Detection of Sentinel Lymph Nodes in Breast Cancer
GA Noble, MC Kolios, CJ Kumaradas
Department of Physics, Ryerson University.
Research is underway to develop opto-acoustic techniques for tracking magnetite nanoparticles injected around breast
tumors to the sentinel lymph nodes. Currently magnetite nanoparticles are being used as a visual contrast agent for the
surgical removal of sentinel lymph nodes. The magnetite preparation is injected near the tumor and is taken up by the
lymph system and accumulates in the sentinel lymph nodes. Magnetite can also be heated to ~45 ° C using alternating
magnetic fields. At these temperatures cell death occurs due to thermal therapy (hyperthermia). This type treatment
would be minimally invasive. For effective hyperthermia treatment, the magnetic nano-particles must be
tracked/monitored to determine the optimum time after injection that maximum accumulation in lymph nodes occurs
and to determine the amount of normal tissue involvement. Preliminary research has demonstrated that opto-acoustic
techniques can detect magnetite nano-particles. Opto-acoustic imaging refers to detection of an acoustic pressure wave
in tissue created from a pulse light source incident on that tissue. The monitoring will ensure that the particles are
isolated in the proper region at a known time after injection. This aims of this research are: 1) to develop opto-acoustic
imaging techniques for the detection of magnetite nano-particles in tissue at depths appropriate for breast
imaging/treatment, and 2) to develop computational techniques and carry out experimental work to determine the
optimal parameters for imaging magnetite nano-particles in breast tissue.
P75
Updates from the construction of the fast nonlinear microscope
Masood Samim and Virginijus Barzda
Department of Physics, University of Toronto
Recent developments towards high-speed imaging capabilities have
gained significant notice in the field of microscopy for rapid dynamics
of important biological processes. Three-dimensional video-rate
scanning capability is essential to the investigation of rapid dynamics in
organisms, such as their muscle contractions. The multi-contrast,
multi-foci nonlinear optical microscope with fast scanning capabilities
is seen as the indispensable solution. We have developed such a tool in
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
80
our lab that has integrated a Xilinx Vertix-5 FPGA board, a pair of deformable mirrors, and the galvo/scanning mirrors.
The optical wavefronts of a novel Yb:KGW femto-second (1028 nm) laser-beams are monitored by the microlens-array
Shack-Hartmann wavefront sensor. The setup is capable of capturing simultaneous images of orthogonal polarization
from distinct optical sections, in both forward and backward directions. We demonstrate the ability of the 39-actuator
deformable mirrors in reshaping the wavefront and correcting for the optical aberrations to achieve the diffraction-
limited focal volume for video-rate scanning.
P76
Numerical analysis of third harmonic generation in nonlinear microscopy
Daaf Sandkuijl, Virginijus Barzda
Department of Physics, Institute for Optical Sciences and Department of Chemical and Physical Sciences, University of
Toronto Mississauga
Adaptive optics and focal spot engineering are projected to play an important role in the future of nonlinear microscopy.
Applying new optical techniques to arbitrarily shape and optimize the focal spot can lead to enhanced signal generation
and new contrast mechanisms, which in turn enables endoscopic application of nonlinear microscopy in the medical
environment and provides nonlinear microscopy with new investigative possibilities.
In the context of applying focal spot engineering to nonlinear microscopy, it is extremely important to be able to predict
the nonlinear signal generated by a tailored input beam. In this work we focused on numerically calculating the electric
field at the focus of a microscope objective for an arbitrary input beam, calculating the nonlinear signal generated at the
focus by an arbitrary material or multilayer structure, and the far field radiation intensity that would be observed.
Third harmonic generation (THG), a common mode of nonlinear microscopy, is mostly generated at interfaces between
media of different refractive indexes or nonlinear susceptibility, due to cancellation of the THG signal in bulk media. In
this work we show numerical calculations on THG with a higher theoretical level than preceding work. These numerical
calculations have been generalized to include second harmonic generation (SHG) as well. We present results on THG
from interface structures and glass wedges, where we observe THG signal enhancement from wedge structures, and THG
signal reminiscent of Maker’s fringes focused with high numerical aperture objectives. We show that the generation of
Maker’s fringes depends mainly on the optical dispersion of the material wedged between glass slabs. The new findings
will be used to investigate multilayer biological structures such as mitochondria and chloroplasts.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
81
P77
Unit Sphere Representation of the First Hyperpolarizability Tensor
Adam Tuer1,2,3
, Serguei Krouglov2, Richard Cisek
1,2,3, Danielle Tokarz
1,4, Virginijus Barzda
1,2,3,4
1Department of Chemical and Physical Sciences, University of Toronto Mississauga
2Department of Physics, University of
Toronto 3Institute for Optical Sciences, University of Toronto
4Department of Chemistry, University of Toronto
With the increased popularity in investigating biological materials with polarization
dependent second harmonic generation (SHG) microscopy, the need to develop tools with
which to understand and interpret observed SHG properties has arisen. Quantum
mechanical calculations of the rank-3 first hyperpolarizability tensor have become a popular
method for understanding the SHG properties of molecules. The unit sphere representation
visualizes the incident and SHG polarizations with respect to the molecule by utilizing an
effective dipole generated from the double scalar product between the first
hyperpolarizability tensor and the dyadic product of the incident electric
fields ),(),(ˆ: ϕθϕθββ EEeff
)rr= . The representation was developed to provide insight and intuition regarding the
relationship between SHG properties and molecules. The visualization tool has the potential to facilitate the design of
novel nonlinear optical materials and may prove valuable in deducing the structural organization of molecules in
biological samples imaged with polarization dependent SHG microscopy.
P78
Innovative Nanostructures as Harmonophores for Third Harmonic Generation Microscopy
Danielle Tokarz1,2
, Richard Cisek2,3
, Adam Tuer2,3
, Virginijus Barzda1,2,3
, and Ulrich Fekl1,2
1Department of Chemistry, University of Toronto.
2Department of Chemical and Physical Sciences, University of Toronto
Mississauga. 3Department of Physics and Institute for Optical Sciences, University of Toronto.
Third harmonic generation (THG) microscopy is a valuable technique used for imaging biological systems without
staining. THG is produced at an interface where a difference in the refractive index or third-order nonlinear susceptibility
is present. Therefore, biological membranes and multilamellar structures are readily visualized with THG microscopy.
Although highly valued for noninvasive structural visualization without labeling, THG microscopy is not specific to
particular biomolecules or cellular organelles. Structural specificity can be achieved by designing molecular labels that
demonstrate large third-order optical nonlinearities. Several molecules have been shown to label and enhance THG signal
intensity of stained intracellular structures. Such compounds have been designated the term, harmonophores. Recently,
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
82
we have found that cancerous tissue when stained with hematoxylin solution, a label used by pathologists for better
contrast under white light microscopy, also demonstrated intense THG due to the aggregation of hemalum particles,
inorganic aluminum oxide oligomers formed in tissue under specific pH conditions. Therefore, we have reasoned that
labels for THG microscopy should occur in an aggregated form.
Since this discovery, we have been able to synthesize various sizes of hemalum nanoparticles. By controlling synthesis
conditions, transmission electron microscopy revealed the growth of sea-urchin-like hemalum nanostructures. Using THG
microscopy, we have tested the size dependence of the hemalum nanostructures on their ability to generate third
harmonic signal. These findings revealed the optimal size of the hemalum for maximal ability to generate THG. In parallel,
we have also studied the ability of hemalum nanoparticles to label cells via endocytosis. The discovery of hemalum
nanoparticles opened new perspectives for developing high brightness bleach-free harmonophore labels for the study of
cellular structures with nonlinear microscopy.
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
_________________________
Forest View Restaurant466-468 Dundas St. W
Saturday Evening Banquet
Lash Miller Chemical Laboratories
80 St. George St.
Leslie L. Dan Pharmacy Building
144 College St.
Pogue Mahone777 Bay Street
Friday Evening Social
200 metres
Subway StopsM
CBP would not be possible without a little help from our friends:
Thank you for your generosity and support.