8th international meeting biomolecules under pressure (imbp) · andrea kreusel samy al-ayoubi...
TRANSCRIPT
8th
INTERNATIONAL MEETING ON
BIOMOLECULES UNDER PRESSURE (IMBP)
- Towards New Horizons in High Pressure Molecular Biosciences -
15th
-17th
February, 2016 TU Dortmund University
Dortmund, Germany
2
Contents
Objectives ...................................................................................................... 3
Sponsors ........................................................................................................ 3
Organization .................................................................................................. 4
Location ......................................................................................................... 5
Conference Program ...................................................................................... 7
Oral Presentations........................................................................................ 11
Poster Presentations. ................................................................................... .45
List of Participants...................................................................................... 69
Objectives
3
Objectives
The IMBP ("International Meeting on Biomolecules under Pressure")
meetings have a long tradition meanwhile, with meeting places in Japan,
France, the USA and Germany. The IMBP meetings deal with fundamental
questions regarding high pressure molecular bioscience, including topics
revolving around hydration and conformational fluctuations in proteins,
fundamentals of volume, compressibility and expansivity, the free energy
landscape & high-energy conformers on the pressure axis, pressure
perturbation of intermolecular interactions and associations, high-pressure
enzymology, cosolvent and crowding effects, corresponding topics on lipid
membranes and nucleic acids, and related deep sea biology. This time we
are planning to expand the participating community, bringing together
people from the "traditional" IMBP group, researchers from our DFG
Research Unit FOR 1979 "Exploring the Dynamical Landscape of
Biomolecular Systems by Pressure Perturbation", colleagues interested in
Solvation Science (from RESOLV, located at the Ruhr-University of
Bochum) and in high pressure molecular sciences in general.
Sponsors
Organization
4
Organization
Conference Chair
Prof. Dr. Roland Winter (TU Dortmund)
Local Organizing Committee
Andrea Kreusel
Samy Al-Ayoubi
Süleyman Cinar
Paul Hendrik Schummel
Ralf Maserski
Conference Webpage
http://www.ccb.uni-dortmund.de/fb03/de/Forschung/PC/Winter/index.html
Scientific Advisory Board
Prof. Dr. Catherine Royer (Rensselaer Polytechnic Institute)
Prof. Dr. Dr. Hans-Robert Kalbitzer (University of Regensburg)
Prof. Dr. Kazuyuki Akasaka (Kinki University)
Prof. Dr. Tigran Chalikian (University of Toronto)
Site map of TU Dortmund
5
Location
Site map of TU Dortmund
6
Conference Program
7
Conference Program
Monday, February 15th
, 2016
8:00 Registration
8:50 Roland Winter Opening Remarks
Session 1: Chair: R. Winter
9:00 Thomas
Kiefhaber
Reaction and Activation Volumes for Fast
Conformational Transitions in Proteins Measured by
High-Pressure Triplet-Triplet Energy Transfer
Experiments
9:30 Jochen
Balbach
Pressure-Temperature Phase Diagrams of Proteins
Probed by High-Pressure NMR
10:00 Kazuyuki
Akasaka
Cavities Control Function through Coupled Motions
in the Excited States: T4 Lysozyme
Coffee Break (10:30-11:00)
Session 2: Chair: K. Weise
11:00 Robert
Macgregor
The Effect of Loops on the ΔV of the Unfolding of
Structures Formed by Human Telomeric Sequence
11:30 Tigran V.
Chalikian
Hydration and Volumetric Properties of G-
quadruplexes
12:00 Narendra
Kumar
Molecular Dynamics Study of Pressure-induced
Effects on Ribozyme Catalysis
Lunch (12:30-13:30)
Session 3: Chair: W. Kremer
13:30 Christian
Roumestand
Exploring the Unfolding Landscape of ∆+PHS
Staphylococcal Nuclease with High-Pressure NMR:
Effect of Cavity Creation
14:00 Toshiko Ichiye
Pressure and Temperature Dependence of Enzyme
Flexibility: Molecular Dynamics Simulations
14:30 Martin
Hofmann
Investigation of Pressure Effects on Modular Peptidic
Organocatalysts
Conference Program
8
Coffee Break (15:00-15:30)
Session 4: Chair: K. Akasaka
15:30 Hans Robert
Kalbitzer
Detection of Rare Conformational States by High
Pressure NMR Spectroscopy
16:00 Michael
Spoerner
Characterization of Intrinsic Conformational
Equilibria in Ras-like Proteins by High Pressure
NMR: Identifying Targets for Novel Signaling
Modulators
16:30 Werner
Kremer
Rare Excited States of Human IAPP and Short α/β-
Peptide Catalysts Studied by High Pressure NMR
Spectroscopy
17:00 Mariano
Dellarole
Evolutionarily Conserved Pattern of Interactions in a
Protein Revealed by Local Thermal Expansion
Properties
Poster Session (17:30-19:30)
Tuesday, February 16th
, 2016
Session 5: Chair: C. Czeslik
09:00 Judith Peters Neutron Techniques for the Investigation of
Molecular Dynamics under High Hydrostatic Pressure
09:30 Phil M. Oger Bridging the Gap Between Physiological and
Biophysical Insights on Molecular Adaptation in the
High-Hydrostatic Pressure Adapted Archaeon T.
barophilus
10:00 Nick Brooks
Triggering Dynamic Structural Changes in Model
Lipid Membranes
Coffee Break (10:30-11:00)
Session 6: Chair: D. Horinek
11:00 Stefan M. Kast Electronic Structure and Interactions at High
Hydrostatic Pressure
11:30 Nico van der
Vegt
Mechanism of Hydrophobic Polymer Collapse in
Miscible Good Solvents
Conference Program
9
12:00 Sho Imoto Pressure Effects on Solvation Structure and
Vibrational Spectroscopy of Aqueous TMAO
Solutions
12:30 Lukas Knake
The Impact of TMAO and Urea on the H-Bond
Dynamics under High Pressure
Lunch (13:00-14:00)
Session 7: Chair: S. Kast
14:00 Julia
Nase
The Solid/liquid Interface under Conditions of High
Hydrostatic Pressure
14:30 Dominik
Horinek
Pressure Effects on an Alkane SAM/Water Interface
15:00 Claus Czeslik Combined Effects of Pressure and Interfaces on
Enzymatic Activity
Coffee Break (15:30-16:00)
Session 8: Chair: T. Chalikian
16:00 László Smeller
FTIR studies on Biomolecules under Pressure
16:30 Arvi Freiberg
Pressure Tuning of Primary Photochemistry
17:00 Vytautas
Petrauskas
Determination of the Protein-Ligand Binding Volume
by High-Pressure Spectrofluorimetry
Conference Dinner (19:30)
Hövels-Brewery (Hoher Wall 5, Dortmund)
Wednesday, February 17th
, 2016
Session 9: Chair: H.-R. Kalbitzer
9:30 Masayoshi
Nishiyama
High-Pressure Microscopy for Studying Molecular
Motor and Cytoskeleton
10:00 Elena
Boldyreva
Pressure Effects on Amino Acids and their Salts in the
Crystalline State
10:30 Guilherme A.
P. de Oliveira
Alpha-Synuclein Fibrils Triggered by Pressure and
the Seeding Mechanism in Parkinson Disease
10:50 Sunilkumar P.
Narayanan
Activation of Auto-Inhibited Twitchin Kinase by
Compressive Force - a High Pressure NMR Study
Conference Program
10
11:10 Maksym
Golub
Combined SANS-QENS Studies of Low-Density
Lipoprotein Under High Hydrostatic Pressure
11:30 Julian Schulze
Phase Behavior of Dense Lysozyme Solutions
11:50 Mimi Gao Actin Polymerization and Bundling: Exploring their
Temperature and Pressure Limits
Lunch (12:10-13:30)
- End of Conference -
11
Oral Presentations
Oral Presentations
12
Reaction and Activation Volumes for Fast Conformational Transitions in
Proteins Measured by High-Pressure Triplet-Triplet Energy Transfer
Experiments
Thomas Kiefhaber, Sabine Neumaier Martin-Luther-Universität Halle-Wittenberg, Institute of Biochemistry und Biotechnology, Kurt-Mothes-
Strasse 3, 06108 Halle (Saale), Germany
We investigated conformational fluctuations in peptides and proteins on the nanoseconds to
microseconds time scale using triplet-triplet energy transfer (TTET), which is a diffusion-
controlled process that requires van-der-Waals contact between a triplet donor and an acceptor
group. The photochemical processes involved in TTET occur on the picoseconds time scale which
enables measurements of rate constants for diffusional processes on the time scale of 10
picoseconds to 100’s of microseconds. Coupling TTET to a conformational equilibrium in folded
structures gives site-specific information on dynamic and equilibrium properties of conformational
fluctuations in protein secondary structures, folding intermediates and native proteins. We
performed TTET experiments at pressures between 1 and 4000 bar to investigate local stability and
dynamics in -helical peptides and in the native state of the villin headpiece subdomain to gain
information on reaction and activation volumes of fast conformational transitions in proteins.
TTET experiments showed that the volume of a 21-amino acid alanine-based helical peptide
decreases upon helix formation. Thus, helices (in contrast to native proteins) become more stable
with increasing pressure explaining the frequently observed helical structures in pressure-unfolded
proteins. The reaction volume for adding a single residue to a helix is small and negative (-0.23
cm3/mol = -0.38 Å
3/molecule) implying that intrahelical H-bonds have a slightly smaller volume
than peptide-water H-bonds. Both helix folding and unfolding become slower with increasing with
activation volumes of 2.2 cm3/mol (3.7 Å
3/molecule) for adding and 2.4 cm
3/mol (4.0 Å
3/molecule)
for removing a single residue. The larger volume of the transition state may be due to the presence
of unsatisfied hydrogen bonds, although steric effects may also be involved.
A dry molten globule (DMG) state has been observed as a transient intermediate in protein
unfolding and as an alternative native state for several proteins. The DMG state has a solvent
inaccessible core but shows increased side-chain flexibility and reduced strength of side-chain
interactions compared to the native state and was proposed to have a larger volume than the native
state. TTET experiments discovered two native states in the villin headpiece subdomain (HP35)
and one of them shows the properties of a DMG. High-pressure studies revealed that the two native
states have a similar volume but the transition state separating them has a largely increased volume.
The properties of the alternative native state indicate that it represents a compact DMG state,
whereas the transition state for interconverting between the two native states represents the
originally proposed expanded DMG state.
References:
[1] Neumaier, S., Büttner, M., Bachmann, A. & Kiefhaber, T. Transition state and ground state
properties of the helix-coil transition in peptides deduced from high pressure studies. Proc.
Natl. Acad. Sci. USA 110 (2013) 20988–20993
[2] Reiner, A., Henklein, P. & Kiefhaber, T. An Unlocking/Relocking Barrier in Conformational
Fluctuations of Villin Headpiece Subdomain. Proc. Natl. Acad. Sci. USA 107 (2010) 4955-
4960
[3] Neumaier, S. & Kiefhaber, T. Redefining the dry molten globule state of proteins. J. Mol. Biol.
426 (2014) 2520-2528
Oral Presentations
13
Pressure-temperature phase diagrams of proteins probed by high-
pressure NMR
Jochen Balbach
Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, Germany;
The accessible free energy landscape is a generic property of proteins, which determines both their
protein folding pathways and their biological function. This landscape can be explored by
determining the thermodynamic stability of proteins at different pressures and temperatures. We
combine these variations with NMR spectroscopy to gain molecular resolution. Pressure-
temperature phase diagrams of three different proteins (Bs-CspB, Bc-Csp R3E L66E and Kti11)
will be presented and the determination of changes in volume, thermal expansion, and
compressibility upon unfolding. A residue-by-residue analysis reveals pressure sensitive sections
along the peptide chain, which correspond with functional properties of the respective protein.
Oral Presentations
14
Cavities control function through coupled motions in the excited states:
T4 lysozyme
Akihiro Maeno1,2
, Ryo Kitahara3, Renee Otten
4, Frederick W. Dahlquist
5, Shigeyuki Yokoyama
6,
Frans A. A. Mulder7, Kazuyuki Akasaka
1,8
1 - High Pressure Protein Research Center, Institute of Advanced Technology, Kinki University, 930
Nishimitani, Kinokawa, Wakayama 649-6493, Japan
2 - RIKEN SPring-8 Center Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan
3 - College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
4 - Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4,
9747 AG Groningen, The Netherlands
5 - The Department of Chemistry and Biochemistry and the Department of Molecular, Cellular and
Developmental Biology, University of California Santa Barbara, Santa Barbara CA 93106-6105, USA
6 - RIKEN Systems and Structural Biology Center, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045,
Japan
7 - Department of Chemistry and Interdisciplinary Nanoscience Center iNANO, University of Aarhus, Gustav
Wieds Vej 14, DK-8000 Aarhus C, Denmark
8 - Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho,
Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522, Japan
To find and characterize functionally-relevant motions in enzymes is not only a fundamental, but
experimentally a challenging problem. This is because, in enzymes, these motions are relatively
small in amplitude and occur relatively rarely, making their detection extremely difficult with most
conventional techniques. To overcome this problem, our strategy is to enhance the amplitude of
such motions by applying pressure and detect them sensitively as changes in the 1H,
13C/
1H and/or
15N/
1H NMR spectra. Here we apply the method to wild-type T4 lysozyme and provide evidence
that cavities are the independent source of motions in the protein and that these motions are
dynamically coupled to the functionally-relevant motions in the distant catalytic region, through the
excited states of the protein.
Oral Presentations
15
The effect of loops on the ΔV of the unfolding of structures formed by
human telomeric sequence
Yang Li, Francisco Wong Chung, Karen Leung, and R. B. Macgregor, Jr.* Department of Pharmaceutical Sciences, University of Toronto, 144 College St., Toronto, Ontario, M5S 3M2
Canada
In aqueous solutions containing Na
+ or K
+, oligodeoxyribonucleotides (ODNs) rich in guanine
form non-canonical DNA structures called G-quadruplexes (GQ), which are destabilized at
elevated pressure. We have used pressure to investigate the volumetric changes arising from the
formation of GQ structures. GQs display a great deal of structural heterogeneity that depends on
the stabilizing cation as well as the oligonucleotide sequence. Using UV melting at different
pressures, we have investigated the volume change of the helix-coil equilibrium of ODNs whose
sequence is related to the G-rich human telomeric sequence. The sequence of the ODNs used in
this study are based on that of HTel (d[A(GGGTTA)3GGG]), which contains four repeats of the
human telomeric sequence. The experiments are conducted in aqueous buffer, pH 7.4, containing
either 100 mM NaCl or KCl. The GQs stabilized by Na+ are more sensitive to pressure perturbation
than those stabilized by K+. The molar volume change (ΔV) of the unfolding transition of these
GQs is large and negative. A large fraction of the measured ΔV value arises from the re-hydration
of the cations released from the interior of the folded structure. However, the differences in the
measured ΔV values demonstrate that variations in the structure of each ODN, arising from
differences in the sequence of the loops, contribute significantly to the total volume change and
presumably the hydration of the folded structures. Depending on the sequence of the loop, the
magnitude of the measured volume changes can be larger and smaller than that of HTel in solutions
containing either sodium or potassium ions.
Oral Presentations
16
Hydration and Volumetric Properties of G-quadruplexes
Tigran V. Chalikian
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144
College Street, Toronto, Ontario M5S 3M2, Canada
Guanine-rich DNA sequences that may form G-quadruplexes are located in strategic DNA loci
with the ability to regulate biological events. G-quadruplexes have been under intensive scrutiny
owing to their potential to serve as novel drug targets in emerging anticancer strategies.
Thermodynamic characterization G-quadruplexes is an important and necessary step in developing
predictive algorithms for evaluating the conformational preferences of G-rich sequences in the
presence or the absence of their complementary C-rich strands. We used a combination of
spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding
transitions of two human telomeric DNA sequences with the potential form intramolecular G-
quadruplexes – the 22-meric d[A(G3T2A)3G3] (Tel22) and the 26-meric d[A3G3(T2AG3)3A2]
(Tel26) oligomeric sequences. In the presence of Na+ ions, the former adopts an antiparallel G-
quadruplex conformation. On the other hand, the latter forms, in the presence of K+ ions, the
hybrid-1 G-quadruplex structure, a tightly packed structure with an unusually small number of
solvent-exposed atomic groups. The Na+- or K
+-induced folding of the G-quadruplex at room
temperature is a slow process that involves significant accumulation of an intermediate at the early
stages of the transition. The two G-quadruplexes we studied are characterized by larger volumes
and compressibilities and smaller expansibilities compared to their respective coil states. These
results are in qualitative agreement with each other all suggesting significant dehydration to
accompany the G-quadruplex formation. Based on our volumetric data, we estimate that 103 ± 44
and 432 ± 19 water molecules become released to the bulk upon the G-quadruplex formation by the
Tel22 and Tel26 DNA sequences. These large numbers suggest DNA dehydration may not be
limited to water molecules in direct contact with the regions that become buried but may involve a
general decrease in solute-solvent interactions all over the surface of the folded structure.
Oral Presentations
17
Molecular Dynamics Study of Pressure-induced Effects on Ribozyme
Catalysis
Narendra Kumar and Dominik Marx
Lehrstuhl für Theoretische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Email: [email protected]
Ribozymes, a very important class of non-coding RNA molecules, catalyze site-specific self-
cleavage reactions of phosphodiester bonds and play prominent roles in several biological
processes such as RNA splicing, controlling of gene expression, and processing of tRNA. What
remains largely unexplored, however, is the pressure-response of ribozymatic activity. Large-scale
replica exchange molecular dynamics simulations including explicit solvent and ions were utilized
to explore pressure effects on the self-cleavage reaction of a ribozyme. Our results provide strong
support for the involvement of trapped water molecules.
Oral Presentations
18
Exploring the Unfolding Landscape of ∆+PHS Staphylococcal Nuclease
with High-Pressure NMR: Effect of Cavity Creation.
Julien Roche1, Mariano Dellarole
1, Jose A. Caro
2, Angel E. Garcia
3, Bertrand Garcia-Moreno E
2.,
Catherine A. Royer1,3
and Christian Roumestand1.
1Centre de Biochimie Structurale, INSERM U554, CNRS UMR 5048, Universités de Montpellier,
France. 2Department of Biophysics, Johns Hopkins University, Baltimore MD USA.
3Department of
Physics and Applied Physics and Center for Biotechnology and Interdisciplinary Studies,
Rensselaer Polytechnic Institute, Troy NY USA
Staphylococcal nuclease (SNase) has long served as a model system for protein folding. It is a
globular protein of moderate complexity, consisting of two structural sub-domains (SubD1 and
SubD2) and the interface between this two domains (IntD). To identify the structural and energetic
determinants of its folding free energy landscape we have used high pressure NMR to examine the
consequences of cavity creating mutations in each of the two sub-domains of SNase (1, 2). Cavity
creation in different regions of the reference protein, despite equivalent effects on global stability,
had very distinct consequences on the complexity of the folding free energy landscape.
The folding pathway of ∆+PHS SNase as seen through
computation based on HP-NMR data.
To address the effect of cavity
creation on SNase folding
kinetics, and thus to the
Transition State Ensemble
(TSE), we also performed
pressure-jump relaxation studies
on these proteins (3). Real-time 1H-
15N 2D correlation peak
intensity profiles were collected
at over 100 residues for SNase
and several cavity containing
variants as a function of
pressure. For SNase, the
intermediate with a folded
SubD1 and a disordered SubD2
is populated not only at
equilibrium under certain
conditions, but also transiently.
Pressure therefore facilitates the identification and characterization of the multiple conformations
on a folding landscape, and has provided crucial information for understanding the sequence and
structural determinants of this complex process.
References:
(1) J. Roche et al. (2012). Cavities Determine the Pressure Unfolding of Proteins. Proc. Natl. Acad.
Sci. USA 109 (18), 6945-50.
(2) J. Roche et al. (2012). Remodeling of the folding free-energy landscape of staphylococcal
nuclease by cavity-creating mutations. Biochemistry 51(47), 9535-46.
(3) J. Roche et al. (2013). Effect of Internal Cavities on Folding Rates and Routes Revealed by
Real-Time Pressure-Jump NMR Spectroscopy. J. Am. Chem. Soc. 135(39), 14610-14618.
Oral Presentations
19
Pressure and temperature dependence of enzyme flexibility: Molecular
dynamics simulations
Jocelyn Rodgers, Qi Huang, Kelly Huang, and Toshiko Ichiye*
Department of Chemistry, Georgetown University, Washington, D.C. 20057, USA
Extremophiles must maintain enzyme activity under extreme conditions to grow and flourish.
Enzymes from thermophiles and psychrophiles generally have similar flexibility at growth
temperatures as that of homologs from mesophiles at normal temperature but must also maintain
their three-dimensional structures [1]. Thermophile enzymes need to be more thermostable than
their mesophile homologs, which also prevents them from being too flexible at growth
temperatures since flexibility increases with temperature. In contrast, psychrophile enzymes tend to
be less stable than their mesophile homologs to be flexible enough at their growth pressures, and
are sometimes only marginally stable. Enzymes from piezophiles may have the same conflict as
those from psychrophiles since flexibility decreases with increasing pressure. However, unraveling
the effects of pressure and temperature is difficult since many of the known piezophiles were
isolated from deep cold ocean environments. Here, flexibility matching is examined in Escherichia
coli and Photobacterium profundum adenylate kinase and in E. coli and Moritella profunda
dihydrofolate reductase using molecular dynamics simulations. In addition, structure and
fluctuations at high pressures in microsec timescale simulations of Clostridium acidurici ferredoxin
are examined.
References:
[1] Georlette, D., V. Blaise, T. Collins, S. D'Amico, E. Gratia, A. Hoyoux, J.-C. Marx, G. Sonan,
G. Feller, C. Gerday, FEMS Microbiology Reviews 28 (2004) 25-42
Oral Presentations
20
Investigation of pressure effects on modular peptidic organocatalysts
M. Hofmann[a]
, L. Pilsl[a]
, B. Ertinger[a]
, A. Haag[a]
, O. Reiser[a]
, S. Puthenpurackal[a], W. Kremer[a]
,
H.-R. Kalbitzer[a]
, C. R. Botines[b]
, O. I. Soler[b]
, R. M. Ortuño[b]
[a] Universität Regensburg, Universitätsstraße 31, 93053 Regensburg/D
[b] Universitat Autònoma de Barcelona, Placa Cívica, 08913 Bellaterra, Barcelona/ESP
Ever since its resurrection at the turn of the century, organocatalysis is developing into an
important tool for organic chemistry besides metal- and biocatalysis, as it provides access to a
multitude of chemo-, regio- and stereoselective transformations [1-3]. The often stated low
reactivity of these catalysts can be counteracted when a secondary activation mode like high
hydrostatic pressure (HHP) is employed [4,5]. The use of HHP greatly accelerates organocatalyzed
reactions and even promotes reactions, which are not feasible under ambient conditions [6,7].
Interestingly, this combination of organocatalysis and HHP has not been investigated to a great
extent up until today. The effect of HHP on the relationship between catalyst structure,
conformational freedom of its scaffold, and catalytic performance is a subject of investigation
which has yet to be explored. To shed light onto this problem, small catalytically active tripeptides
with the structural motif H-Pro-ucAA-Pro-OH were chosen as model systems [8-10]. The use of
unnatural cyclic amino acids (ucAA) enables to fine-tune the conformational freedom of the
catalyst system and thereby the possible effects of high pressure on their conformational and
catalytic behavior. Various tripeptides have been synthesized and their abilities as asymmetric
catalysts were investigated under ambient as well as HHP conditions. These results were then
compared with high pressure NMR investigations of the same peptides, which have been conducted
to gain insight into their conformational behavior and the resulting changes upon pressurization.
This should allow for a deeper insight into the influence of HHP on organocatalyzed reactions.
References:
[1] D. W. C. MacMillan, Nature 2008, 304.
[2] E. N. Jacobsen, D. W. C. MacMillan, PNAS 2010, 107, 20618.
[3] J. Seayad, B. List, Org. Biomol. Chem. 2005, 3, 719.
[4] J. G. Hernández, E. Juaristi, Chem. Commun. 2012, 48, 5396.
[5] G. Jenner, Tetrahedron 2002, 58, 5185.
[6] K. Matsumoto, T. Uchida, Chem. Lett. 1981, 10, 1673.
[7] A. Sera, K. Takagi, H. Katayama, H. Yamada, J. Org. Chem. 1988, 53, 1157.
[8] V. D’Elia, H. Zwicknagl, O. Reiser, J. Org. Chem. 2008, 73, 3262.
[9] L. Pilsl, Dissertation, 2014, Universität Regensburg.
[10] C. R. Botines, Master Thesis, 2014, Universitat Autònoma de Barcelona.
Oral Presentations
21
Detection of rare conformational states by high pressure NMR
spectroscopy
Hans Robert Kalbitzer, Claudia E. Munte, Italo A. Cavini, Michael Spoerner, Markus Beck Erlach,
Joerg Koehler, Werner Kremer
Institute of Biophysics and Physical Biochemistry, University of Regensburg, 93053 Regensburg, Germany
Rare conformational states are often crucial for the understanding of function and catalytic
mechanism of biological macromolecules. In addition to the elucidation of the classical three-
dimensional (ground state) structure of proteins there is gaining interest in the dynamic properties
of proteins and the characterization of intrinsic equilibria between conformers. Applying high
pressure to such biomolecules allows the shift of intrinsic conformational equilibria up to the
stabilization of higher energy conformers according to their smaller specific volume. The
combination of high pressure conditions with NMR spectroscopy as detection method allows the
investigation of such intrinsic equilibria of conformations and the characterization of rare
conformers up to atomic resolution, respectively [1]. Rare (exited) conformational states allow to
define a novel type of allosteric inhibitors, the intrinsic allosteric inhibitors, in drug design [2].
They are most probably also involved in the fibrillation of pathogenic proteins such as the Amyloid
peptide Aand the prion protein. The talk will focus on the pressure response of the intrinsically unfolded peptide A. A fibrils
are found in the brains of patients with Alzheimer’s disease. The focus will be on the possible
detection of excited states during fibril formation and their thermodynamics. In addition, the
pressure response of random-coil model peptides will be reported.
References:
[1] H. R. Kalbitzer, High Pressure NMR Methods for Characterizing Functional Substates of
Proteins. In “High Pressure Bioscience - Basic Concepts, Applications and Frontiers” (K. Akasaka
and H. Matsuki, eds, 2015, pp. 179-198), Springer, Heidelberg, Germany
[2] H. R., Kalbitzer, I. C. Rosnizeck, C. E Munte, S. Puthenpurackal Narayanan, V. Kropf, and M.
Spoerner (2013) Intrinsic Allosteric Inhibition of Signaling Proteins by Targeting Rare Interaction
States Detected by High-Pressure NMR Spectroscopy. Angew. Chem. Int. Ed. 52, 14242 –14246.
[3] C. E. Munte, M. Beck Erlach, W. Kremer, J. Koehler, and H. R. Kalbitzer (2013) Distinct
Conformational States of the Alzheimer -Amyloid Peptide can be Detected by High Pressure
NMR Spectroscopy. Angew. Chem. Int. Ed. 52, 8943 –8947
Oral Presentations
22
Characterization of Intrinsic Conformational Equilibria in Ras-like
Proteins by High Pressure NMR: Identifying Targets for Novel Signaling
Modulators
Michael Spoerner, Pedro Lopes, Sunilkumar P. Narayanan, Ina Rosnizeck, Hans Robert Kalbitzer
Institute of physical Biochemistry and Biophysics, University of Regensburg, 93053 Regensburg,
Germany
In addition to the structure of proteins there is gaining interest in their dynamic properties and the
characterization of intrinsic equilibria between conformers because of their importance for
function. Applying high pressure to such biomolecules allows the shift of intrinsic conformational
equilibria up to the stabilization of higher energy conformers according to their smaller specific
volume. The combination of high pressure conditions with NMR spectroscopy as detection method
allows the investigation of such intrinsic equilibria of conformations and the characterization of
rare conformers up to atomic resolution, respectively [1].
Our molecules of interest are guanine nucleotide-binding proteins (GNBP) which regulate various
essential cellular responses and transport processes. Dysfunction of these important proteins
contributes in many cases to tumour formation or other diseases. Therefore, modulation of their
signalling activity is an important topic in academia as well as in the pharmaceutical industry [2].
Beside the general switching between the GDP-bound “off“-state and GTP-bound “on“-state
several conformational substates could be detected so far for the active form [see e.g. 3]. This
phenomenon is expected in particular for proteins acting in a regulation cycle in which a variety of
different interaction states with regulators and effectors are essential. Depending on the phase
within the activation/inactivation cycle single conformations predominate, whereas the others exist
only in lower population according to higher Gibbs free energies [4].
We present data on GNBPs in terms of conformational equilibria together with the functional
consequence. Typical functional properties are the affinity to effector molecules or regulators, as
well as enzymatic activities i.e. GTPase activity [5]. Beside 31
P NMR experiments detecting the
phosphates of the nucleotides in free or protein bound form we use [1H,
15N] HSQC experiments to
characterize conformational equilibria in the resolution of amino acid level [4].
The identification and characterization of the equilibria between selected states is the basis for the
development of a novel class of allosteric acting drugs targeting these intrinsic equilibria, and thus
modulating the signalling activity [4]. So far, we could identify small compounds which are able to
perturb the Ras-effector interaction by selective stabilization of a weak effector-binding
conformation [6-8].
References:
[1] High Pressure Bioscience in Subcellular Biochemistry 72 Ed. A. Kazuyuki and A. Matsuki,
Springer Verlag 2015
[2] P.M. Cromm, J. Spiegel, T.N. Grossmann, H. Waldmann, Angew. Chem. Int. Ed. 54 (2015) 2-
24
[3] M. Spoerner, A. Wittinghofer, H.R. Kalbitzer FEBS Lett. 578 (2004) 305-310
[4] H.R. Kalbitzer, I.C. Rosnizeck, C.E. Munte, S.N. Narayanan, V. Kropf, M. Spoerner Angew.
Chem. Int. Ed. 52 (2013) 14242-14246
[5] M. Spoerner, C. Hozsa, J. Poetzl, K. Reiss, P. Ganser, M. Geyer, H.R. Kalbitzer J. Biol. Chem.
285 (2010) 39768-39778
[6] I.C. Rosnizeck, T. Graf, M. Spoerner, J. Tränkle, D. Filchtinski, C. Herrmann, L. Gremer, I.R.
Vetter, A. Wittinghofer, B. König, H.R. Kalbitzer Angew. Chemie Int. Ed. 49 (2010) 3830-3833
[7] I.C. Rosnizeck, M. Spoerner, T. Harsch, D. Filchtinski, C. Herrmann, D. Engel, B. König, H.R.
Kalbitzer Angew. Chem. Int. Ed. 51 (2012) 10647-1065
[8] I.C. Rosnizeck, D. Filchtinski, R.P. Lopes, B. Kieninger, C. Herrmann, H.R. Kalbitzer,
M. Spoerner Biochemistry 53 (2014) 3867-3878
Oral Presentations
23
Rare excited states of human IAPP and short /-peptide catalysts
studied by high pressure NMR spectroscopy
Werner Kremera, Markus Beck-Erlach
a, Jörg Koehler
a, Janine Seeliger
b, Roland Winter
b ,
Martin Hofmannc, Oliver Reiser
c and Hans Robert Kalbitzer
a
a Institute of Biophysics and Physical Biochemistry, Center of Magnetic Resonance in Chemistry and
Biomedicine (CMRCB), University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
e-mail: [email protected] b
Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU
Dortmund University, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany c
Institute of Organic Chemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg,
Germany
The human islet amyloid polypeptide, hIAPP, is a small peptide of 37 amino acids length. As a
hormone, it is secreted by the pancreatic beta cells along with glucagon and insulin. In humans, a
balanced ratio of insulin and IAPP controls the glucose metabolism. Patients diagnosed with type-II
diabetes display a disturbance of this balance, and hIAPP is the main component of an amyloid
deposition in the pancreas. In these amyloids, IAPP is organized in a cross--sheet fibrillar
structure. The origin of the structural conversion of native, soluble monomeric IAPP into insoluble
amyloid fibrils and the pathways possible are still largely unknown. Here we apply high hydrostatic
pressure (HHP) together with high field high resolution NMR spectroscopy to uncover the
conformational substates of hIAPP, including the ones that are potentially prone to initiate the
aggregation and subsequent fibrillation reaction of hIAPP. High resolution HHP-NMR
spectroscopy is the most powerful technique to observe the structural properties at a residue
specific level and capture the transient species at the onset of the nucleation and aggregation
process. The catalytic activity of the /-peptide catalysts depends on their three-dimensional structures and
possible rare conformations. Here we apply high resolution HHP-NMR spectroscopy to identify
rare conformational states and to determine their structure when they are stabilized by pressure.
The effects on the Aldol reaction will be discussed.
Oral Presentations
24
Evolutionarily Conserved Pattern of Interactions in a Protein Revealed
by Local Thermal Expansion Properties.
Dellarole M(1), Caro JA(2), Roche J(1), Fossat M(1), Barthe P(1), García-Moreno E B(2), Royer
CA(1), Roumestand C(1). (1)†Centre de Biochimie Structurale, CNRS UMR5048, INSERM U554, Université Montpellier 1, 29 rue de
Navacelles, Montpellier, France 34090.
(2)‡T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St.. Baltimore,
Maryland 21218, United States.
The way in which the network of intramolecular interactions determines the cooperative folding
and conformational dynamics of a protein remains poorly understood. High-pressure NMR
spectroscopy is uniquely suited to examine this problem because it combines the site-specific
resolution of the NMR experiments with the local character of pressure perturbations. Here we
report on the temperature dependence of the site-specific volumetric properties of various forms of
staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as
measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-
induced unfolding arises from poorly understood differences in thermal expansion between the
folded and unfolded states. A significant inverse correlation was observed between the global
thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds,
as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of
the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein
family suggests that the architecture of the interactions detected in the NMR experiments could be
linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-
specific volume changes of unfolding yielded residue-specific differences in expansivity and
revealed how mutations impact intramolecular interaction patterns. These results show that
intramolecular interactions in the folded states of proteins impose constraints against thermal
expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the
patterns of strong intramolecular interactions and other local determinants of protein stability,
cooperativity, and potentially also of function.
Oral Presentations
25
Neutron techniques for the investigation of molecular dynamics under
high hydrostatic pressure
Judith Peters a,b
aUniv. Grenoble Alpes, LiPhy, CS 10090, 38044 Grenoble, France
bInstitut Laue-Langevin, CS 20156, 38042 Grenoble cedex 9, France
Adaptation mechanisms of biological systems to high hydrostatic pressure conditions are a debated
question and many factors, as a genetic adaptation and/or structural and dynamical changes, have to
be taken into account for a better understanding. Neutron scattering studies are a suited tool for
such investigations. Therefore new sample cells adapted for high pressure and high temperature
experiments1
were recently developed at the ILL and in-situ tests and results of this approach
applied to biological systems will be presented.
The influence of high hydrostatic pressure on the internal sub-nanosecond dynamics of highly
concentrated lysozyme in aqueous solutions was studied by Elastic Incoherent Neutron Scattering
(EINS) up to pressures of 4 kbar2. We have found, with increasing pressure, a reduction in the
dynamics of H-atoms of folded lysozyme, suggesting a loss in protein mobility that follows a
change in the local energy landscape upon the increase in packing density. Moreover, the amplitude
of the protein fluctuations depends drastically on the protein concentration, and protein structural
and interaction parameters as well as the dynamical properties are affected by pressure in a
nonlinear way.
Many prokaryotes are living near hot vents in the deep sea, at very high temperatures and
in anaerobic environments experiencing conditions that are very different to what we can
observe on the surface of Earth. Our present work focuses on three different micro-
organisms: E. coli which natural habitat is the human gut, T. kodakarensis that can be
found in hot sulfur springs at the surface of the Earth and finally T. barophilus that lives in
the bottom of the oceans near hot vents. In vivo whole proteome dynamics measurements
under pressure show striking differences between these organisms3 and could help us to
explain how these bacteria cope with extreme conditions.
Furthermore we investigated, by means of EINS, the pressure dependence of Mean Square
Displacements (MSD) of hydrogen atoms of deeply cooled water confined in the pores of a
3-dimensional disordered SiO2 xerogel4. The “pressure anomaly” typical of supercooled
water (i.e. a MSD increase with increasing pressure) is observed in our sample at all the
temperatures investigated; however, contrary to previous simulation results, the pressure
effect is much smaller at 210 K than at 250 K. EINS data are complemented by differential
scanning calorimetry data that put in evidence, besides the second order-like glass
transition at about 170 K, a first order-like transition occurring at about 235 K that, in view
of the neutron scattering results, can be attributed to a liquid-liquid phase transition. Taken
together our results give convincing experimental evidence of the existence of a Liquid-
Liquid Phase Transition in deeply cooled confined water, from a Low Density Liquid
(LDL) phase predominant at 210 K to a High Density Liquid (LDL) phase predominant at
250K.
References:
[1] J. Peters, M. Trapp, D. Hughes, S.Rowe, B. Demé, J.-L. Laborier, C. Payre, J.P.
Gonzales, S. Baudoin, N. Belkhier and E. Lelievre-Berna, High Pressure Res. 32
(2011) 97–102.
[2] M. Erlkamp, J. Marion, N. Martinez, C. Czeslik, J. Peters, and R. Winter, J. Phys.
Chem. B 119 (2015), 4842 – 4848.
[3] J. Peters, N. Martinez, G. Michoud, A. Cario, B. Franzetti, M. Jebbar, P. Oger, Z.
Phys. Chem. 228 (2014) 1121-1133.
[4] A. Cupane, M. Fomina, I. Piazza, J. Peters, G. Schirò, Phys. Rev. Lett. 113 (2014)
215701.
Oral Presentations
26
Bridging the gap between physiological and biophysical insights on
molecular adaptation in the high-hydrostatic pressure adapted archaeon
T. barophilus
Phil M. Oger*, A. Cario, N. martinez, P. Vannier, M. Barba, V. Daubin, J. Peters, B. Franzetti, M.
Jebbar
*Laboratoire de Géologie de Lyon, Ecole Normale Supérieure de Lyon, [email protected]
HHP has numerous effects on organisms and cellular components, resembling that of an increase or
a decrease in temperature, such as protein denaturation, membrane destabilization, alteration of
transcription and translation. HHP is however required for optimal activity of deep-environment
adapted microbes (piezophiles). Experimental evidence on macromolecules shows that HHP has a
different impact depending of the biological macromolecule. DNA and lipids are stabilized, while
multimeric proteins tend to be destabilized. For these three types of macromolecules, HHP has a
similar negative impact on cellular functions. In piezosensitve organisms, such as Escherichia coli,
HHP inhibits cell division at ca. 500bars, replication and translation at ca. 700 bars. In contrast,
500bars are optimal conditions for deep hydrothermal vent archaea such as Thermococcus
barophilus.
In thermophilic piezophiles several lines of evidence show that the adaptation of HHP involves the
regulation of the transcription of the genome as well as the expression of specific genes under HHP
[2]. It also involves a specific membrane structure [3] as well as the HHP-dependent accumulation
of osmolytes to maintain proper protein folding and activity [4]. We have direct and indirect
evidence for the structural adaptation of the proteome [5,6], although the specific signature at the
genome level still remains elusive. Using molecular dynamics, we are currently investigating the
protein and membrane structure of Thermococcus barophilus to further characterize HHP
adaptation at the molecular level.
References:
[1] Oger P, Jebbar M (2010) The many ways of coping with pressure. Res Microbiol. 161:799-809
[2] Vannier P, Michoud G, Oger P, Marteinsson Vþ, Jebbar M (2015) Genome expression
of Thermococcus barophilus and Thermococcus kodakarensis in response to different
hydrostatic pressure conditions. Research in Microbiology 166(9):717-725.
[3] Cario A, Grossi V, Schaeffer P, Oger P (2015) Membrane homeoviscous adaptation in
the piezo-hyperthermophilic archaeon Thermococcus barophilus. Frontiers in
Microbiology DOI: 10.3389/fmicb.2015.01152.
[4] Cario A, Mizgier A, Thiel A, Jebbar M, Oger P (2015) Restoration of the di-myo-
inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus
barophilus. Biochimie 118(11):288-293.
[5] Cario A, Jebbar M, Kervadec N, Oger P (2015) Accumulation of mannosylglycerate in
Thermococcus barophilus, a piezo-hyperthermophilic archaeon, in response to salt and
heat stresses. Nature Communications (in preparation)
[6] Peters J, Martinez N, Michoud G, Carlo A, Franzetti B, Oger P, Jebbar M (2014) Deep
Sea Microbes Probed by Incoherent Neutron Scattering Under High Hydrostatic
Pressure. Zeitschrift Fur Physikalische Chemie 228:1121-1133.
Oral Presentations
27
Triggering Dynamic Structural Changes in Model Lipid Membranes
Nicola McCarthy, Hanna Barriga, Arwen Tyler, Sowmya Purushothaman and Nick Brooks*
Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
Lipid membrane structural dynamics and micromechanics are vitally important to a wide range of
cellular processes including mediating protein activity, signaling, material transport and apoptosis
(programmed cell death). Developing model systems to study the structural and energetic behavior
of membranes, and methods to trigger changes in these parameters are essential to understanding
the contributions of the many components that make up biological membranes.
We have recently developed a range of novel instruments for studying soft matter and biological
system at non-ambient conditions and out-of-equilibrium. Amongst these, our new platforms for
high pressure and pressure-jump microscopy, small angle X-ray diffraction (SAXS) and
spectroscopy have led to a series of exciting studies of the pressure dependence of key
micromechanical membrane parameters and membrane structural dynamics.
Of particular interest are recent first measurements of the bending rigidity of lipid membranes
under pressure, generation and control of highly swollen interconnected cubic lipid structures, and
rapid triggering of domain growth in mixed lipid membranes.
Figure 1. Pressure induced lipid phase separation in a giant unilamellar vesicle (GUV)
References:
[1] N. McCarthy, O. Ces, R. Law, J. Seddon and N. Brooks, Chem. Commun. 51 (2015) 8675-
8678
[2] H. Barriga, A. Tyler, N. McCarthy, E. Parsons, O. Ces, R. Law, J. Seddon and N. Brooks,
Soft Matter 11 (2015) 600-607
[3] A. Tyler, H. Barriga, E. Parsons, N. McCarthy, O. Ces, R. Law, J. Seddon and N. Brooks,
Soft Matter 11 (2015) 3279-3286
[4] S. Purushothaman, P. Cicuta, O. Ces and N. Brooks, J. of Phys. Chem. B 119 (2015) 9805-
9810
[5] H. Barriga, R. Law, J. Seddon, O. Ces and N. Brooks Phys. Chem. Chem. Phys. (2015) DOI:
10.1039/C5CP04239A
Oral Presentations
28
Electronic structure and interactions at high hydrostatic pressure
Stefan M. Kast*1, Patrick Kibies
1, Roland Frach
1, Saraphina Böttcher
1, Tim Pongratz
1, Franziska
Hoffgaard1, Dominik Horinek
2
1Fakultät für Chemie und Chemische Biologie, TU Dortmund, 44227 Dortmund, Germany
2Institut für Physikalische und Theoretische Chemie, Universität Regensburg, 93040 Regensburg, Germany
Applying high hydrostatic pressure to biomolecules has substantial impact on their free energy
surfaces that govern structure, function, dynamics, and thermodynamics. This poses a challenge to
computational modeling approaches since the applicability of conventional empirical molecular
interaction functions (force fields) is not known. As a step toward clarifying the situation, we need
to account for high pressure in quantum-chemical calculations. A suitable methodology is provided
by molecular integral equation theories, in particular the “embedded cluster reference interaction
site model” (EC-RISM) [1,2] that combines statistical-mechanical 3D RISM integral equation
theory and quantum-chemical calculations self-consistently. In this context the impact of pressure
is naturally accounted for since the solvent susceptibility function that enters the theory contains
the pure solvent correlation functions at the pressure chosen, derived from either an integral
equation theory or molecular simulations. Here we describe the theoretical basis and illustrate the
methodology for several benchmark applications in a pressure range of 1 bar up to 10 kbar. In
particular, we study the effect of pressure perturbation on the dipole moment of TMAO in aqueous
solution from which an improved force field can be derived. The quality of electronic structure
calculations is examined by computing pressure-dependent chemical shifts to be compared with
experimental NMR reference data obtained for N-methylacetamide (H.-R. Kalbitzer, unpublished).
The results indicate a pressure-related baseline for interpreting NMR spectra recorded to examine
pressure-induced conformational changes of peptides and proteins.
Fig. 1. Pressure-dependent chemical shifts of the NMA amide group nuclei
in water from GIAO/EC-RISM/6-
31+G(d,p) calculations (top, susceptibilities taken from simulation in blue and HNC in orange) and
experiment (middle) along with deviations between theory and experiment (bottom row).
References:
[1] T. Kloss, J. Heil, S. M. Kast, J. Phys. Chem. B 112 (2008) 4337-4343
[2] R. Frach, S. M. Kast, J. Phys. Chem. A 118 (2014) 11620-11628
Oral Presentations
29
Mechanism of Hydrophobic Polymer Collapse in Miscible Good Solvents
Nico van der Vegt, Francisco Rodriguez-Ropero
Eduard-Zintl-Institut für Anorganische und Physikalische Chemie, Technische Universität Darmstadt
I will discuss interaction mechanisms of osmolytes and cosolvents in relation to their effect on the
hydrophobic collapse transition of thermo-responsive polymers in aqueous solution. The physical
principles that underlie cosolvent-induced collapse or re-entry (denaturation) of macromolecules
remain largely unknown, even for systems that have been known in the literature since several
decades. In my talk, some recent results obtained with molecular simulations will be discussed,
including simple hydrophobic polymers in aqueous solutions with trimethylamine-N-oxide
(TMAO), poly(N-isopropylacrylamide) (PNiPAM) in methanol/water miscible good solvents, and
PNiPAM in urea/water mixtures. Several unresolved questions are discussed: what drives the
collapse and the re-entrance of PNiPAM in miscible good solvents? Why does urea stabilize the
compact globular state of PNiPAM? Why do subtle changes in polymer chemistry shift the balance
from collapsed to highly extended swollen states?
References:
[1] F. Rodriguez-Ropero, N. F.A. van der Vegt, J. Phys. Chem. B (2014) 118, 7327-7334
[2] F. Rodriguez-Ropero, N. F.A. van der Vegt, PCCP (2015) 17, 8491-8498
[3] F. Rodriguez-Ropero, T. Hajari, N. F. A. van der Vegt, J. Phys. Chem. B (2015) doi:
10.1021/acs.jpcb.5b10684
Oral Presentations
30
Pressure Effects on Solvation Structure and Vibrational Spectroscopy of
Aqueous TMAO Solutions
Sho Imoto*, Harald Forbert and Dominik Marx Lehrstuhl für Theoretische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany
Although the effects of pressure perturbations on aqueous biomolecules and proteins are not well
understood, pressure is an essential thermodynamic variable like temperature and concentration.
Recent experiments revealed that the local solvation shell structure around proteins is strongly
altered upon compressing to high hydrostatic pressures (HHP) in the kilobar regime. Especially the
properties of trimethylamine N-oxide (TMAO) solutions at HHP conditions are of great interest
because the molecule stabilizes proteins against pressure denaturation. The TMAO molecule has
strongly hydrophilic and hydrophobic groups at its opposite ends and, thus, water molecules around
the hydrophilic and hydrophobic groups are supposed to show different responses against pressure
perturbations. We analyzed the structure [1] as well as the intermolecular (THz) and intramolecular
(mid-IR) vibrational spectra of TMAO in water at 10 kbar compared to ambient pressure by using
ab initio molecular dynamics simulations.
Reference:
[1] Sho Imoto, Harald Forbert and Dominik Marx, Phys. Chem. Chem. Phys. 17 (2015) 24224.
Oral Presentations
31
The Impact of TMAO and Urea on the H-Bond Dynamics under High
Pressure
Lukas Knake, Hendrik Vondracek, Gerhard Schwaab and Martina Havenith
Physical Chemistry II, Ruhr-University Bochum, D-44801 Bochum, Germany
It is well known that life can withstand extreme conditions regarding pressure and temperature.
Organic osmolytes such as amino acids, sugars and trimethylamine-N-oxide (TMAO) have been
found to be accumulated under pressure and thermal stress [1]. TMAO has been found to be
enriched in deep sea animals living under high pressure (up to 1 kbar) conditions, counteracting the
denaturing effect of high pressure [2]. Previous studies suggest that the stabilizing mechanism of
TMAO is based on a solvent-mediated effect, e.g. a modification of the hydrogen-bond network
and an enhancement of the water structure [3]. Although at ambient conditions the influence of
TMAO on the water network is known, the impact of high pressure on the solvent dynamics is still
an open question.
In our study, we use broadband Terahertz (THz) absorption spectroscopy to study the molecular
details of changes in the fast (sub-ps) hydrogen bond network dynamics around TMAO and Urea
up to 14 kbar. Based upon a detailed analysis we reveal new insights into the solvent dynamics of
these solvated biomolecules when changing from ambient to high pressure conditions.
References:
[1] P.H. Yancey, J. Exp. Biol. 208 (2005) 2819-2830
[2] P. Yancey, M. Gerringer, J. Drazen, et al., Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 4461–4465
[3] F. Meersman, D. Bowron, A. Soper, M. Koch, Biophys. J. 97 (2009) 2559-2566
Oral Presentations
32
The solid/liquid interface under conditions of high hydrostatic pressure
F. Wirkert, M. Paulus, P. Salmen, C. Sternemann, M. Tolan, and J. Nase
TU Dortmund, Fakultät Physik/DELTA, 44221 Dortmund, Germany
Studying the response of matter to high hydrostatic pressure (HHP) has a long-standing tradition.
On the one hand, applying HHP is a good method to impose stress to a system without increasing
the internal energy, as for example done by an increased temperature. HHP is thus a very gentle
way to disturb a system. On the other hand, it is known that life can exist at extreme conditions, as
for example in deep sea regions or in hot vents. The knowledge on life at extreme conditions can be
broadened if biological or model systems are investigated under HHP.
Proteins tend to adsorb to almost any interface, with a strong inclination for hydrophobic
substrates. If one wants to study protein adsorption at HHP, then also knowledge on the exact
structure of the substrate is crucial for the correct interpretation of experimental data.
Next to hydrophobic interfaces in contact with water, the existence of thin water layer with
decreased electron density, compared to bulk water, was controversially discussed in the literature
[1-5]. The structure of this so-called hydrophobic gap on a molecular level has been studied for
many years now both in experiments and molecular dynamics simulations. While experimental and
numerical studies confirmed the existence of the density gap, nothing is known about the influence
of HHP on this region.
In this contribution, we will present an X-ray reflectivity study on the behavior of the hydrophobic
gap region at pressures of up to 5 kbar. Experiments were performed in a custom-built HHP-XRR
cell [6]. First results indicate that the hydrophobic gap is surprisingly resistant to pressure.
References:
[1] M. Mezger et al., PNAS 103 (2006) 18401
[2] S. Chattopadhyay et al., Physical Review Letters 105 (2010) 037803
[3] M. Mezger et al., JACS 132 (2010) 6735
[4] M. Maccarini et al., Langmuir 23 (2007) 598
[5] A. Poynor et al., Physical Review Letters 97 (2006) 266101
[6] F. Wirkert et al., Journal of Synchrotron Radiation 21 (2014) 76
Oral Presentations
33
Pressure Effects on an Alkane SAM/Water Interface
Dominik Horinek, Christoph Hölzl
Institut für Physikalische und Theoretische Chemie, Universität Regensburg, 93040 Regensburg
The structure and thermodynamics of water interfaces has been of continuous scientific interest for
a long time. In recent years, much progress has been achieved through studies of self-assembled
monolayers (SAMs) by reflectivity studies and by molecular simulations. In this talk, we present
simulations of hydrophobic OTS monolayers in contact with water (Fig. 1) subject to high
hydrostatic pressures.
Different approaches for the
calculation of the depletion
layer thickness and its
variation with pressure are
determined and discussed in
relation to experimental work.
We also address the influence
of inhomogeneities within the
SAM, a factor that has
previously seen little attention
in simulation studies.
Fig. 1: MD snapshot of SAM water interface.
Oral Presentations
34
Combined effects of pressure and interfaces on enzymatic activity
Vitor Schuabb, Süleyman Cinar, Artem Levin, Claus Czeslik*
TU Dortmund University, Department of Chemistry and Chemical Biology, D-44221 Dortmund, Germany
Enzymes are often immobilized on solid surfaces. In this way, enzymatic activity can be analyzed
by surface-sensitive techniques. Furthermore, enzymes can be recovered from the reaction mixture,
when they are adsorbed on carrier particles. However, enzyme activity at aqueous-solid interfaces
might be lowered due to partial denaturation, by restricted dynamics or by a simple blocking of the
active site. Pressure is known to affect the catalytic rate of enzymes in both directions [1]. Pressure-
induced activation or deactivation of enzymes is associated with negative or positive activation
volumes, respectively. We have investigated the enzymatic activity of -chymotrypsin (-CT) and
horseradish peroxidase (HRP) at a series of interfaces as a function of pressure using TIRF
spectroscopy and the stopped-flow technique [2,3]. Polar, nonpolar, positively and negatively
charged surfaces as well as polyelectrolyte brushes were used as immobilizing surface.
Remarkably, both enzymes can be activated by pressure, when they are adsorbed on particular
surfaces. We have also observed that -CT shows a non-constant activation volume adsorbed at
polar interfaces and free in aqueous solution, which suggests different compressibilities of the
volumes of the enzyme-substrate complex and the transition state. Moreover, it is interesting to
note that large activities of -CT and HRP can be measured at those interfaces that are associated
with negative activation volumes. Overall, the results found so far clearly indicate that the volume
profile along the catalytic path of an adsorbed enzyme strongly depends on the kind of interface
used for immobilization.
References:
[1] M. J. Eisenmenger, J. I. Reyes-De-Corcuera, Enzyme Microbial Technol. 45 (2009) 331-347.
[2] V. Schuabb, C. Czeslik, Langmuir 30 (2014) 15496-15503.
[3] V. Schuabb, S. Cinar, C. Czeslik, Colloids Surf. B 140 (2016) 497-504.
Oral Presentations
35
FTIR studies on biomolecules under pressure 1Judit Somkuti,
2Tamás Oláh,
2László Smeller
1Hungarian Academy of Sciences-Semmelweis University, Molecular Biophysics Research Group, Budapest
2Dept. Biophysics and Radiation Biology, Semmelweis University, Budapest
FTIR spectroscopy is a useful method to follow structural changes in proteins, nucleic acids and
lipids.
Amide I band of the proteins allows following their structural change under pressure. Pressure and
temperature stability of proteins are governed by several factors. From structural point of view,
proteins are stabilized by several factors1as disulfide bridges, prosthetic groups (e.g. porphyrine for
myoglobin and horseradish peroxidase)2, stabilizing ions (e.g. Ca
2+ in case of parvalbumin)
3. The
molecular environment plays also a very important role for the point of view of stability. The
cellular environment is very crowded which makes a wide range of interactions possible.
We investigated the orange variant of the green fluorescent protein (GFP), which is further
modified to have a large stokes shift3. To our knowledge this is the most pressure stable protein
studied so far. It did not unfold at 20 kbar pressure at 40ºC. Besides its high pressure stability its
heat unfolding temperature is also above 100ºC. Although the secondary structure disrupts only
under very extreme conditions, distortions in the secondary and tertiary structure are clearly visible
in the elastic range. The fluorescence spectrum reflects these elastic changes, indicating the overall
swelling of the beta barrel structure.
To study the effect of crowded environment on the stability of the protein, crowded environment
was created by dextran and ficoll. Bovine serum albumin was used for this study. Stabilizing effect
of the crowding was obtained by higher concentrations (>15%) of the crowding agents. Since FTIR
needs a high concentration, self-crowding can also happen, which makes these experiments more
cell-mimicking compared to the otherwise sensitive fluorescence techniques.
Investigating membrane bound processes is also possible utilizing the infrared spectroscopy. We
deposited a lipid layer on the ATR crystal of the spectrometer. This is useful to detect membrane
bound proteins and nucleic acids. A small aptamer and different proteins were attached to the
membrane. Their binding can be detected by infrared spectroscopy. This method is a very
promising one for sensory purposes.
References:
[1] L. Smeller, Biochim. Biophys. Acta - Protein Struct. Molec. Enzymol. 1595, 11 (2002).
[2] L. Smeller, and J. Fidy, Biophysical Journal 82, 426 (2002).
[3] D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. J. Gadella and V. V. Verkhusha, J. Am.
Chem. Soc. 134, 7913 (2012).
Oral Presentations
36
Pressure Tuning of Primary Photochemistry
Manoop Chenchilyan,a Liina Kangur,
a Kõu Timpmann,
a and Arvi Freiberg
a,b
aInstitute of Physics, University of Tartu, Ravila 14c, Estonia
bInstitute of Molecular and Cell Biology, Tartu University, Tartu, Estonia
Charge transfer processes are ubiquitous in biology. The bacterial reaction center (RC) protein
complex from Rhodobacter sphaeroides constitutes an ideal model system for understanding how
the protein structure affects the photoinduced electron transfer in membrane proteins, as a number
of crystal structures from native and mutant RC samples are available. Furthermore, the RC protein
contains several pigmented cofactors that cover significant part of the protein volume. These
individual chromophores establish a series of intrinsic molecular probes that allow convenient
monitoring of the localized structural changes when examined by spectroscopic methods.
In the present contribution, high hydrostatic pressure optical barospectroscopy is used to obtain
new insights into the mechanisms that govern the nano-scale electron transport in bacterial RCs. It
was universally detected by picosecond time-resolved fluorescence that compression of the RC
complex with pressures reaching 1 GPa led to significant (several-fold) acceleration of the primary
electron transfer rate. By steady state absorption and fluorescence spectroscopy evidence was
obtained for a number of local reorganizations in the binding site of the primary electron donor, a
special pair of bacteriochlorophyll a molecules, between 1 atm and 0.6 GPa in different samples.
The effects were generally reversible in a sense that the initial spectral characteristics of the
samples were recovered upon the pressure release. Basic analysis of these experimental data
suggests that the observed increase of the primary electron transfer rate is a combined effect of an
enhancement of the driving force for electron transfer and of modification of the relative geometry
of the electron donor and acceptor sites. Either of these factors applied separately does not provide
satisfactory account of the experimental data. In progress is a more advanced analysis, which
considers the complex internal structure of the special pair. Even minor variations of the electron
donor geometry may induce significant changes of its electronic structure, with probable
consequence of mixing of singlet exciton and charge-transfer states. According to ref. [1], the
pressure-induced deformation of the special pair structure is most likely anisotropic, involving
interfacial compression and shear of the special pair.
References:
[1] K. Leiger, A. Freiberg, M. G. Dahlbom, N. S. Hush, J. R. Reimers, The Journal of Chemical
Physics 126 (2007) 215102
Oral Presentations
37
Determination of the protein-ligand binding volume by high-pressure
spectrofluorimetry
G. Skvarnavičius, M. Grigaliūnas, Z. Toleikis, J. Smirnovienė, P. Cimmperman, D.
Matulis, and V. Petrauskas*
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius,
Lithuania
A majority of high pressure studies were devoted to reveal the thermodynamics of protein
unfolding/refolding reaction and the dissociation of multimeric proteins under pressure. However,
relatively little attention has been paid to the volume changes resulting from the interaction
between a protein and small molecule [1-9]. The change in protein volume observed upon protein-
ligand interaction (termed as the protein-ligand binding volume) is an important but largely
neglected thermodynamic parameter from the perspective of both fundamental science and
potential applications in the development of specific protein ligands.
High pressure spectrofluorimetry has been extensively used during the past several decades
[10] and helped to reveal various aspects of protein folding and stability. Here we describe how
high pressure fluorescence could be used to determine protein-ligand binding volume. We continue
the development and validation of the method on several isoforms of human carbonic anhydrase
(CA) – a protein involved in cancer progression and therapy. The degree of protein unfolding at
elevated pressures was monitored by an intrinsic tryptophan fluorescence. Different approaches of
experimental fluorescence spectra analysis are described and the impact on the quality of
thermodynamic parameters are discussed.
References:
[1] T. M. Li, J. W. Hook, H. G. Drickamer, G. Weber, Biochemistry 15 (1976) 3205–3211.
[2] T. M. Li, J. W. Hook, H. G. Drickamer, G. Weber, Biochemistry 15 (1976) 5571–5580.
[3] K. Heremans, Annu Rev Biophys 11 (1982) 1–21.
[4] C. A. Royer, G. Weber, T. J. Daly, K.S. Matthews, Biochemistry 25 (1986) 8308–8315.
[5] J. L. Silva, G. Weber, Annu Rev Phys Chem 44 (1993) 89–113.
[6] T. V. Chalikian, K. J. Breslauer, Biopolymers 39 (1996) 619–626
[7] K. J. Frye, C. A. Royer, ProteinSc 7 (1998) 2217–2222.
[8] Z. Toleikis, P. Cimmperman, V. Petrauskas, D. Matulis, Analytical Biochemistry 413 (2011)
171–178.
[9] V. Petrauskas, J. Gylytė, Z. Toleikis, P. Cimmperman, D. Matulis, European Biophysics
Journal 42 (2013) 355–362.
[10] C. A. Royer, Chemical Reviews 106 (2006) 1769–1784.
Oral Presentations
38
High-pressure microscopy for studying molecular motor and
cytoskeleton
Masayoshi Nishiyama
The HAKUBI Center for Advanced Research, Kyoto University, Kyoto 606-8501, Japan
Movement is a fundamental characteristic of all living things. This biogenic function is carried out
by various nanometer-sized molecular machines. Molecular motor is a typical molecular machinery
in which the characteristic features of proteins are integrated; these include enzymatic activity,
energy conversion, molecular recognition and self-assembly. These biologically important
reactions occur with the association of water molecules that surround the motors. Application of
pressure is a powerful method for modulating intermolecular interactions between protein and
wáter molecules. To visualize the pressure-induced changes in the structure and function of
molecular motors, we have developed a high-pressure microscope [1] (Fig. 1). The developed
system enables us to acquire high–resolution microscopic images. The maximum pressure is about
1.5-fold higher than that of the deepest part of the Mariana Trench (~11,000 m in depth), which is
the highest pressure found outside the crust of the earth. This ability to withstand pressure at such a
high level is sufficient for studying almost all biological activity on earth. The high-pressure
microscope enables us to modulate the unidirectional motion of molecular motors such as kinesin
[2], F1-ATPase [3] and bacterial flagellar motors [4]. Here, we extended the developed system to
visualize and manipulate the cellular architecture and activity.
References:
[1] M. Nishiyama, High–Pressure Microscopy for Studying Molecular Motors. In “High Pressure
Bioscience - Basic Concepts, Applications and Frontiers” (K. Akasaka and H. Matsuki, eds, 2015,
pp. 593-611), Springer, Heidelberg, Germany
[2] M. Nishiyama, Y. Kimura, Y. Nishiyama and M. Terazima, (2009) Pressure-Induced Changes
in the Structure and Function of the Kinesin-Microtubule Complex. Biophys. J. 96, 1142–1150
[3] D. Okuno, M. Nishiyama and H. Noji, (2013) Single-Molecule Analysis of the Rotation of F1-
ATPase under High Hydrostatic Pressure. Biophys. J. 105, 1635–1642
[4] M. Nishiyama, Y. Sowa, Y. Kimura, M. Homma, A. Ishijima and M. Terazima, (2013) High
Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli
Flagellar Motor. J. Bacteriol. 195, 1809-1814
Fig. 1 High-pressure microscope. (a) High-pressure chamber and separator. The copy of the
chamber is commercially available (PMC-100-2-0.6-630, Syn Corporation, Japan). (b) High-
pressure pump.
Oral Presentations
39
Pressure effects on amino acids and their salts in the crystalline state
Elena Boldyreva*
Institute of Solid State Chemistry and Mechanochemistry, Siberian Branch of Russian Academy of Sciences,
ul. Kutateladze, 18, Novosibirsk 630128 Russia ([email protected])
Amino acids - the elementary building blocks of peptides – can form extended periodic structures
in which molecules link via a network of hydrogen bonds. If a second component is added, a salt,
solvate or co-crystal (with neutral components) can be formed. These crystalline structures can
undergo various structural changes on compression and decompression, ranging from distortion of
hydrogen bonds, and changes in molecular conformations to radical changes in molecular packing.
These processes can be followed in fine detail by single-crystal X-ray diffraction and Raman
spectroscopy. Despite an obvious difference between the properties of amino acids as individual
molecules in crystals and as fragments in a polypeptide chain, there are common properties and
features shared by the two. In particular, the dynamics of the amino acid side chains and the
characteristics of the hydrogen bonds in which they are involved have many similarities.
Quantitative high-pressure investigations of amino acid hydrogen bonds offer invaluable
information, useable for understanding and modeling complex biopolymers. Such information
allows empirical parameterization of the compressibility of different types of hydrogen bonds and
insights into the pressures at which molecular fragments change their conformation. The response
of bi-layered amino acid crystal structures, particularly of those with hydrophobic side chains, can
be indicative of the response biological membranes may under similar conditions. The interaction
of crystals with pressure-transmitting fluids, in particular, solvent-assisted structural
transformations or solvate formation, can give better insight into the effect of liquids on
conformational changes in peptides and proteins. Structural transformations in crystals with layered
or helical structures, or three-dimensional hydrogen-bonded frameworks can be compared to
conformational transitions between helices, layers and folds in biomolecules. Data regarding
structural changes in multi-component crystals can be helpful in understanding the interactions of
biomolecules with different substrates in biological systems.
The author acknowledges support from RSF (grant 14-13-00834).
References:
[1] E.V. Boldyreva, In: High-Pressure Crystallography. From Novel Experimental Approaches to
Applications in Cutting-Edge Technologies (Eds. E. Boldyreva, P. Dera), Springer: Dordrecht,
2010, 533-543.
[2] E.V. Boldyreva, In: Models, Mysteries, and Magic of Molecules, Ed. J. C. A. Boeyens & J. F.
Ogilvie, Springer Verlag, 2007, 169 – 194.
[3] B.A. Zakharov, N.A. Tumanov and E.V. Boldyreva, CrystEngComm, 17 (2015) 2074 – 2079.
[4] E.A. Kapustin, V.S. Minkov, E.V. Boldyreva, Acta Crystallogr. B, 70 (2014) 517-532.
[5] B.A. Zakharov & E.V. Boldyreva, J. Mol. Struct., 1078 (2014) 151-157.
[6] E.V. Boldyreva, Z. Kristallogr., 229 (2014), 236-245.
[7] V.S. Minkov & E.V. Boldyreva, J. Phys. Chem. B, 117 (2013) 14247–14260.
[8] B.A. Zakharov & E.V. Boldyreva, Acta Crystallogr. B, 69 (2013) 271-280.
[9] B.A. Zakharov, E.A. Losev, E.V. Boldyreva, CrystEngComm, 15 (2013) 1693 – 1697.
[10] N.A. Tumanov & E.V. Boldyreva, Acta Crystallogr. B, 68 (2012) 412-423.
[11] B.A. Zakharov, B.A. Kolesov & E.V. Boldyreva, Acta Crystallogr. B, 68 (2012) 275-286.
[12] N.A. Tumanov, E.V. Boldyreva, B.A. Kolesov, A.V. Kurnosov, R.Q. Cabrera, Acta
Crystallogr. B, 66 (2010) 458-471.
[13] E.V. Boldyreva, Phase Transitions, 82 (2009) 303-321.
Oral Presentations
40
Alpha-synuclein fibrils triggered by pressure and the seeding mechanism
in Parkinson disease
Guilherme A. P. de Oliveira1, Mayra de A. Marques
1, Carolina S. Cruzeiro
1, Yraima Cordeiro
2,
Mônica S. de Freitas1 and Jerson L. Silva
1
1Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Instituto Nacional de
Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 2 Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Parkinson Disease (PD) is a devastating neurological disease in which aggregated forms of the
alpha-synuclein (αS) protein believed to participate in regulatory pathways of synaptic vesicle
release and trafficking, are found in the substantia nigra pars compacta. There is a direct and well-
accepted link between αS oligomerization and fibrillation and the citopathological and
neuropathological features of PD brains. High hydrostatic pressure (HHP) is a powerful
physicochemical strategy to understand protein folding, ligand interaction and the assembly of
supramolecular structures like amyloids. In this study, we asked whether we were able to contribute
for the understanding of the molecular mechanisms of αS fibril disassembly and remodeling upon
HHP challenge. We demonstrate that the major species released from HHP-disturbed fibrils are
structurally modified monomers in which conformational exchange motions in the µs-ms timescale
are present at the non-amyloidogenic core (NAC) and acidic C-terminal region of the protein. In
addition, we show at atomic level the remodeling of HHP-disturbed fibril core and how these
species contribute to seed αS aggregation. Our findings explain the key role that HHP can achieve
in populating invisible αS species and fibril remodeling and the association of this physicochemical
approach to help future therapeutics focused on the blockage of de novo aggregation and seeding
that may represent an effective strategy to ameliorate PD progression.
Oral Presentations
41
Activation of auto-inhibited twitchin kinase by compressive force - a high
pressure NMR study.
Sunilkumar Puthenpurackal Narayanan1, Michael Spoerner
1, Markus Beck Erlach
1, Jörg
Köhler1, Werner Kremer
1, Olga Mayans
2 and Hans Robert Kalbitzer
1
1Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstr. 31, D- 93040,
Regensburg, Germany. 2Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool
L69 7ZB, United Kingdom
The giant proteins of the titin-like family (0.7–4MDa) are emerging as key force sensors in muscle.
Proteins from this family include titin and obscurin in mammals; twitchin, the obscurin homolog
UNC-89 and the small TTN-1 titin in nematodes and mollusks; projectin and stretchin in insects.
Titin-like proteins contain numerous Ig like domains, Fn3 like domains and conserved kinase
domain near their C-terminus. The C-terminal kinase domain is supposed to be responsible for the
force sensing (1, 2). This study reports the activation of the auto-inhibited kinase domain by
pressure (compressive force), taking twitchin kinase as a representative from the titin-like family.
The ATP binding site of twitchin kinase is blocked by the N-terminal (NL) and C-terminal (CRD)
tails (3). The active site becomes accessible to the ATP molecule, only when the tails are removed.
The kinase domain together with N-terminal and C-terminal tails (NL-Kin-CRD) has no detectable
kinase activity. When the tails were removed it showed kinase activity (3). Present study reports
the activation of auto-inhibited twitchin kinase (NL-Kin-CRD) by the application of compressive
force (pressure). High pressure NMR technique is applicable to NL-Kin-CRD, because pressure
can shift the equilibrium towards a conformational state (with smaller partial molar volume) similar
to that produced by stretching force.
References:
[1] B. Bullard, et al. J Muscle Res Cell Motil 23 (2002) 435–447.
[2] A. Kontrogianni-Konstantopoulos, et al. Physiol Rev 89 (2009)1217–1267.
[3] Eleonore von Castelmur, et al. PNAS, 109 (34) (2012) 13608–13613.
Oral Presentations
42
Combined SANS-QENS studies of low-density lipoprotein under high
hydrostatic pressure
M. Goluba,b
, B. Lehoferc, K. Kornmueller
c, M. Kriechbaum
d, N. Martinez
a,b, H.
Amenitschd, R. Prassl
c and J. Peters
a,b
aUniv. Grenoble Alpes, IBS, 71 avenue des Martyrs, 38044 Grenoble, France
bInstitut Laue-Langevin, 71 avenue des Martyrs, 38044 Grenoble, France
cMedical University of Graz, Institute of Biophysics, Harrachgasse 21/VI, 8010 Graz, Austria
dGraz University of Technology, Inst. of Inorganic Chemistry, Stremayrg. 9, 8010 Graz, Austria
Low-density lipoprotein (LDL) is a natural nano-particle, whose main function is the transportation
of cholesterol molecules from the liver to the peripheral tissues [1]. The composition of LDL
includes the apolipoprotein B100, phospholipids, cholesterol, cholesteryl esters and triglycerides.
Although intensive studies by SAXS and EM microscopy of LDL were undertaken [2], its structure
is not yet completely clear and even less is known about the dynamical behavior of these particles.
However, composition and structure is known to be modified in case of certain diseases as
hyperlipidemia, where LDL is enriched with cholesterol or fat. In addition, minimally oxidized
LDL particles created by self-oxidation permit to mimic LDL particles seen in atherosclerotic
plaques. We report here recent studies about three different types of LDL (native, oxidized and triglyceride
enriched) exposed to high hydrostatic pressure up to 3000bar to investigate the influence of this
thermodynamic parameter. We used a combined analysis of SANS-QENS data sets, which were
collected at the Paul-Scherrer-Institut (Switzerland) and at the Institut Laue-Langevin (France)
neutron research facilities. The SANS data, which was analyzed with a model of several elliptical
cylinders, reveals a stronger overall shape asymmetry for oxidized LDL protein compared to the
other samples. Moreover, the difference in shape becomes more pronounced at higher pressure
values. At the same time the analysis of the QENS data, which were collected on two spectrometers
at the ILL, IN5 and IN6, sensitive to motions at different time scales, reveals a clear difference in
the dynamics of bound water for the different types of LDL particles. Our findings are essential for
a better understanding of the LDL functionality and of the investigated modifications.
References:
[1] R. Prassl, P. Laggner, Eur. Biophys. J. 38 (2009) 145-158.
[2] P. Laggner et al., Hoppe-Seyler's Z. Physiol. Chem. 358 (1977) 771-778; P. Laggner,
G. M. Kostner, G. Degovics, D. L. Worcester, Proc. Natl. Acad. Sci. USA 81 (1984)
4389-4393; R. Vanantwerpen, M. Labelle, E. Navratilova, R. M. Krauss, J. Lipid Res.
40 (1999) 1827-1836; E. V. Orlova et al., Proc. Natl. Acad. Sci. USA 96 (1999) 8420-
8425.
Oral Presentations
43
Fig. 1: LLPS phase boundaries obtained from (a)
SAXS, b) light transmission measurements for 4
different lysozyme concentrations in the
presence of 500 mM NaCl at selected pressures
and temperatures. Below the curves, the system
is always in the LLPS region, above in the
homogenous phase.
Phase behavior of dense lysozyme solutions
J. Schulze a, J. Möller
b, M. Paulus
a, J. Nase
a, M. Tolan
a, and R. Winter
c a Fakultät Physik/Delta, Technische Universität Dortmund, 44221 Dortmund, Germany
b ESRF - The European Synchrotron, 38043 Grenoble, France
c Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, 44221 Dortmund,
Germany
The influence of temperature, pressure and salt concentration on protein stability, aggregation
propensity, intermolecular interactions as well as crystallization and phase behavior of dense
protein solutions has been in the focus of protein science in recent years. As small-angle X-ray
scattering (SAXS) is an ideal tool for the investigation of proteins and their interactions, this
technique has been used in previous studies to determine the intermolecular interaction potential of
proteins in aqueous solution under the influence of varying conditions such as hydrostatic pressure,
temperature, and ionic strength. A non-monotonous correlation between the strength, J(p), of the
attractive part of the protein-protein interaction and the hydrostatic pressure was found with a
minimum at about 2 kbar, which is probably related to changes in the water structure at elevated
pressures [1,2]. Adding 0.5 M NaCl leads to more prominent short-range interactions [3,4], and a
phase transition to the so-called LLPS (Liquid-Liquid Phase Separation) region can be observed at
high lysozyme concentrations and low temperatures. In the LLPS phase, lysozyme forms small
droplets of high concentration within the more
dilute liquid phase. At elevated hydrostatic
pressures, however, this phase separation is
suppressed. Due to the non-monotony of the attrac-
tive part, J(p), a further pressure increase leads to a
re-entrant LLPS phase.
In this contribution, we will discuss the phase
behavior of lysozyme solutions as a function of
concentration, c, pressure and temperature in the
presence of 500 mM NaCl, as determined by SAXS
measurements. Complementary turbidity measure-
ments were employed to determine the phase
boundaries of the LLPS phases. Both methods
allow, for the first time, the construction of a
concentration-temperature-pressure phase diagram
of dense lysozyme solutions over a wide range of
temperatures, pressures and protein concentrations.
References:
[1] M. A. Schroer, J. Markgraf, D. C. F. Wieland, C. J. Sahle, J. Möller, M. Paulus, M. Tolan,
R. Winter, Phys. Rev. Lett. 106 (2011) 178102
[2] M. A. Schroer, Y. Zhai, D. C. F. Wieland, C. J. Sahle, J. Nase, M. Paulus, M. Tolan, R. Winter,
Angew. Chem. Intern. Ed. 123 (2011) 11615
[3] J. Möller, S. Grobelny, J. Schulze, A. Steffen, S. Bieder, M. Paulus, M. Tolan, R. Winter,
Phys. Chem. Chem. Phys. 16 (2014) 7423
[4] J. Möller, M. A. Schroer, M. Erlkamp, S. Grobelny, M. Paulus, S. Tiemeyer, F. J. Wirkert,
M. Tolan, R. Winter, Biophys. J. 102 (2012) 2641
[5] J. Möller, S. Grobelny, J. Schulze, S. Bieder, A. Steffen, M. Erlkamp, M. Paulus, M. Tolan,
R.Winter, Phys. Rev. Lett. 112 (2014) 28101
Oral Presentations
44
Actin Polymerization and Bundling: Exploring their Temperature and
Pressure Limits
Mimi Gao and Roland Winter
Physical Chemistry I – Biophysical Chemistry, Faculty of Chemistry, TU Dortmund University, 44227
Dortmund, Germany
Today’s living systems are organized in highly dynamic and structured functional units providing a
platform for life. Despite the origin of life took place under extreme environmental conditions, few
higher organisms can still be found in regions with extreme conditions of pressure and temperature.
In vivo experiments revealed that, compared to other cellular components, the temperature and
pressure stability of the cytoskeleton is rather limited. Actin, a key protein for cell shape and
movement, is highly conserved and the most abundant protein in eukaryotes. Its polymerization
reaction is essential to provide driving force for cellular motility and mechanical resistance for cell
shape. Upon polymerization, actin filaments (F-actin) can be further organized into different
architectures including crosslinked and bundled networks. In this study, using the examples of actin
polymerization and bundling we illustrate the importance of actin-binding proteins for maintaining
the stability and dynamics of the cytoskeleton in a pressurized world. Using preformed gelsolin-
actin nuclei and applying stopped-flow methodology, we quantitatively studied the polymerization
process of actin as a function of temperature and pressure and found that the temperature-pressure
sensitivity of its kinetics is essentially due to the initial de novo nucleation event rather than the
elongation reaction of F-actin, highlighting the need of actin nucleation factors to bypass the
energetically costly and pressure-sensitive de novo nucleation in vivo ensuring formation of the
microfilament (1). Furthermore, using small-angle X-ray scattering and transmission electron
microscopy we compare the temperature-pressure stability of actin bundles formed by the protein
fascin and Mg2+
and show that the naturally occurring bundles are more adapted to apply
mechanical forces, also under high pressure conditions (2).
References: (1) M. Gao, R. Winter (2015) ChemPhysChem 16:3681 (2) M. Gao, M. Berghaus, Julian von der Ecken, Stefan Raunser, R. Winter (2015) Angew. Chemie
Int. Ed. 54:11088
Poster Presentations
45
Poster Presentations
Poster Presentations
46
High pressure simulations using a pressure-dependent force field for
TMAO
Christoph Hölzl and Dominik Horinek
Institute of Physical and Theoretical Chemistry, University of Regensburg, 93040 Regensburg, Germany
Osmolytes are a class of small organic molecules, which accumulate in the cells of many
organisms. Their function, besides regulating the intracellular osmotic pressure, is also to influence
the stability of proteins [1]. As an example of a protecting osmolyte, trimethylamine-N-oxide
(TMAO) has been shown to stabilize proteins against chemical, thermal, and pressure denaturation
[1,2].
For quite some time it has been of interest to create a classical model of TMAO in order to be able
to investigate these effects using force field molecular dynamics: After the first flexible model was
developed by Kast et al. using ab initio calculations and experimental data [3], it was modified by
scaling the contact distance of carbon and the partial charges of oxygen and nitrogen [4]. This
model by Schneck et al. was optimized with respect to activity coefficients in aqueous solutions
and the transfer free energy of a polyglycine model peptide. However, none of the existing models
give a
good description of the density of aqueous solutions, a quantity that is very important when
investigating the effects of pressure on these systems.
We present a new set of pressure-dependent force field parameters for TMAO that reproduce the
experimental densities of aqueous solutions (using the TIP4P/2005 water model[5]) up to kilobar
pressures and the activity coefficients at normal pressure. Most importantly, the change of the
solvation shell of water around TMAO with pressure is described correctly, which is achieved by
scaling the partial charges depending on the pressure.
Furthermore, the new model was applied to determine the free energies of transfer of periodic
model peptides into TMAO solutions at different pressures. In addition, we show how the transfer
free energies scale with the solvent-accessible surface area of the peptides, as predicted by the
Transfer Model [6,7].
References:
[1] T. Arakawa, L. Timasheff, Biophys. J. 47 (1985) 411-414
[2] P. H. Yancey, J. F. Siebenaller, J. Exp. Biol. 202 (1999) 3597-3603
[3] K. M. Kast, J. Brickmann, S. M. Kast, R. S. Berry, J. Phys. Chem. A 26 (2003) 5342-5351
[4] E. Schneck, D. Horinek, R. R. Netz., J. Phys. Chem. B 117 (2013) 8310-8321
[5] J. Abascal, C. Vega, J. Chem. Phys. 123 (2005) 234505
[6] M. Auton, D. Bolen, Biochemistry 43 (2004) 1329-1342
[7] B. Moeser, D. Horinek, J. Phys. Chem. B 118 (2014) 107-114
Poster Presentations
47
Activation volumes of enzymes at polyelectrolyte brushes
Artem Levin, Claus Czeslik
TU Dortmund University, Department of Chemistry and Chemical Biology, D-44221 Dortmund, Germany
Polyelectrolyte brushes can provide a native-like environment for the immobilization of proteins at
aqueous-solid interfaces. In particular, poly(acrylic acid) (PAA) brushes have been shown to
preserve the secondary structure of proteins and maintain enzyme activity to a high degree [1,2].
Moreover, the degree of protein adsorption at a PAA brush can be controlled by the ionic strength
of the protein solution [3]. In view of these interesting properties, we have investigated the
structure and activity of -chymotrypsin (-CT) as a function of pressure using TIRF
spectroscopy, neutron reflectometry, and ATR-FTIR spectroscopy. -CT is hydrolyzing peptide
bonds, and its activity can be enhanced by pressure [4,5]. We have found that this pressure
response is largely maintained, when -CT is adsorbed at a PAA brush, i.e. pressure still increases
the catalytic rate of -CT. From ATR-FTIR measurements, no significant changes of the secondary
structure of -CT can be observed upon adsorption confirming the benign properties of the brush.
Furthermore, the density profile of -CT at a PAA brush has been determined as a function of
pressure from neutron reflectivities. The profiles indicate a strong interaction between -CT and
the PAA chains and little pressure effects on the interfacial structure. Overall, a PAA brush seems
to be a favorable surface modification for the immobilization of enzymes that can even be activated
by pressure.
References:
[1] C. Reichhart, C. Czeslik Langmuir 25 (2009) 1047-1053.
[2] C. Reichhart, C. Czeslik Colloids and Surfaces B 75 (2010) 612-616
[3] O. Hollmann, C. Czeslik Langmuir 22 (2006) 3300-3305
[4] M. J. Eisenmenger, J. I. Reyes-De-Corcuera, Enzyme Microbial Technol. 45 (2009) 331-347.
[5] V. Schuabb, C. Czeslik, Langmuir 30 (2014) 15496-15503.
Poster Presentations
48
Effect of interfacial properties on the activation volume of adsorbed
enzymes
Vitor Schuabb, Süleyman Cinar, Claus Czeslik
TU Dortmund University, Department of Chemistry and Chemical Biology, D-44221 Dortmund, Germany
We have studied the enzymatic activities of α-chymotrypsin (α-CT) and horseradish peroxidase
(HRP) that are adsorbed on various chemically modified planar surfaces under aqueous solution.
The enzymes were adsorbed on bare quartz, hydrophobic poly(styrene) (PS), positively charged
poly(allylamine hydrochloride) (PAH), and negatively charged poly(styrene sulfonate) (PSS).
Activation volumes of the enzymes at the aqueous-solid interfaces were determined by using high-
pressure total internal reflection fluorescence (TIRF) spectroscopy. Apparently, the pressure
response of the adsorbed enzymes strongly depends on the interfacial properties. α-CT can be
activated by pressure (increasing enzymatic rate) on negatively charged surfaces like quartz and
PSS, whereas HRP is activated by pressure on hydrophobic PS. Corresponding negative activation
volumes of -29 mL mol-1 for α-CT on quartz, -23 mL mol-1 for α-CT on PSS, and -35 mL mol-1
for HRP on PS are found. In addition, the absolute activities of α-CT and HRP on quartz, PS, PAH
and PSS were determined by UV absorption at ambient pressure. Remarkably, large activities are
found on those surfaces that are associated with negative activation volumes. However, Fourier
transform infrared (FTIR) spectra collected in attenuated total reflection (ATR) mode do not
indicate major adsorption induced conformational changes of the enzymes at any interface studied.
Overall, the results of this study show that the activity of immobilized enzymes can largely be
enhanced by the right combination of adsorbent material and applied pressure.
Poster Presentations
49
The local structure of concentrated yttrium(III) chloride aqueous
solutions under high hydrostatic pressure
Mirko Elbers*, Karin Julius*, Michael Paulus*, Christian Sternemann*, Florian Wirkert*, Julia
Nase*, Paul Salmen*, Göran Surmeier*, Ralph Wagner** and Metin Tolan*
*Fakultät Physik/DELTA, Tu Dortmund, Germany
**Fachbereich C – Physik, Bergische Universität Wuppertal, Germany
We present an extended X-ray absorption fine structure (EXAFS) study on the hydration properties
of yttrium(III) chloride (YCl3) under high hydrostatic pressures. In order to take a closer look at
ion-ion interactions, aqueous salt solutions with different concentrations were investigated. In
nature, the interaction between macromolecules or nanoparticles is mediated by the surrounding
aqueous phase. Thus, changes in the water structure, e.g. by the application of pressure or the
addition of ions, have a direct impact on the particle-particle interaction potential. For example, in a
pressure dependent small angle X-ray scattering study of dense aqueous lysozyme solutions, we
found a minimum of the attractive interaction strength at a hydrostatic pressure of 2 kbar [1]. This
effect was assigned to a collapse of the second hydration shell of the surrounding water, which
might be affected by the additions of ions. Hence, we studied the pressure dependence of the local
structure of salt solutions by EXAFS measurements between 1 bar and 5 kbar at concentrations up
to 3M.
References:
[1] M. A. Schroer, J. Markgraf, D.C.F. Wieland, Ch.J. Sahle, J. Möller, M. Paulus, M. Tolan, and
R. Winter, Physical Review Letters 106 (2011) 178102
Poster Presentations
50
Electronic structure and interactions at high hydrostatic pressure
Stefan M. Kast*1, Patrick Kibies
1, Roland Frach
1, Saraphina Böttcher
1, Tim Pongratz
1, Franziska
Hoffgaard1, Dominik Horinek
2
1Fakultät für Chemie und Chemische Biologie, TU Dortmund, 44227 Dortmund, Germany
2Institut für Physikalische und Theoretische Chemie, Universität Regensburg, 93040 Regensburg, Germany
Applying high hydrostatic pressure to biomolecules has substantial impact on their free energy
surfaces that govern structure, function, dynamics, and thermodynamics. This poses a challenge to
computational modeling approaches since the applicability of conventional empirical molecular
interaction functions (force fields) is not known. As a step toward clarifying the situation, we need
to account for high pressure in quantum-chemical calculations. A suitable methodology is provided
by molecular integral equation theories, in particular the “embedded cluster reference interaction
site model” (EC-RISM) [1,2] that combines statistical-mechanical 3D RISM integral equation
theory and quantum-chemical calculations self-consistently. In this context the impact of pressure
is naturally accounted for since the solvent susceptibility function that enters the theory contains
the pure solvent correlation functions at the pressure chosen, derived from either an integral
equation theory or molecular simulations. Here we describe the theoretical basis and illustrate the
methodology for several benchmark applications in a pressure range of 1 bar up to 10 kbar. In
particular, we study the effect of pressure perturbation on the dipole moment of TMAO in aqueous
solution from which an improved force field can be derived. The quality of electronic structure
calculations is examined by computing pressure-dependent chemical shifts to be compared with
experimental NMR reference data obtained for N-methylacetamide (H.-R. Kalbitzer, unpublished).
The results indicate a pressure-related baseline for interpreting NMR spectra recorded to examine
pressure-induced conformational changes of peptides and proteins.
Fig. 1. Pressure-dependent chemical shifts of the NMA amide group nuclei
in water from GIAO/EC-RISM/6-
31+G(d,p) calculations (top, susceptibilities taken from simulation in blue and HNC in orange) and
experiment (middle) along with deviations between theory and experiment (bottom row).
References:
[1] T. Kloss, J. Heil, S. M. Kast, J. Phys. Chem. B 112 (2008) 4337-4343
[2] R. Frach, S. M. Kast, J. Phys. Chem. A 118 (2014) 11620-11628
Poster Presentations
51
High Pressure Induced Rupture of Hydrogen Bonds in Membrane
Proteins: The Case of the Reaction Center from Rhodobacter sphaeroides
Liina Kangur a , Marit Puusepp
a, Arvi Freiberg
a,b*,
aInstitute of Physics, Tartu University, Tartu, Estonia
bInstitute of Molecular and Cell Biology, Tartu University, Tartu, Estonia
Protein function is defined by its folded structure, while denatured conformations result in
disorders. Understanding and quantifying the protein stability with respect to unfolding is thus
equally important for solving fundamental problems as well as for practical (e.g., medical)
applications. We have previously demonstrated that the bacteriochlorophyll (BChl) binding light-
harvesting pigment-protein complexes, LH1 and LH2, from purple photosynthetic bacteria are
convenient model systems to examine the poorly understood role of hydrogen bonds as stabilizing
factors of membrane protein complexes [1, 2]. In the present contribution, we expand to another
membrane component of the photosynthetic apparatus of purple bacteria, the photo-chemical
reaction center (RC). The RC complex from Rhodobacter sphaeroides is composed from three
subunits. Two of them called L and M have quite similar structure and involve five membrane
spanning helices connected by shorter helixes; the third, H, subunit is located in the cytoplasmic
side and has a single membrane spanning helix. L and M subunits comprise 6 cofactors, 4 BChls
and 2 pheophytins, which together arrange an electron transfer chain. Two out of four BChls are
closely attached, forming a special entity called special pair. The special pair also serves as a
primary electron donor. Taking the cofactors as intrinsic and local optical probes the high pressure
induced rupture of hydrogen bonds in their binding pockets of wild type and mutant RC complexes
was monitored by characteristic discontinuous shifts and broadenings of the electronic absorption
and fluorescence spectra. In the wild type complex the special pair has uniquely only a single
hydrogen bond to the surrounding protein. Since the spectral effects were well reversible, the free
energy change corresponding to the rupture of this single bond could be evaluated as follows: 11±2
kJ/mol, while the accompanying volume change was -43±11 ml/mol. These values are quite
comparable with those estimated for hydrogen bonds in the LH1 and LH2 complexes when counted
for single hydrogen bonds.
References:
[1] A. Freiberg, L. Kangur, J. D. Olsen, C. N. Hunter, Biophysical Journal 103 (2012) 2352-2360
[2] L. Kangur, K. Leiger, A. Freiberg, Journal of Physics: Conference Series 121 (2008) 112004
Poster Presentations
52
Lysozyme at the solid – liquid interface under pressure
Paul Salmen , Michael Paulus, Florian J. Wirkert, Metin Tolan, Julia Nase
Fakultät Physik/DELTA, TU Dortmund, 44221 Dortmund, Germany
The behavior of Lysozyme under pressure at the solid/liquid interface was studied by x-ray
reflectometry. In our custom-built cell for x-ray reflectivity (XRR) measurements [1], we were able
to apply pressures up to 5 kbar and study the solid/liquid interface in-situ with Ångstrom
resolution. As solid, hydrophobic substrate, silicon wafers covered with octadecyltrichlorosilane
(OTS) were used. Lysozyme was dissolved in 20 mM BisTris buffer (pH 7.1) at a concentration of
0.1 mg/ml. The measurements were performed at the synchrotron light sources DELTA
(Dortmund, Germany), ESRF (Grenoble, France) and SLS (Villigen, Switzerland) using high
energy x-ray radiation.
At all pressures, a double layer system consisting of denatured lysozyme with native lysozyme on
top was found at the solid/liquid interface. We also compare the effects of different denaturants like
Urea or a high dose of X-ray’s on the lysozyme layer.
[1] F. J. Wirkert, M. Paulus, J. Nase, J. Möller, S. Kijawski, C. Sternemann, M. Tolan, Journal of
Synchrotron Radiation 21 (2014) 76-81
Poster Presentations
53
Lipid membranes under pressure - An x-ray reflectivity study at the
solid-liquid interface
Benedikt Nowak1, Michael Paulus
1, Julia Nase
1, Paul Salmen
1, Florian J. Wirkert
1,
Patrick Degen2, Metin Tolan
1
1Fakultät Physik / DELTA, Technische Universität Dortmund, 44221 Dortmund, Germany
2Fakultät Chemie, Physikalische Chemie II, Technische Universität Dortmund, 44221 Dortmund, Germany
Cell membranes are complex structures consisting of lipid bilayers, cholesterol, transport proteins
and structural proteins. They regulate the material exchange between the intra- and extracellular
regions. It is well-known that lipid membranes show pressure-dependent phase transitions. While
these phase transitions were studied in bulk solution in detail, the behaviour of solid-supported
membranes under pressure is widely unknown. As a simple model system for highly complex
membranes, we prepared lipid multilayers composed of phospholipids on hydrophilic silicon
surfaces and studied the interfacial structure of the solid-liquid interface under high hydrostatic
pressure using x-ray reflectometry. The layers’ vertical structures were analyzed up to a maximum
pressure of 4500 bar. With increasing pressure, a gradual filling of the sublayers between the
hydrophilic head groups with water was observed. We show that high pressure can trigger the
formation of multilayer structures on lipid bilayers.
Poster Presentations
54
The Structure of Water under Extreme Conditions
Hendrik Vondracek, Lukas Knake and Martina Havenith
Ruhr-Universität Bochum, LS Physikalische Chemie II, Bochum,
Germany.
Studies of water under extreme conditions (high and low temperatures, extreme pressures) are of
particular scientific interest. Unravelling the properties of water under extreme conditions is a
fundamental prerequisite for a better understanding of geological and biological processes as well
as the exploitation of various technical applications. Furthermore, it is also widely believed that the
acquired knowledge will be fundamental for deeper insights into the structure of water under
ambient conditions [1].
Under high pressures and temperatures to the supercritical regime, the structure of water and the
hydrogen bond network show peculiar features, e.g. clustering [2].
THz absorption spectroscopy is an ideal tool to study the structural properties of water as it allows
for a direct investigation of the intermolecular hydrogen-bond network. The principle of this
spectroscopic technique and specific experimental challenges will be explained. Furthermore, first
results of spectroscopic measurements of water under high pressure conditions will be presented.
References:
[1] A. Nilsson, L.G.M. Pettersson Chem. Phys. 389 (2011) 1-34
[2] Q. Sun, Q. Wang and D. Ding. J. Phys. Chem. B 118 (2014) 11253-11258
Poster Presentations
55
Pressure Dependence of 15
N, 1H and
13C Random Coil Chemical Shifts in
the Tetrapeptide Ac-GGXA-NH2
Markus Beck Erlach, Joerg Koehler, Werner Kremer, Hans Robert Kalbitzer
Institute of Biophysics and Physical Biochemistry and Centre of Magnetic Resonance in Chemistry and
Biophysics, University of Regensburg, 93040 Regensburg, Germany
The importance of the knowledge of random coil chemical shifts has been shown and proven over
the last decades. From first systematic investigations of random coil chemical shits [1] to the
establishment of shift-structure correlations [2] up to the development of the chemical shift index
(CSI)[3, 4] the usefulness of random coil datasets has been shown. The same now is true for the
evaluation of high pressure data, where datasets of high pressure random coil values can
significantly help in understanding complex pressure responses of proteins. As a fundamental basis
we have measured all chemical shifts of the tetrapeptide Ac-GGXA-NH2 where X is one of the 20
canonical amino acids. We gathered pressure data for the 15
N and HN from high resolution 2D-
HSQC Spectra [5, 6], 1H and
13C data of high resolution 1D experiments on an 800 MHz Bruker
Avance spectrometer with cryoprobe (TCI). This work now summarizes the pressure effect of all
the nuclei leading to a high quality set of high pressure coefficients. In addition the correlation
between the pressure effects of the nuclei has been investigated.
References:
[1] R. Richarz, K. Wuethrich, Biopolymers 17 (21978) 2133-2141
[2] D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222 (1991) 311-333
[3] D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31 (1992) 1647-1651
[4] D. S. Wishart, B. D. Sykes, J. Biomol NMR 4 (1994) 171-180
[5] M. R. Arnold, W. Kremer, H.-D. Luedemann, H. R. Kalbitzer, Biophys. Chem. 96 (2002)
129-140
[6] J. Koehler, M. Beck Erlach, E. Crusca Jr., W. Kremer, C. E. Munte, H. R. Kalbitzer, Materials
5 (2012) 1774-1786
Poster Presentations
56
Phase Behavior of Plasma Membrane Vesicles under Extreme Conditions
Nelli Erwin , Janine Seeliger ,Katrin Weise , Roland Winter *
Physical Chemistry I – Biophysical Chemistry, Faculty of Chemistry, TU Dortmund University, 44227
Dortmund, Germany
High hydrostatic pressure has been found to significantly affect all levels of cellular physiology,
and biological membranes seem to be one of the most pressure sensitive cellular components.
Pressure-induced perturbations of membranes can cause structural changes and thus influence their
functional properties. Even though this poses a serious challenge for the biological cell, it has not
prevented organisms from surviving in the cold and high pressure habitats of marine depths where
pressures up to the 110 MPa level are reached.1, 2
The basic structural element of biological membranes consist of lamellar lipid bilayers that display
various phase transitions including a chain melting (gel-to-fluid) transition. Upon hydrostatic
compression of a lipid bilayer, an increase in bilayer thickness and conformational order of lipid
chains is observed. This is accompanied by a decrease in cross-sectional area per molecule owing
to lipid chain condensation. In addition biological membranes contain proteins embedded in the
lipid bilayer. The function of these membrane proteins can ceases at pressures of a few hundred
MPa.3, 4
Although the pressure effects on natural membranes are still elusive, it is already clear that
the membrane's physical-chemical properties markedly influence the lipid-protein interaction,
activity and the pressure stability of the membrane proteins.
In this study, the temperature- and pressure-dependent structure and phase behavior and lateral
organization of giant plasma membrane vesicles isolated from mammalian cells has been
investigated in the absence and presence of membrane proteins using a combined spectroscopic and
microscopic approach. Phase separation into extended liquid-ordered and liquid-disordered
domains is observed over a wide range of temperatures and pressures. Only at pressures beyond
200 MPa a physiologically prohibited all-ordered lipid phase-state is reached at ambient
temperature. This is in fact the pressure range where membrane-protein function has generally been
observed to cease, thereby shedding new light on the possible origin of this observation.5
References:
[1] I. Daniel, P. Oger and R. Winter, Chem. Soc. Rev. 35, 858 (2006)
[2] D. H. Bartlett, Biochim. Biophys. Acta 1595, 367 (2002)
[3] K. Heremans, L. Smeller, Biochim. Biophys. Acta 1386, 353 (1998)
[4] H. M. Ulmer, H. Herberhold, S. Fahsel, M. G. Gänzle, R. Winter and R. F. Vogel, Appl.
Environ. Microbiol. 68, 1088 (2002)
[5] J. Seeliger, N. Erwin, C. Rosin, M. Kahse, K. Weise, and R. Winter, Phys. Chem. Chem. Phys.
17, 7507 (2015)
Poster Presentations
57
Secondary structure and folding stability of proteins adsorbed on silica
particles – Pressure versus temperature denaturation
Süleyman Cinar, Claus Czeslik*
TU Dortmund University, Department of Chemistry and Chemical Biology, D-44221 Dortmund, Germany
We present a systematic study of the pressure and temperature dependent unfolding behavior of
proteins that are adsorbed on silica particles. Hen egg white lysozyme and bovine ribonuclease A
(RNase) were used as model proteins, and their secondary structures were resolved by Fourier
transform infrared (FTIR) spectroscopy in the temperature range of 10–90 °C and the pressure
range of 1–16,000 bar. Apparently, the secondary structures of both proteins do not change
significantly when they are adsorbing on the silica particles. Remarkably, the changes of the
secondary structure elements upon protein unfolding are very similar in the adsorbed and the free
states. This similarity could be observed for both lysozyme and RNase using both high pressures
and high temperatures as denaturing conditions. However, the pressures and temperatures of
unfolding of lysozyme and RNase are drastically lowered upon adsorption indicating lower folding
stabilities of the proteins on the silica particles. Moreover, the temperature ranges, where changes
in secondary structure occur, are broadened due to adsorption, which is related to smaller enthalpy
changes of unfolding. For both proteins, free or adsorbed, pressure-induced unfolding always leads
to less pronounced changes in secondary structure than temperature-induced unfolding. In the case
of lysozyme, high pressure also favors a different unfolded conformation than high temperature.
Overall, the results of this study reveal that adsorption of proteins on silica particles decreases the
folding stability against high pressures and temperatures, whereas the unfolding pathways are
mainly preserved in the adsorbed state. [1].
References:
[1] S. Cinar, C. Czeslik, Colloids and Surfaces B: Biointerfaces, 129 (2015) 161–168
Poster Presentations
58
Cosolvent and crowding effects on the temperature and pressure stability
of monomeric actin
Paul Hendrik Schummel and Roland Winter
Physical Chemistry I – Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund
University, 44227 Dortmund, Germany
Actin can be found in nearly all eukaryotic cells and is responsible for many different cellular
functions. The polymerization process of actin has been found to be among the most pressure
sensitive processes in vivo. In this study, we explored the effects of chaotropic and kosmotropic
cosolvents, such as urea and the compatible osmolyte trimethylamine-N-oxide (TMAO), as well as
the crowding agent Ficoll® PM 70 on the temperature and pressure stability of global actin (G-
actin). The temperature and pressure of unfolding as well as thermodynamic parameters upon
unfolding, such as volume and enthalpy changes, have been determined by fluorescence
spectroscopy over a wide range of temperatures and pressures, ranging from 10-80 °C and 1-3000
bar, respectively. Different from the chaotropic agent urea, TMAO increases both, the temperature
and pressure stability for the protein most effectively. In mixtures of these osmolytes, urea
counteracts the stabilizing effect of TMAO to some extent. To create a more cell-like environment,
Ficoll® PM 70 was added as macromolecular crowding agent as well. Addition of the crowding
agent increases the temperature and pressure stability even further, thereby allowing sufficient
stability of the protein at the temperature and pressure conditions encountered under extreme
environmental conditions on Earth.
Poster Presentations
59
Crystallographic structures of Ras under high hydrostatic pressure
N. Colloc’h1, E. Girard
2, P. Lopes
3, A.C. Dhaussy
4, T. Prangé
5, M. Spoerner
3, H.R. Kalbitzer
3
1 CERVOxy team, ISTCT, UMR 6301 CNRS CEA UNICAEN, GIP Cyceron, Caen, France
2 IBS, UMR 5075 CEA CNRS UJF, Grenoble, France
3. University of Regensburg, Regensburg, Germany
4. CRISTMAT UMR 6508 CNRS ENSICAEN, Caen, France
5. LCRB UMR 8015 CNRS Université Paris Descartes, Paris, France
The guanine nucleotide binding (GNB) protein Ras is involved in cellular signal transduction
pathways inducing proliferation, differentiation and apoptosis of cells. It functions as a molecular
switch cycling between an inactive GDP-bound state and an active GTP-bound state. Only Ras
complexed with GTP is able to bind different effectors such as Raf-kinase or RalGDS with high
affinity. In solutions of Ras complexed with GTP-analogs GppNHp or GppCH2p, NMR allows to
detect two major conformations (state 1(T) and state 2(T)) with similar populations at atmospheric
pressure (37 and 63% resp.) that coexist in a dynamical equilibrium with exchange correlation
times in the millisecond range. State 1(T) is a weak-binding state for effectors and corresponds to a
guanine exchange factor (GEF) interacting state1. State 2(T) is the strong effector binding state.
Solution HPNMR data show that pressure shifts the conformational equilibrium1 towards state 1(T)
so that at 50 MPa, the fraction of state 1(T) should equal to state 2(T). A detailed analysis of the
HPNMR data2 shows that in solution additional conformational states coexist in low population at
ambient pressures, state 3(T), the interaction state with the GTPase activating protein GAP and the
nucleotide release state 1(0). At 150 MPa state 3(T) dominates, a thermodynamic analysis predicts
that at 500 MPa the protein exists mainly in state 1(0). The observed conformational equilibrium
can be exploited to devise a new type of state specific inhibitors of the Ras-effector interaction thus
interrupting the signal transduction in oncogenic Ras-mutants2.
In contrast to NMR, X-ray
crystallography at ambient pressure shows a well-defined structure corresponding to state 2(T)3.
Therefore, a direct observation of these states by high pressure macromolecular crystallography 4.5
could also have a strong impact on drug design for fighting Ras-dependent tumor formation. A
large number of data sets have thus been collected at ambient pressure and at different high
pressure. The switch II loop (residues 61-67) seems to be the most sensitive to pressure (high
elevation of B-factor, high r.m.s.). The comparison between the different structures will be
discussed.
References:
[1] H.R. Kalbitzer, M. Spoerner, P. Ganser, C. Hozsa, W. Kremer, J. Am. Chem. Soc. 131 (2009)
16714–16719
[2] H.R. Kalbitzer, I.C. Rosnizeck, C.E. Munte, S.P. Narayanan, V. Kropf, M. Spoerner, Angew.
Chem. Int. Ed. 52 (2013) 14242-14246
[3] E.F. Pai, U. Krengel, G.A. Petsko, R.S. Goody; W. Kabsch, A. Wittinghofer, EMBO J. 9 (1990)
2351-2359
[4] R. Fourme, E. Girard, A.C. Dhaussy, K. Medjoubi, T. Prangé, I. Ascone, M. Mezouar, R. Kahn
J. Synchr. Rad. 18 (2011) 31-36
[5] R. Fourme, E. Girard, K. Akasaka, Curr. Opin. Struc. Biol. 5 (2012) 636-642
Poster Presentations
60
Detecting the functional conformations of active Ras protein by high
pressure NMR spectroscopy
Pedro Lopes, Michael Spoerner, Sunilkumar P. Narayanan, Ina Rosnizeck, Andreas Huberth,
Werner Kremer and Hans Robert Kalbitzer*
Institute of physical Biochemistry and Biophysics, University of Regensburg, 93053 Regensburg,
Germany
The small GTPase Ras is the prototype member of the guanine nucleotide binding (GNB) proteins
superfamily and it is one of the central proteins of signal transduction pathways within the cell. It
cycles between two main structural states stabilized by GDP and GTP, acting as a molecular switch
[1]. The active, Ras-GTP, protein exists in a dynamic equilibrium between two different
conformational states. State 1(T) is characterized by having high affinity towards activating
proteins (GEFs) and state 2(T) has high affinity towards effector proteins. Both states can be
directly observed by 31
P NMR when Ras is complexed with the non-hydrolysable GTP analogue,
GppNHp. A third 3(T) and a fourth 1(0) state in activated Ras were detected by high pressure (HP)
[1H,
15N]- HSQC NMR, corresponding to the interaction with GAP proteins that end the activation
cycle and a nucleotide free state with GDP.Pi bound, respectively [2].
In the present work we demonstrate the importance of high pressure NMR as a novel technique
capable of detecting new functional conformations on Ras that can be targeted by small molecules,
making them available for virtual drug screening and drug design. Using HP 31
P NMR a decrease
on the population of the effector binding state 2(T) and an increase of GEF state 1(T) can be
detected. Simultaneously a shift of the conformation towards state 3(T) is also observed. Titration
experiments with GAP showed furthermore that the obtained chemical shift value for state 3(T)
corresponds to the value obtained for the Ras-GAP complex [3-4].
References:
[1] I. R. Vetter, A. Wittinghofer, Science 294 (2001) 1299-1304
[2] H.R. Kalbitzer, I.C. Rosnizeck, C.E. Munte, S.N. Narayanan, V. Kropf, M. Spoerner, Angew.
Chem. Int. Ed. 52 (2013) 14242-14246
[3] H.R. Kalbitzer, M. Spoerner, P. Ganser, C. Hozsa, W. Kremer, J. Am. Chem. Soc. 131 (2009)
16714-16719
[4] M. Spoerner, A. Wittinghofer, H.R. Kalbitzer, FEBS Lett. 578 (2004) 305-310
Poster Presentations
61
Pressure Modulation of the Enzymatic Activity of Phospholipase A2
S. Suladze, S. Cinar, B. Sperlich, R. Winter
Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU
Dortmund University, 44227 Dortmund, Germany
Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane
phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we
used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties
to reveal new mechanistic information about the membrane association and subsequent enzymatic
reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and
integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions
operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of
HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee
venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform
infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results
show that PLA2 binding to model biomembranes is not significantly affected by pressure and
occurs in at least two kinetically distinct steps. Followed by fast initial membrane association,
structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface.
FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid
hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under
extreme environmental conditions, such as those encountered in the deep sea where pressures up to
the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being
a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.
Poster Presentations
62
Exploring the effects of temperature and pressure on the structure and
stability of a small RNA hairpin
Caroline Schuabb, Salomé Pataraia, and Roland Winter*
TU Dortmund University, Department of Chemistry and Chemical Biology, D-44227 Dortmund, Germany
Small RNA hairpins (sRNAHp) are secondary structure elements that are integral components of
RNAs such as ribozymes and transfer RNAs. They function as nucleation sites for RNA folding
and ligand binding. Theoretical calculations showed that the sRNAHp's free energy landscape
might be represented by an elliptically-shaped temperature-pressure (p-T) stability diagram with
multiple conformational states.1 Furthermore, these studies showed that they have pronounced
thermodynamic stability due to noncanonical interactions.2 Aiming to establish the p-T stability
diagram and correlate it with theoretical predictions, UV-vis, Fourier-transform infrared (FTIR)
and fluorescence resonance energy transfer (FRET) experiments were carried out over a wide range
of temperatures and pressures. The combined results reveal characteristic conformational changes
as a function of temperature and pressure. The thermal melting analysis revealed a broad, non-two-
state melting transition between 40 and 60oC. Combined high-pressure FRET and UV results
indicate that below the melting temperature pressure perturbs stem interactions and increases the
population of non-native conformations. The high-pressure FTIR data showed that at ambient
temperature, pressure is able to destabilize native stem interactions, without leading to complete
unfolding, however. At high temperatures, e.g. 70 oC, in the unfolded state, pressure does not lead
to significant refolding. The combined structural analysis seems to be compatible with a non-two-
state elliptically shaped p-T stability diagram.
References:
(1) Garcia A. E.; Paschek D.J., J. Am. Chem. Soc. 2008, 130, 815-817
(2) Chakraborty D., et al., J. Am. Chem. Soc. 2014, 136, 18052-18061
Poster Presentations
63
Combined temperature, pressure, and cosolvent effects on enzyme
activity
Trung Quan Luong, Roland Winter
Department of Chemistry and Chemical Biology, Biophysical Chemistry, TU Dortmund University, D-44221
Dortmund, Germany
We studied the combined effects of pressure (0.1-200 MPa), temperature (20-40 °C) and cosolvents
on the enzyme activity of α-chymotrypsin upon the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-
nitroanilide using a high-pressure stopped-flow system. A kosmotropic osmolyte (TMAO) and a
chaotropic agent (urea) and mixtures thereof were used as cosolvents. High pressure enhances the
hydrolysis rate as a consequence of a negative activation volume for all solution conditions (-2
to -4 mL mol
-1). The enhancement is most significant at 20 °C and smaller at high temperatures.
Kinetic constants, such as the rate constant of the catalysis (kcat) and the Michaelis constant (KM),
were determined as a function of pressure. Compared to the pure buffer solution, addition of 1 M
TMAO has minor effects on the kinetic constants, while upon addition of 2 M urea kcat increases by
35% and KM increases 6-fold. In the TMAO:urea 1:2 mixture, the urea-effect on kcat and KM is
compensated to some extent, and, remarkably, pressure is found to have no effect on the rate of the
enzyme reaction. Our data clearly show that by a combination of temperature, pressure and
cosolvents, enzyme activity can be effectively modulated and optimized according to changes in
environmental conditions.
Poster Presentations
64
Near-surface behavior of a bicontinuous microemulsion under high
hydrostatic pressure conditions
Melanie Berghaus1, Michael Paulus
2, Paul Salmen
2, Samy Al-Ayoubi
1, Metin Tolan
2,
and Roland Winter1
1 Physikalische Chemie I - Biophysikalische Chemie, TU Dortmund, D-44227 Dortmund, Germany
2 Fakultät Physik/DELTA, TU Dortmund, D-44221 Dortmund, Germany
The transition from a fluid lamellar phase to a bicontinuous lipid phase plays, for example, an
important role in biomembrane phase transitions such as vesicle fusion. This transition has been
studied for several lipids forming highly ordered cubic lipid phases, [1–3]
and can be initiated using
the pressure-jump methodology. What is lacking so far, though of high biological relevance, is
knowledge on the effect of interfaces and conformational disorder on such kind of mesophase
transitions. In order to mimic these effects, we studied the pressure-dependent phase behavior of a
disordered bicontinuous microemulsion (BME) in the presence of a solid interface by X-ray
reflectometry (XRR). BMEs are ternary systems consisting of water, oil and a surfactant occupying
the interface between the latter. Interestingly, some BMEs show a transition from a lamellar phase
close to the surface to a bicontinuous phase when approaching the bulk, as revealed by neutron
reflectometry, recently.[4,5]
We investigated how high hydrostatic pressure (HHP) influences the
structure of this transition region. HHP is not only an important feature in marine environments and
biotechnological applications, but can also be used as a physical parameter to study the kinetics and
mechanism of lipid phase transitions and to continuously tune the lattice constant of lipid phases.
Our results show that bicontinuous microemulsions form a lamellar phase close to hydrophilic
interfaces, which are markedly compressible. Pressure increases the lamellar order, but does not
significantly extend the correlation length of lamellar order induced by the presence of the
hydrophilic interface. Possible biological implications are discussed.
References:
[1] V. Cherezov, D. P. Siegel, W. Shaw, S. W. Burgess, M. Caffrey, J. Membr. Biol. 195 (2003)
165–182.
[2] J. Lendermann, R. Winter, Phys. Chem. Chem. Phys. 5 (2003) 1440–1450.
[3] C. Conn, O. Ces, X. Mulet, S. Finet, R. Winter, J. Seddon, R. Templer, Phys. Rev. Lett. 96
(2006) 108102.
[4] M. Kerscher, P. Busch, S. Mattauch, H. Frielinghaus, D. Richter, M. Belushkin, G. Gompper,
Phys. Rev. E 83 (2011) 030401.
[5] X.-L. Zhou, L.-T. Lee, S.-H. Chen, R. Strey, Phys. Rev. A 46 (1992) 6479–6489.
Poster Presentations
65
Insights into the intramolecular coupling between the N- and C-domains
of skeletal troponin C
Mayra de A. Marques‡, Guilherme A. P. de Oliveira
‡, Cristiane B. Rocha
§, Yraima Cordeiro
δ,
Martha M. Sorenson‡, Jerson L. Silva
‡*, Débora Foguel
‡ and Marisa C. Suarez
‡*
‡ Programa de Biologia Estrutural, Instituto de Bioquímica Médica UFRJ,
§ UNIRIO - Universidade Federal do Estado do Rio de Janeiro
δ Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Troponin C (TnC), the Ca2+
-binding component of the troponin complex of vertebrate skeletal
muscle, consists of two structurally homologous domains, N and C, connected by an exposed a-
helix. Mutants of whole TnC and of its isolated domains have been constructed using site-directed
mutagenesis to replace different Phe residues by Trp. Previous studies using these mutants and high
hydrostatic pressure have shown that the C-domain apo form is less stable than the N-domain and
that the N-domain has no effect on the stability of the C-domain [Rocha, C. B., Suarez, M. C., Yu,
A., Ballard, L., Sorenson, M. M., Foguel, D., Silva, J. L. (2008) Biochemistry 47, 5047-5058]. Here
we analyze the stability of intact F29W TnC by structural approaches and against urea and pressure
denaturation using a fluorescent mutant with Phe 29 replaced by Trp, located in the N-domain.
From these experiments we calculate the thermodynamic parameters (DV and DG°atm) that govern
the folding of the intact F29W TnC in the absence and presence of Ca2+
. We find that the C-domain
has only a small effect on the structure of the N-domain in the absence of Ca2+
. However, using
fluorescence spectroscopy we showed a significant decrease in the stability of the N-domain in the
Ca2+
-bound state when Ca2+
is bound to sites III and IV of the C-domain. An accompanying
decrease in the thermodynamic stability of the N-domain generates a reduction of DDG°atm in
absolute terms and affects the Ca2+
affinity of N-domain in whole TnC. Cross-talk between C- and
N-domains may be mediated by the central helix, which has a smaller volume and probably greater
rigidity and stability upon Ca2+
-binding to the EF-hand sites, as determined by our reconstruction
of low-resolution 3D models from SAXS.
Poster Presentations
66
Exploring conformational changes of the villin headpiece subdomain
monitored by high pressure NMR spectroscopy
Paul Becker, Jeremy Sloan, Andi Klamt, Thomas Kiefhaber, Jochen Balbach
Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, Germany; [email protected]
The villin headpiece subdomain (HP-35) is a natural occurring, monomeric polypeptide that folds
autonomously into a specific and thermostable structure. HP-35 consists of 35 amino acids, which
makes it a considerable and well studied model system concerning protein folding and protein
dynamics. The unfolding of HP-35 induced by high pressure or guanidinium chloride observed by
triplet-triplet-energy transfer (TTET) experiments revealed an unlocked state which was classified
as an dry molten globule state. To characterize the conformational changes residue by residue
resolution during the unfolding of HP-35, we will present the analysis of high pressure NMR data
recorded with HP-35 up to 200 MPa.
Poster Presentations
67
Protein-protein interactions in crowded lysozyme solutions
Karin Julius*, Michael Paulus*, Melanie Berghaus**, Nico König*,
Roland Winter** and Metin Tolan*
*Fakultät Physik / DELTA, Technische Universität Dortmund, 44221 Dortmund, Germany
**Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, 44221 Dortmund
Germany
Inside cells, proteins are surrounded by different macromolecules, including proteins themselves,
which cover approximately 30% of the available volume. It has been shown that this reduction of
free space by macromolecules has a significant impact on the stability of proteins, rendering them
more resistant to temperature or pressure denaturation [1], the so called crowding effect. However,
the influence of crowding on the protein-protein interaction potential that is mediated by the
solvent is still unknown. The final goal of this project is the investigation of the pressure dependent
interaction potential between proteins in aqueous solutions as a function of the crowder
concentration, mimicking intracellular solution conditions. For this purpose, small-angle
X-ray scattering (SAXS) under high hydrostatic pressure was applied. As we focus on the effect of
crowding, the well characterized model protein lysozyme was used at a concentration of 5 - 10 wt.-
% in combination with the macromolecular crowder Ficoll PM 70 and its monomeric subunit
sucrose.
References:
[1] M. Erlkamp, S. Grobelny, and R. Winter, Phys. Chem. Chem. Phys. 16 (2014) 5965-5976
Poster Presentations
68
Exploring folding cooperativity of a repeat protein folding by 2D-NMR detected
pressure perturbation
Martin J. Fossat
1,2, Angel Garcia
3, Doug Barrick
4, Christian Roumestand
2 and Catherine Royer
1
1 Department of Biological Sciences Rensselaer Polytechnic Institute, Troy, NY USA
2 Centre de Biochimie Structurale CNRS-Université Montpellier, Montpellier, France
3 Department of Physics Rensselaer Polytechnic Institute, Troy, NY USA
4 T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore MD, USA
Most natural proteins fold cooperatively to form their tertiary structure. Repeat proteins offer a
good model for the study of the molecular determinants of folding cooperativity because of their
linear nature. In this study, we measured the pressure-induced unfolding of the leucine-rich repeat
protein, pp32, at four different temperatures, using high pressure 2D NMR spectroscopy. Pressure
leads to protein unfolding because internal cavities in the folded state are lost upon unfolding,
thereby decreasing the molar volume of the protein-solvent system. From the pressure dependence
of the individual HSQC peak intensities we obtained residue specific pressure denaturation curves.
Deviations from cooperative unfolding were manifested by differences in the apparent
thermodynamic parameters (ΔVu, ΔGu) extracted from fits of the curves from each residue to a 2-
state transition. We found the apparent unfolding cooperativity to be strongly temperature
dependent. Unfolding was highly cooperative at 293°K, but deviations from two-state behavior
were significant at higher and lower temperature. Then, we used fractional contact map analysis to
visualize the structural basis of the unfolding heterogeneity. The fraction of contact of each residue
pair was used to generate experimentally biased Structure Based Modeling (SBM) ensembles under
different conditions, yielding an approximation of the folding free energy landscape. We found that
at both high and low temperatures, several partially folded intermediates were populated in which
the N-terminal repeats unfolded at lower pressures on average than the C-terminal repeats.
69
List of Participants
70
Kazuyuki Akasaka
Kyoto Prefectural University
1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto,
Kyoto 606-8522, Japan
Nathalie Colloc'h
ISTCT UMR 6301 CNRS UNICAEN
CERVOxy team, ISTCT, UMR 6301 CNRS
CEA UNICAEN, GIP Cyceron, Caen,
France
Samy R. Al-Ayoubi
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Claus Czeslik
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Jochen Balbach
Martin-Luther-Universität Halle-Wittenberg
Betty-Heimann-str. 7
D-06120 Halle (Saale), Germany
Mariano Dellarole
Institut Pasteur
25-28 Rue du Dr Roux
FRA- 75015 Paris, Frankreich
Markus Beck-Erlach
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Mirko Elbers
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Paul Becker
Martin-Luther-Universität Halle-Wittenberg
Betty-Heimann-str. 7
D-06120 Halle (Saale), Germany
Nelli Erwin
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Melanie Berghaus
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Martin Fossat
Rensselaer Polytechnic Institute
110 8th Street,
Troy, NY 12180, USA
Elena Boldyreva
Siberian Branch of Russian Academy of Sciences,
ul. Kutateladze, 18,
RUS- 630128 Novosibirsk, Russia
Roland Frach
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Nick Brooks
Imperial College London,
South Kensington Campus,
London SW7 2AZ, UK
Arvi Freiberg
University of Tartu
Ülikooli 18
EST-50090 Tartu, Estland
Süleyman Cinar
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Monica Freitas
Federal University of Rio de Janeiro
Av. Pedro Calmon, 550
BRA- 21941-901 Rio de Janeiro, Brasilien
71
Mimi Gao
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Karin Julius
Technische Universität Dortmund
Otto-Hahn-Str. 4
D-44227 Dortmund, Germany
Alfons Geiger
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Hans Robert Kalbitzer
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany [email protected]
Maksym Golub
University Grenoble Alpes
71 avenue des Martyrs
F-38044 Grenoble, France
Liina Kangur
University of Tartu
Ülikooli 18
EST-50090 Tartu, Estland
Martina Havenith
Ruhr-Universität Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
Stefan M. Kast
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Martin Hofmann
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Patrick Kibies
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Christoph Hölzl
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Thomas Kiefhaber
Martin-Luther-Universität Halle-Wittenberg
Universitätsplatz 10
D-06108 Halle, Germany
Dominik Horinek
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Lukas Knake
Ruhr-Universitaet Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
Toshiko Ichiye
Georgetown University
3700 O St NW
20057 Washington, DC, USA
Inga Kolling
Ruhr-Universität Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
Sho Imoto
Ruhr-Universität Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
Werner Kremer
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
72
Narendra Kumar
Ruhr-Universität Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
Phil Oger
Ecole Normale Supérieure de Lyon
15 parvis René Descartes
FRA-69342 Lyon, France
Artem Levin
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Guilherme A.P. de Oliveira
Federal University of Rio de Janeiro
Av. Pedro Calmon, 550
BRA- 21941-901 Rio de Janeiro, Brasilien
Pedro Lopes
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Judith Peters
Univ. Grenoble Alpes, LiPhy, CS 10090,
38044 Grenoble, France
Institut Laue-Langevin, CS 20156, 38042
Grenoble cedex 9, France
Trung Quan Luong
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Vytautas Petrauskas
Vilnius University
V.A. Graičiūno 8,
LT-02241 Vilnius, Lithuania
Robert Macgregor
University of Toronto
144 College St., Toronto, Ontario,
M5S 3M2 Canada
Tim Pongratz
Technische Universität Dortmund
Otto-Hahn-Str. 4
D-44227 Dortmund, Germany
Mayra Marques
Federal University of Rio de Janeiro
Av. Pedro Calmon, 550
BRA- 21941-901 Rio de Janeiro, Brasil
Sunilkumar Puthenpurackal Narayanan
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Dominik Marx
Ruhr-Universität Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
Marit Puusepp
Institute of Physics, Tartu University, Tartu,
Estonia
Julia Nase
Technische Universität Dortmund
Otto-Hahn-Str. 4
D-44227 Dortmund, Germany
Oliver Reiser
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Masayoshi Nishiyama
Kyoto University
Yoshida-Honmachi Sakyo-ku,
JPN-606-8501 Kyoto, Japan
Francisco Rodríguez Ropero
Technische Universität Darmstadt
Alarich-Weiss-Straße 10
64287 Darmstadt, Germany
73
Christian Roumestand
Universités de Montpellier,
INSERM U554, CNRS UMR 5048, France
Michael Spoerner
Universität Regensburg
Universitätsstraße 31
D-93053 Regensburg, Germany
Catherine Royer
Rensselaer Polytechnic Institute
110 8th Street,
Troy, NY 12180, USA
Metin Tolan
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Paul Salmen
Technische Universität Dortmund
Otto-Hahn-Str. 4
D-44227 Dortmund, Germany
Tigran V. Chalikian
University of Toronto,
144 College Street, Toronto,
Ontario M5S 3M2, Canada
Vitor Schuabb
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Nico van der Vegt
Technische Universität Darmstadt
Alarich-Weiss-Straße 10
64287 Darmstadt
Caroline Schuabb
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Hendrik Vondracek
Ruhr-Universität Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
Julian Schulze
Technische Universität Dortmund
Otto-Hahn-Str. 4
D-44227 Dortmund, Germany
Katrin Weise
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Paul Hendrik Schummel
Technische Universität Dortmund
Otto-Hahn-Str. 4
D-44227 Dortmund, Germany
Roland Winter
Technische Universität Dortmund
Otto-Hahn-Str. 4a
D-44227 Dortmund, Germany
Gerhard Schwaab
Ruhr-Universität Bochum
Universitätsstraße 150
D- 44801 Bochum, Germany
László Smeller
Semmelweis University
1444 Budapest, Pf 263, Hungary
74