Biophysics of Macromolecules

Transcription

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

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

3 Thermophoresis Molecules in a temperature gradient

4 Thermophoresis Molecules in a temperature gradient

5 Thermophoresis Molecules in a temperature gradient

6 Thermophoresis Molecules in a temperature gradient

7 Thermophoresis Molecules in a temperature gradient

8 Thermophoresis

9 Thermophoresis

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

15 Philipp Baaske; Stefan Duhr

16 Philipp Baaske; Stefan Duhr

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22 Publications using Thermophoresis in 2014

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29 Thermophoretic Immunology? Analytical Chemistry 84, (2012)

30 Thermophoretic Immunology? Analytical Chemistry 84, (2012)

31 Thermophoretic Immunology? Analytical Chemistry 84, (2012)

32 Basis of Thermophoresis

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34 Local Equilibrium

35 Local Equilibrium

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

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

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

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

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

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

42 Multiwell-Plates

43 Angewandte Chemie 53, (2014)

44 Angewandte Chemie 53, (2014)

45 Angewandte Chemie 53, (2014)

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

47 Inside Living Cells

48 Cell Cell

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50 Lower thermophoretic mobility and slower diffusion

51 Reaction Kinetics in a Cell

52 Hybridization Kinetics Is Different Inside Cells Proc. Natl. Acad. Sci. USA 106: (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

53 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

54 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

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

56 Temperature Reference Calibration of Cy5 Dye Complex Fit with Transfer Function for a first-order transition Intracellular Delivery Spatial Temperature Kinetics 10µm 10µm

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

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

59 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

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

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

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

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

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

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

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

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

68 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?

69 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

70 Viability of Cells

71 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

72 Reaction Amplitude

73 Subcellular Resolution scale bars: 5 µm

74 Which Effect Is Probe Specific?

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

76 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

77 Life as we know it

78 Life as we know it - Replication

79 Life as we know it - Replication of Genetic Information

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

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

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

83 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

84 Thermal Molecule Traps

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86 Thermal Trap

87 Thermal Trap

88 Thermal Trap Accumulation of bp DNA

89 Light driven Microfluidics to drive a thermal trap

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

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

92 Create a crowded Environment

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

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

95 Create a crowded Environment Optical Driven Trap

96 Create a crowded Environment Optical Driven Trap

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

98 Dynamic Gel in Thermophoretic Trap 100µm (unpublished data)

99 Dynamic Gel in Thermophoretic Trap (unpublished data)

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

101 Sequence Sorting in Gel

102 Sequence Sorting in Gel

103 Sequence Sorting in Gel

104 Replication of Genetic Information

105 Replication of Genetic Information

106 Replication of Genetic Information Tyranny of the Shortest

107 Selection

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

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

110 Selection and Replication

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

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

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

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

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

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

117 Polymerization No trap Gelation trap PNAS 2013 submitted

118 Selection Nature Chemistry 2015 Translation PRL 2012

119 Life Early Earth Simons Foundation

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