Biophysics of Macromolecules - uni-muenchen.de · Biophysics of Macromolecules 6. July 2015. Crowding alters Biochemical Equilibria Crowding alters the ... Energy Transfer (FRET)

How to create methods to probe macromoleculesin vivo ?

Lecture 18: In vivo Methods

Braun/Lipfert SS 2015

Biophysics of Macromolecules

6. July 2015

Crowding alters Biochemical Equilibria

Crowding alters the Kinetics within Cells

Excluded volume forces Binding statistics

Slowing of diffusion

ThermophoresisMolecules in a temperature gradient





Thermophoresis

Thermophoresis

Why does the steady state depletion say something about the probability of binding?

=> Two State model (black board)

Philipp Baaske; Stefan Duhr

Philipp Baaske; Stefan Duhr

Publications using Thermophoresis in 2014

Thermophoretic Immunology?

Analytical Chemistry 84, 3523–3530 (2012)





Basis of Thermophoresis

Local Equilibrium

Local Equilibrium

Local Equilibrium

Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)

Local Equilibrium


Local Equilibrium

Capacitor


Local Equilibrium

Capacitor Seebeck


Capacitor

Seebeck Contribution


Seebeck

Capacitor

Seebeck Contribution


Multiwell-Plates

Angewandte Chemie53, 7948–7951 (2014)




Problem: Need to hit center otherwise droplet moves

away from the focus

Inside Living Cells

Cell Cell

Lower thermophoretic mobility and slower diffusion

Reaction Kinetics in a Cell

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)

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

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

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

Temperature Reference

Complex Fit with Transfer Functionfor a first-order transition

Calibration of Cy5 Dye

Intracellular Delivery

Spatial Temperature Kinetics

10µm 10µm

Simulated Temperature Characteristics

finite element simulation- solve Fourier-transformed heat conduction equations (FEMLAB)- transfer function fit as in experiment

calibration against temperature kineticsof the measurement chamber

Data Analysis and Interpretation

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

DNA Hybridization Kinetics in vivo

f = 1...200Hz

10 µm

Donor and FRET Signals yielded consistent Time Constants.

10 µm


f = 1...200Hz

10 µm


10 µm


f = 1...200Hz

10 µm


10 µm

Reaction Speed in Cellular Compartments

Hybridization Reaction inside Nucleus was faster than in Cytoplasm.

10 µm10 µm

Kinetics in vivo versus in vitro

PBS solution

1 4of DNA on ofkckkτ− =+

ACCELERATION


PBS solution


ACCELERATION


PBS solution


ACCELERATION SLOWING DOWN

Effects of Divalent Ions and Crowding Agents in vitro

Mg2+ ACCELERATED the kinetics

Crowding DID NOT CHANGE the kinetics

30% (w/v)

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?

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

Viability of Cells

Calibration Dye Brightness

possible quenchingemission senesitivity to 5 mM glutathione and 0.2 mM ascorbic acid

solution of known concentrationsmulti-point confocal images

Reaction Amplitude

Subcellular Resolution

scale bars: 5 µm

Which Effect Is Probe‐Specific?

Can we recreateautonomous

Darwinian Evolution(a.k.a. Life)in the lab ?

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 ?

Life as we know it

Life as we know it- Replication

Life as we know it- Replication of Genetic Information

Life as we know itReplication of Genetic Information ...

… to create Proteins from Genes ...



… in a crowded Soup of Nutrients ...



… in a crowded Soup of Nutrients ...… far from Equilibrium.



… in a crowded Soup of Nutrients ...

The big Puzzleof Biogenesis

… far from Equilibrium.

Thermal Molecule Traps

Thermal Trap

Thermal Trap

Thermal TrapAccumulation of 100-1000bp DNA

Light driven Microfluidicsto drive a thermal trap

Light driven Microfluidics

PRL 100, 164501 (2008); JAP 104, 104701 (2008)

Light driven Microfluidics

PRL 100, 164501 (2008); JAP 104, 104701 (2008)

Create a crowdedEnvironment

ChristofMast

Length Concentration

trapping

elongation

PNAS 110, 80308035 (2013)


PolymerizationMachine

ChristofMast


trapping

elongation

PNAS 110, 80308035 (2013)


Optical Driven Trap


Optical Driven Trap


ChristofMast


trapping

elongation

PNAS 110, 80308035 (2013)


Dynamic Gel in Thermophoretic Trap

100µm

(unpublished data)

Dynamic Gel in Thermophoretic Trap

(unpublished data)

Gel only forms with sticky ends

(unpublished data)

therm

oph

ore

tic

trap

pin

g

PNAS2013

Sequence Sorting in Gel



Replication of Genetic Information


Tyranny ofthe Shortest


Selection …

Selection …

Nature Chemistry (2015) doi:10.1038/nchem.2155

Selection …

Nature Chemistry (2015) doi:10.1038/nchem.2155

Selection and Replication

Mast & Braun, PRL, 104, 188102 (2010)


Feeding


Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015


Feeding

Tyranny ofthe shortest



Selection of the Largest!



Setting looks like an Evolution Machine ?Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015

Accumulation Replication

PNAS 2006, PNAS 2007 PRL 2002, PRL 2010

Polymerization Gelation

PNAS 2013 submitted

No trap trap

Selection Translation

PRL 2012Nature Chemistry 2015

Earl

y E

art

h

Life

SimonsFoundation

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Biophysics of Macromolecules - uni-muenchen.de · Biophysics of Macromolecules 6. July 2015. Crowding alters Biochemical Equilibria Crowding alters the ... Energy Transfer (FRET)