Biophysics of Macromolecules - PDF Free Download

Biophysics of Macromolecules Lecture 18: In vivo Methods Braun/Lipfert SS 2015 How to create methods to probe macromolecules in vivo? 6. July 2015

Crowding alters Biochemical Equilibria Excluded volume forces Binding statistics Crowding alters the Kinetics within Cells Slowing of diffusion

Thermophoresis Molecules in a temperature gradient

Thermophoresis

Why does the steady state depletion say something about the probability of binding? => Two State model (black board)

Philipp Baaske; Stefan Duhr

Publications using Thermophoresis in 2014

Thermophoretic Immunology? Analytical Chemistry 84, 3523 3530 (2012)

Basis of Thermophoresis

Local Equilibrium

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

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

Local Equilibrium Capacitor Seebeck Reichl, Herzog, Götz, and Braun, PRL 112, 198101 (2014)

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

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

Multiwell-Plates

Angewandte Chemie 53, 7948 7951 (2014)

Problem: Need to hit center otherwise droplet moves away from the focus Angewandte Chemie 53, 7948 7951 (2014)

Inside Living Cells

Cell Cell

Lower thermophoretic mobility and slower diffusion

Reaction Kinetics in a Cell

Hybridization Kinetics Is Different Inside Cells Proc. Natl. Acad. Sci. USA 106: 21649-21654 (2009) 10µm Ingmar Schön, Hubert Krammer and Dieter Braun Systems Biophysics, LMU München, Germany 54th Annual Meeting of the Biophysical Society San Francisco, California February 21, 2010

How fast do DNA Stands find each other inside a Cell? Molecular Crowding Minton, J.Cell Sci., 2006 Specific Interactions (w/ Proteins) Sugiyama et al., PNAS, 1998

Experimental Approach Principle: Perturbe Equilibrium Analyze Relaxation Detection:Fluorescence Resonance Energy Transfer (FRET) TOOL Microscopy Delivery: Lipofection into HeLa Cells (Temperature Oscillation Optical Lock-In) Braun & Libchaber, Appl. Phys. Lett., 2003

Data Analysis and Interpretation stroboscopic illumination phase-locked relative to perturbation quantum efficiency illumination 0 90 collect fluorescence by slow CCD (low-pass filtering) 180 270 fit with transfer function for a first-order transition

Temperature Reference Calibration of Cy5 Dye Complex Fit with Transfer Function for a first-order transition 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

Data Analysis and Interpretation calibration against temperature kinetics of the measurement chamber

Preparation Procedure microscope slides - chromium layer for IR adsorption - silicon substrate for fast cooling cell culture - sterilization - surface coating - cell seeding measurement chamber poly-d-lysine

DNA Hybridization Kinetics in vivo f = 1...200Hz 10 µm 10 µm Donor and FRET Signals yielded consistent Time Constants.

Reaction Speed in Cellular Compartments 10 µm 10 µm Hybridization Reaction inside Nucleus was faster than in Cytoplasm.

Kinetics in vivo versus in vitro 4 τ 1 =+ kckk of PBS solution ACCELERATION DNA on of

Kinetics in vivo versus in vitro 4 τ 12=+ kckk of PBS solution ACCELERATION DNA on of

Kinetics in vivo versus in vitro 4 τ 12=+ kckk of DNA on of PBS solution ACCELERATION SLOWING DOWN

Effects of Divalent Ions and Crowding Agents in vitro 30% (w/v) Mg2+ ACCELERATED the kinetics Crowding DID NOT CHANGE the kinetics

Possible Origin of Different Kinetics in vivo Reasons for ACCELERATION Reasons for DECELERATION - Crowding (Excluded Volume): not observed in vitro - Crowding (Hindered Diffusion): not observed in vitro - Recombination Mediator Proteins - Background Interactions: (e.g. Rad52) Stationary Buffering of ssdna or dsdna - High Divalent Ion Concentrations by DNA-Binding Proteins (unlikely) (Reduced Effective Concentration) - Background Hybridization with RNA/DNA (Enhanced Effective Concentration): Inconsistent with Qualitative Trend OPEN QUESTIONS - Which Proteins are involved? - Which Effect is Probe Specific? - What Mediates Specifity: Oligo Length? Sequence?

DNA Probe Construct Intracellular Delivery R hg 5 -C A G G TTA C TA TC G TAT T C -3 ROX 5 -C A AT A C G ATA G TA A C C T C -3 C = L-enantiomeric cytosin Melting Characteristics excitation at 488nm scale bars: 10 µm

Viability of Cells

Calibration Dye Brightness solution of known concentrations multi-point confocal images possible quenching emission senesitivity to 5 mm glutathione and 0.2 mm ascorbic acid

Reaction Amplitude

Subcellular Resolution scale bars: 5 µm

Which Effect Is Probe Specific?

Can we recreate autonomous Darwinian Evolution (a.k.a. Life) in the lab?

Can we recreate autonomous Darwinian Evolution (a.k.a. Life) in the lab? Let's try it! Hard puzzles are best approached by doing experiments to test hypothesis

Life as we know it

Life as we know it - Replication

Life as we know it - Replication of Genetic Information

Life as we know it Replication of Genetic Information... to create Proteins from Genes...

Life as we know it Replication of Genetic Information... to create Proteins from Genes... in a crowded Soup of Nutrients...

Life as we know it Replication of Genetic Information... to create Proteins from Genes... in a crowded Soup of Nutrients... far from Equilibrium.

Life as we know it Replication of Genetic Information... to create Proteins from Genes... in a crowded Soup of Nutrients... far from Equilibrium. The big Puzzle of Biogenesis

Thermal Molecule Traps

Thermal Trap

Thermal Trap Accumulation of 100-1000bp DNA

Light driven Microfluidics to drive a thermal trap

Light driven Microfluidics PRL 100, 164501 (2008); JAP 104, 104701 (2008)

Create a crowded Environment

Create a crowded Environment elongation Length Christof Mast Concentration trapping Polymerization Machine PNAS 110, 8030 8035 (2013)

Create a crowded Environment elongation Length Concentration trapping PNAS 110, 8030 8035 (2013) Christof Mast

Create a crowded Environment Optical Driven Trap

Create a crowded Environment elongation Length Concentration trapping PNAS 110, 8030 8035 (2013) Christof Mast

Dynamic Gel in Thermophoretic Trap 100µm (unpublished data)

Dynamic Gel in Thermophoretic Trap (unpublished data)

PNAS 2013 thermophoretic trapping Gel only forms with sticky ends (unpublished data)

Sequence Sorting in Gel

Replication of Genetic Information

Replication of Genetic Information Tyranny of the Shortest

Selection

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

Selection and Replication

Selection and Replication Mast & Braun, PRL, 104, 188102 (2010)

Selection and Replication Feeding Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015

Selection and Replication Tyranny of the shortest Feeding Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015

Selection and Replication Selection of the Largest! Kreysing, Keil, Lanzmich & Braun, Nature Chemistry 2015

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

Accumulation PNAS 2006, PNAS 2007 Replication PRL 2002, PRL 2010

Polymerization No trap Gelation trap PNAS 2013 submitted

Selection Nature Chemistry 2015 Translation PRL 2012

Life Early Earth Simons Foundation