biophysics of macromolecules - uni-muenchen.de · biophysics of macromolecules 6. july 2015....
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How to create methods to probe macromoleculesin vivo ?
Lecture 18: In vivo Methods
Braun/Lipfert SS 2015
Biophysics of Macromolecules
6. July 2015
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Crowding alters Biochemical Equilibria
Crowding alters the Kinetics within Cells
Excluded volume forces Binding statistics
Slowing of diffusion
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ThermophoresisMolecules in a temperature gradient
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ThermophoresisMolecules in a temperature gradient
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ThermophoresisMolecules in a temperature gradient
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ThermophoresisMolecules in a temperature gradient
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ThermophoresisMolecules in a temperature gradient
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Thermophoresis
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Thermophoresis
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Why does the steady state depletion say something about the probability of binding?
=> Two State model (black board)
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Philipp Baaske; Stefan Duhr
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Philipp Baaske; Stefan Duhr
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Publications using Thermophoresis in 2014
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Thermophoretic Immunology?
Analytical Chemistry 84, 3523–3530 (2012)
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Thermophoretic Immunology?
Analytical Chemistry 84, 3523–3530 (2012)
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Thermophoretic Immunology?
Analytical Chemistry 84, 3523–3530 (2012)
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Basis of Thermophoresis
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Local Equilibrium
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Local Equilibrium
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Local Equilibrium
Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)
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Local Equilibrium
Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)
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Local Equilibrium
Capacitor
Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)
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Local Equilibrium
Capacitor Seebeck
Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)
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Capacitor
Seebeck Contribution
Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)
Seebeck
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Capacitor
Seebeck Contribution
Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)
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Multiwell-Plates
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Angewandte Chemie53, 7948–7951 (2014)
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Angewandte Chemie53, 7948–7951 (2014)
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Angewandte Chemie53, 7948–7951 (2014)
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Angewandte Chemie53, 7948–7951 (2014)
Problem: Need to hit center otherwise droplet moves
away from the focus
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Inside Living Cells
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Cell Cell
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Lower thermophoretic mobility and slower diffusion
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Reaction Kinetics in a Cell
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54th Annual Meeting of the Biophysical SocietySan Francisco, California
February 21, 2010
Ingmar Schön, Hubert Krammer and Dieter Braun
Systems Biophysics, LMU München, Germany
Hybridization Kinetics Is Different Inside Cells
10µm
Proc. Natl. Acad. Sci. USA 106: 21649-21654 (2009)
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How fast do DNA Stands find each other inside a Cell?
Molecular Crowding Specific Interactions (w/ Proteins)
Sugiyama et al., PNAS, 1998Minton, J.Cell Sci., 2006
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Experimental Approach
Principle: Perturbe EquilibriumAnalyze Relaxation
Detection:Fluorescence Resonance Energy Transfer (FRET)
Delivery: Lipofection into HeLa CellsTOOL Microscopy
(Temperature Oscillation Optical Lock-In)
Braun & Libchaber, Appl. Phys. Lett., 2003
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quantum efficiency
illumination
stroboscopic illuminationphase-locked relative to perturbation
0° 90° 180° 270°
collect fluorescence by slow CCD(low-pass filtering)
Data Analysis and Interpretation
fit with transfer functionfor a first-order transition
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Temperature Reference
Complex Fit with Transfer Functionfor a first-order transition
Calibration of Cy5 Dye
Intracellular Delivery
Spatial Temperature Kinetics
10µm 10µm
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Simulated Temperature Characteristics
finite element simulation- solve Fourier-transformed heat conduction equations (FEMLAB)- transfer function fit as in experiment
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calibration against temperature kineticsof the measurement chamber
Data Analysis and Interpretation
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Preparation Procedure
microscope slides- chromium layer for IR adsorption- silicon substrate for fast cooling
measurement chamber
cell culture- sterilization- surface coating- cell seeding
poly-D-lysine
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DNA Hybridization Kinetics in vivo
f = 1...200Hz
10 µm
Donor and FRET Signals yielded consistent Time Constants.
10 µm
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DNA Hybridization Kinetics in vivo
f = 1...200Hz
10 µm
Donor and FRET Signals yielded consistent Time Constants.
10 µm
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DNA Hybridization Kinetics in vivo
f = 1...200Hz
10 µm
Donor and FRET Signals yielded consistent Time Constants.
10 µm
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Reaction Speed in Cellular Compartments
Hybridization Reaction inside Nucleus was faster than in Cytoplasm.
10 µm10 µm
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Kinetics in vivo versus in vitro
PBS solution
1 4of DNA on ofkckkτ− =+
ACCELERATION
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Kinetics in vivo versus in vitro
PBS solution
12 4of DNA on ofkckkτ− =+
ACCELERATION
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Kinetics in vivo versus in vitro
PBS solution
12 4of DNA on ofkckkτ− =+
ACCELERATION SLOWING DOWN
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Effects of Divalent Ions and Crowding Agents in vitro
Mg2+ ACCELERATED the kinetics
Crowding DID NOT CHANGE the kinetics
30% (w/v)
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Possible Origin of Different Kinetics in vivo
Reasons for ACCELERATION
- Crowding (Excluded Volume): not observed in vitro
- Recombination Mediator Proteins (e.g. Rad52)- High Divalent Ion Concentrations (unlikely)- Background Hybridization with RNA/DNA(Enhanced Effective Concentration):Inconsistent with Qualitative Trend
Reasons for DECELERATION
- Crowding (Hindered Diffusion): not observed in vitro
- Background Interactions: Stationary Buffering of ssDNA or dsDNAby DNA-Binding Proteins(Reduced Effective Concentration)
OPEN QUESTIONS
- Which Proteins are involved?- Which Effect is Probe Specific?- What Mediates Specifity: Oligo Length? Sequence?
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DNA Probe
RhG|
5’-C AG G TTA CTA TCG TAT T C-3’
RO X|
5’-C AAT ACG ATA G TA ACC T C-3’
C = L-enantiomeric cytosin
Construct Intracellular Delivery
Melting Characteristics
scale bars: 10 µmexcitation at 488nm
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Viability of Cells
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Calibration Dye Brightness
possible quenchingemission senesitivity to 5 mM glutathione and 0.2 mM ascorbic acid
solution of known concentrationsmulti-point confocal images
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Reaction Amplitude
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Subcellular Resolution
scale bars: 5 µm
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Which Effect Is Probe‐Specific?
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Can we recreateautonomous
Darwinian Evolution(a.k.a. Life)in the lab ?
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Let's try it!Hard puzzles are best approached
by doing experiments to test hypothesis
Can we recreateautonomous
Darwinian Evolution(a.k.a. Life)in the lab ?
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Life as we know it
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Life as we know it- Replication
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Life as we know it- Replication of Genetic Information
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Life as we know itReplication of Genetic Information ...
… to create Proteins from Genes ...
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Life as we know itReplication of Genetic Information ...
… to create Proteins from Genes ...
… in a crowded Soup of Nutrients ...
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Life as we know itReplication of Genetic Information ...
… to create Proteins from Genes ...
… in a crowded Soup of Nutrients ...… far from Equilibrium.
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Life as we know itReplication of Genetic Information ...
… to create Proteins from Genes ...
… in a crowded Soup of Nutrients ...
The big Puzzleof Biogenesis
… far from Equilibrium.
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Thermal Molecule Traps
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Thermal Trap
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Thermal Trap
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Thermal TrapAccumulation of 100-1000bp DNA
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Light driven Microfluidicsto drive a thermal trap
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Light driven Microfluidics
PRL 100, 164501 (2008); JAP 104, 104701 (2008)
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Light driven Microfluidics
PRL 100, 164501 (2008); JAP 104, 104701 (2008)
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Create a crowdedEnvironment
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ChristofMast
Length Concentration
trapping
elongation
PNAS 110, 80308035 (2013)
Create a crowdedEnvironment
PolymerizationMachine
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ChristofMast
Length Concentration
trapping
elongation
PNAS 110, 80308035 (2013)
Create a crowdedEnvironment
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Optical Driven Trap
Create a crowdedEnvironment
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Optical Driven Trap
Create a crowdedEnvironment
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ChristofMast
Length Concentration
trapping
elongation
PNAS 110, 80308035 (2013)
Create a crowdedEnvironment
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Dynamic Gel in Thermophoretic Trap
100µm
(unpublished data)
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Dynamic Gel in Thermophoretic Trap
(unpublished data)
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Gel only forms with sticky ends
(unpublished data)
therm
oph
ore
tic
trap
pin
g
PNAS2013
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Sequence Sorting in Gel
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Sequence Sorting in Gel
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Sequence Sorting in Gel
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Replication of Genetic Information
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Replication of Genetic Information
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Tyranny ofthe Shortest
Replication of Genetic Information
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Selection …
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Selection …
Nature Chemistry (2015) doi:10.1038/nchem.2155
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Selection …
Nature Chemistry (2015) doi:10.1038/nchem.2155
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Selection and Replication
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Mast & Braun, PRL, 104, 188102 (2010)
Selection and Replication
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Feeding
Selection and Replication
Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015
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Selection and Replication
Feeding
Tyranny ofthe shortest
Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015
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Selection and Replication
Selection of the Largest!
Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015
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Selection and Replication
Setting looks like an Evolution Machine ?Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015
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Accumulation Replication
PNAS 2006, PNAS 2007 PRL 2002, PRL 2010
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Polymerization Gelation
PNAS 2013 submitted
No trap trap
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Selection Translation
PRL 2012Nature Chemistry 2015
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Earl
y E
art
h
Life
SimonsFoundation
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