Physics of bio-molecules and cells = Physique des biomolécules et des cellules : Les Houches, Session LXXV, 2-27 July 2001

Aimed at those working to enter this rapidly developing field, this volume on biological physics is written in a pedagogical style by leading scientists giving explanations that take their starting point where any physicist can follow and end at the frontier of research in biological physics. These lectures describe the state-of-the-art physics of biomolecules and cells. In biological systems ranging from single biomolecules to entire cells and larger biological systems, it focuses on aspects that require concepts and methods from physics for their analysis and understanding, such as the mechanics of motor proteins; how the genetic code is physically read and managed; the machinery of protein - DNA interactions; force spectroscopy of biomolecules' velopes, cytoskeletons, and cytoplasms; polymerization forces; listeria propulsion; cell motility; lab-on-a-chip nanotechnology for single-molecule analysis of biomolecules; bioinformatics; and coding and computational strategies of the brain. 01.pdf -1 01.pdf 1 Contents 15 1 Introduction 16 1.1 The central dogma and bacterial gene expression 16 1.1.1 Two families 16 1.1.2 Prokaryote gene expression 18 1.2 Molecular structure 21 1.2.1 Chemical structure of DNA 21 1.2.2 Physical structure of DNA 23 1.2.3 Chemical structure of proteins 25 1.2.4 Physical structure of proteins 27 2 Thermodynamics and kinetics of repressor-DNA interaction 29 2.1 Thermodynamics and the lac repressor 29 2.1.1 The law of mass action 29 2.1.2 Statistical mechanics and operator occupancy 32 2.1.3 Entropy, enthalpy, and direct read-out 33 2.1.4 The lac repressor complex: A molecular machine 36 2.2 Kinetics of repressor-DNA interaction 39 2.2.1 Reaction kinetics 39 2.2.2 Debye–Smoluchowski theory 41 2.2.3 BWH theory 43 2.2.4 Indirect read-out and induced fit 45 3 DNA deformability and protein-DNA interaction 47 3.1 Introduction 47 3.1.1 Eukaryotic gene expression and Chromatin condensation 47 3.1.2 A mathematical experiment and White’s theorem 50 3.2 The worm-like chain 53 3.2.1 Circular DNA and the persistence length 55 3.2.2 Nucleosomes and the Marky–Manning transition 55 3.2.3 Protein-DNA interaction under tension 58 3.2.4 Force-Extension Curves 60 3.3 The RST model 63 3.3.1 Structural sequence sensitivity 63 3.3.2 Thermal fluctuations 65 4 Electrostatics in water and protein-DNA interaction 66 4.1 Macro-ions and aqueous electrostatics 67 4.2 The primitive model 69 4.2.1 The primitive model: Ion-free 70 4.2.2 The primitive model: DH regime 70 4.3 Manning condensation 71 4.3.1 Charge renormalization 71 4.3.2 Primitive model: Oosawa theory 72 4.3.3 Primitive model: Free energy 74 4.4 Counter-ion release and non-specific protein-DNA interaction 76 4.4.1 Counter-ion release 76 4.4.2 Nucleosome formation and the isoelectric instability 77 References 80 02.pdf 1 Contents 83 1 Introduction 84 2 Cell motility and motor proteins 85 3 Motility assays 86 4 Single-molecules assays 88 5 Atomic structures 90 6 Proteins as machines 91 7 Chemical forces 93 8 Effect of force on chemical equilibria 94 9 Effect of force on the rates of chemical reactions 95 10 Absolute rate theories 98 11 Role of thermal fluctuations in motor reactions 100 12 A mechanochemical model for kinesin 102 13 Conclusions and outlook 105 03.pdf 1 Contents 109 1 Making a move: Principles of energy transduction 111 1.1 Motor proteins and Carnot engines 111 1.2 Simple Brownian ratchet 112 1.3 Polymerization ratchet 113 1.4 Isothermal ratchets 116 1.5 Motor proteins as isothermal ratchets 117 1.6 Design principles for e.ective motors 118 2 Pulling together: Mechano-chemical model of actomyosin 121 2.1 Swinging lever-arm model 121 2.2 Mechano-chemical coupling 123 2.3 Equivalent isothermal ratchet 124 2.4 Many motors working together 125 2.5 Designed to work 128 2.6 Force-velocity relation 129 2.7 Dynamical instability and biochemical synchronization 131 2.8 Transient response of muscle 132 3 Motors at work: Collective properties of motor proteins 132 3.1 Dynamical instabilities 132 3.2 Bidirectional movement 133 3.3 Critical behaviour 134 3.4 Oscillations 137 3.5 Dynamic buckling instability 138 3.6 Undulation of flagella 140 4 Sense and sensitivity: Mechano-sensation in hearing 142 4.1 System performance 142 4.2 Mechano-sensors: Hair bundles 143 4.3 Active amplification 144 4.4 Self-tuned criticality 146 4.5 Motor-driven oscillations 147 4.6 Channel compliance and relaxation oscillations 149 4.7 Channel-driven oscillations 151 4.8 Hearing at the noise limit 152 04.pdf 1 Contents 158 Part 1: 159 1 Dynamic force spectroscopy. I. Single bonds 159 1.1 Introduction 159 1.1.1 Intrinsic dependence of bond strength on time frame for breakage 160 1.1.2 Biomolecular complexity and role for dynamic force spectroscopy 160 1.1.3 Biochemical and mechanical perspectives of bond strength 162 1.1.4 Relevant scales for length, force, energy, and time 165 1.2 Brownian kinetics in condensed liquids: Old-time physics 166 1.2.1 Two-state transitions in a liquid 167 1.2.2 Kinetics of first-order reactions in solution 168 1.3 Link between force – time – and bond chemistry 170 1.3.1 Dissociation of a simple bond under force 170 1.3.2 Dissociation of a complex bond under force: Stationary rate approximation 171 1.3.3 Evolution of states in complex bonds 175 1.4 Testing bond strength and the method of dynamic force spectroscopy 176 1.4.1 Probe mechanics and bond loading dynamics 177 1.4.2 Stochastic process of bond failure under rising force 180 1.4.3 Distributions of bond lifetime and rupture force 181 1.4.4 Crossover from near equilibrium to far from equilibrium unbonding 184 1.4.5 Effect of soft-polymer linkages on dynamic strengths of bonds 187 1.4.6 Failure of a complex bond and unexpected transitions in strength 189 1.5 Summary 197 References 198 Part 2: 199 2 Dynamic force spectroscopy. II. Multiple bonds 199 2.1 Hidden mechanics in detachment of multiple bonds 199 2.2 Impact of cooperativity 200 2.3 Uncorrelated failure of bonds loaded in series 203 2.3.1 Markov sequence of random failures 203 2.3.2 Multiple-complex bonds 205 2.3.3 Multiple-ideal bonds 206 2.3.4 Equivalent single-bond approximation 207 2.4 Uncorrelated failure of bonds loaded in parallel 210 2.4.1 Markov sequence of random failures 210 2.4.2 Equivalent single-bond approximation 210 2.5 Poisson statistics and bond formation 211 2.6 Summary 215 References 216 06.pdf 1 Contents 227 1 Introduction 228 2 A genuine gel 229 2.1 A little chemistry 229 2.2 Elastic behaviour 231 3 Hydrodynamics and mechanics 231 3.1 Motion in the laboratory frame 231 3.2 Propulsion and steady velocity regimes 232 3.3 Gel/bacterium friction and saltatory behaviour 234 4 Biomimetic approach 236 4.1 A spherical Listeria 236 4.2 Spherical symmetry 237 4.3 Steady state 238 4.4 Growth with spherical symmetry 240 4.5 Symmetry breaking 240 4.6 Limitations of the approach and possible improvements 242 5 Conclusion 245 References 246 07.pdf 1 Contents 249 1 Architecture of composite cell membranes 250 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture 250 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer 253 1.3 The actin cortex: Architecture and function 256 2 Physics of the actin based cytoskeleton 260 2.1 Actin is a living semiflexible polymer 260 2.2 Actin network as viscoelastic body 264 2.3 Correlation between macroscopic viscoelasticity and molecular motional processes 269 3 Heterogeneous actin gels in cells and biological function 271 3.1 Manipulation of actin gels 271 3.2 Control of organization and function of actin cortex by cell signalling 276 4 Micromechanics and microrheometry of cells 278 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways 282 6 On cells as adaptive viscoplastic bodies 285 7 Controll of cellular protrusions controlled by actin/myosin cortex 289 References 293 08.pdf 1 Contents 297 1 Introduction 298 2 Mimicking cell adhesion 303 3 Microinterferometry: A versatile tool to evaluate adhesion strength and forces 305 4 Soft shell adhesion is controlled by a double well interfacial potential 305 5 How is adhesion controlled by membrane elasticity? 308 6 Measurement of adhesion strength by interferometric contour analysis 310 7 Switching on specific forces: Adhesion as localized dewetting process 311 8 Measurement of unbinding forces, receptor-ligand leverage and a new role for stress fibers 311 9 An application: Modification of cellular adhesion strength by cytoskeletal mutations 314 10 Conclusions 314 A Appendix: Generic interfacial forces 315 References 319 09.pdf 1 Contents 322 1 Why micro/nanofabrication? 324 Lecture 1a: Hydrodynamic Transport 328 1 Introduction: The need to control flows in 2 1/2 D 328 2 Somewhat simple hydrodynamics in 2 1/2 D 330 3 The N-port injector idea 337 4 Conclusion 342 References 342 Lecture 1b: Dielectrophoresis and Microfabrication 344 1 Introduction 344 2 Methods 346 2.1 Fabrication 346 2.2 Viscosity 347 2.3 Electronics and imaging 347 2.4 DNA samples 347 3 Results 348 3.1 Basic results and dielectrophoretic force extraction 348 4 Data and analysis 352 5 Origin of the low frequency dielectrophoretic force in DNA 356 6 Conclusion 362 References 363 Lecture 2a: Hex Arrays 365 1 Introduction 365 2 Experimental approach 369 3 Conclusions 373 References 374 Lecture 2b: The DNA Prism 375 1 Introduction 375 2 Design 375 3 Results 376 4 Conclusions 381 References 382 Lecture 2c: Bigger is Better in Rachets 383 1 The problems with insulators in rachets 383 2 An experimental test 384 3 Conclusions 390 References 390 Lecture 3: Going After Epigenetics 391 1 Introduction 391 2 The nearfield scanner 392 3 The chip 393 4 Experiments with molecules 396 5 Conclusions 400 References 400 Lecture 4: Fractionating Cells 401 1 Introduction 401 2 Blood specifics 401 3 Magnetic separation 406 4 Microfabrication 407 5 Magnetic field gradients 408 6 Device interface 410 7 A preliminary blood cell run 415 8 Conclusions 418 References 419 Lecture 5: Protein Folding on a Chip 420 1 Introduction 420 2 Technology 421 3 Experiments 424 4 Conclusions 427 References 427 10.pdf 1 Contents 430 1 Introduction 431 2 New technologies 433 3 Sequence comparison 435 4 Clustering 438 5 Gene regulation 440 References 441 11.pdf 1 Contents 444 1 Enzymatic networks. Proofreading knots: How DNA topoisomerases disentangle DNA 446 1.1 Length scales and energy scales 447 1.2 DNA topology 448 1.3 Topoisomerases 449 1.4 Knots and supercoils 452 1.5 Topological equilibrium 454 1.6 Can topoisomerases recognize topology? 455 1.7 Proposal: Kinetic proofreading 456 1.8 How to do it twice 457 1.9 The care and proofreading of knots 459 1.10 Suppression of supercoils 461 1.11 Problems and outlook 463 1.12 Disquisition 465 2 Gene expression networks. Methods for analysis of DNA chip experiments 465 2.1 The regulation of gene expression 465 2.2 Gene expression arrays 468 2.3 Analysis of array data 471 2.4 Some simplifying assumptions 472 2.5 Probeset analysis 474 2.6 Discussion 478 3 Neural and gene expression networks: Song-induced gene expression in the canary brain 479 3.1 The study of songbirds 480 3.2 Canary song 481 3.3 ZENK 482 3.4 The blush 484 3.5 Histological analysis 484 3.6 Natural vs. artificial 487 3.7 The Blush II: gAP 488 3.8 Meditation 489 References 490 12.pdf 1 Contents 493 1 Introduction 494 2 Photon counting 498 3 Optimal performance at more complex tasks 508 4 Toward a general principle? 525 5 Learning and complexity 545 6 A little bit about molecules 559 7 Speculative thoughts about the hard problems 571 References 580 12.pdf......Page 0 Contents......Page 15 1.1.1 Two families......Page 16 1.1.2 Prokaryote gene expression......Page 18 1.2.1 Chemical structure of DNA......Page 21 1.2.2 Physical structure of DNA......Page 23 1.2.3 Chemical structure of proteins......Page 25 1.2.4 Physical structure of proteins......Page 27 2.1.1 The law of mass action......Page 29 2.1.2 Statistical mechanics and operator occupancy......Page 32 2.1.3 Entropy, enthalpy, and direct read-out......Page 33 2.1.4 The lac repressor complex: A molecular machine......Page 36 2.2.1 Reaction kinetics......Page 39 2.2.2 Debye–Smoluchowski theory......Page 41 2.2.3 BWH theory......Page 43 2.2.4 Indirect read-out and induced fit......Page 45 3.1.1 Eukaryotic gene expression and Chromatin condensation......Page 47 3.1.2 A mathematical experiment and White’s theorem......Page 50 3.2 The worm-like chain......Page 53 3.2.2 Nucleosomes and the Marky–Manning transition......Page 55 3.2.3 Protein-DNA interaction under tension......Page 58 3.2.4 Force-Extension Curves......Page 60 3.3.1 Structural sequence sensitivity......Page 63 3.3.2 Thermal fluctuations......Page 65 4 Electrostatics in water and protein-DNA interaction......Page 66 4.1 Macro-ions and aqueous electrostatics......Page 67 4.2 The primitive model......Page 69 4.2.2 The primitive model: DH regime......Page 70 4.3.1 Charge renormalization......Page 71 4.3.2 Primitive model: Oosawa theory......Page 72 4.3.3 Primitive model: Free energy......Page 74 4.4.1 Counter-ion release......Page 76 4.4.2 Nucleosome formation and the isoelectric instability......Page 77 References......Page 80 Contents......Page 83 1 Introduction......Page 84 2 Cell motility and motor proteins......Page 85 3 Motility assays......Page 86 4 Single-molecules assays......Page 88 5 Atomic structures......Page 90 6 Proteins as machines......Page 91 7 Chemical forces......Page 93 8 Effect of force on chemical equilibria......Page 94 9 Effect of force on the rates of chemical reactions......Page 95 10 Absolute rate theories......Page 98 11 Role of thermal fluctuations in motor reactions......Page 100 12 A mechanochemical model for kinesin......Page 102 13 Conclusions and outlook......Page 105 Contents......Page 109 1.1 Motor proteins and Carnot engines......Page 111 1.2 Simple Brownian ratchet......Page 112 1.3 Polymerization ratchet......Page 113 1.4 Isothermal ratchets......Page 116 1.5 Motor proteins as isothermal ratchets......Page 117 1.6 Design principles for e.ective motors......Page 118 2.1 Swinging lever-arm model......Page 121 2.2 Mechano-chemical coupling......Page 123 2.3 Equivalent isothermal ratchet......Page 124 2.4 Many motors working together......Page 125 2.5 Designed to work......Page 128 2.6 Force-velocity relation......Page 129 2.7 Dynamical instability and biochemical synchronization......Page 131 3.1 Dynamical instabilities......Page 132 3.2 Bidirectional movement......Page 133 3.3 Critical behaviour......Page 134 3.4 Oscillations......Page 137 3.5 Dynamic buckling instability......Page 138 3.6 Undulation of flagella......Page 140 4.1 System performance......Page 142 4.2 Mechano-sensors: Hair bundles......Page 143 4.3 Active amplification......Page 144 4.4 Self-tuned criticality......Page 146 4.5 Motor-driven oscillations......Page 147 4.6 Channel compliance and relaxation oscillations......Page 149 4.7 Channel-driven oscillations......Page 151 4.8 Hearing at the noise limit......Page 152 Contents......Page 158 1.1 Introduction......Page 159 1.1.2 Biomolecular complexity and role for dynamic force spectroscopy......Page 160 1.1.3 Biochemical and mechanical perspectives of bond strength......Page 162 1.1.4 Relevant scales for length, force, energy, and time......Page 165 1.2 Brownian kinetics in condensed liquids: Old-time physics......Page 166 1.2.1 Two-state transitions in a liquid......Page 167 1.2.2 Kinetics of first-order reactions in solution......Page 168 1.3.1 Dissociation of a simple bond under force......Page 170 1.3.2 Dissociation of a complex bond under force: Stationary rate approximation......Page 171 1.3.3 Evolution of states in complex bonds......Page 175 1.4 Testing bond strength and the method of dynamic force spectroscopy......Page 176 1.4.1 Probe mechanics and bond loading dynamics......Page 177 1.4.2 Stochastic process of bond failure under rising force......Page 180 1.4.3 Distributions of bond lifetime and rupture force......Page 181 1.4.4 Crossover from near equilibrium to far from equilibrium unbonding......Page 184 1.4.5 Effect of soft-polymer linkages on dynamic strengths of bonds......Page 187 1.4.6 Failure of a complex bond and unexpected transitions in strength......Page 189 1.5 Summary......Page 197 References......Page 198 2.1 Hidden mechanics in detachment of multiple bonds......Page 199 2.2 Impact of cooperativity......Page 200 2.3.1 Markov sequence of random failures......Page 203 2.3.2 Multiple-complex bonds......Page 205 2.3.3 Multiple-ideal bonds......Page 206 2.3.4 Equivalent single-bond approximation......Page 207 2.4.2 Equivalent single-bond approximation......Page 210 2.5 Poisson statistics and bond formation......Page 211 2.6 Summary......Page 215 References......Page 216 Contents......Page 227 1 Introduction......Page 228 2.1 A little chemistry......Page 229 3.1 Motion in the laboratory frame......Page 231 3.2 Propulsion and steady velocity regimes......Page 232 3.3 Gel/bacterium friction and saltatory behaviour......Page 234 4.1 A spherical Listeria......Page 236 4.2 Spherical symmetry......Page 237 4.3 Steady state......Page 238 4.5 Symmetry breaking......Page 240 4.6 Limitations of the approach and possible improvements......Page 242 5 Conclusion......Page 245 References......Page 246 Contents......Page 249 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture......Page 250 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer......Page 253 1.3 The actin cortex: Architecture and function......Page 256 2.1 Actin is a living semiflexible polymer......Page 260 2.2 Actin network as viscoelastic body......Page 264 2.3 Correlation between macroscopic viscoelasticity and molecular motional processes......Page 269 3.1 Manipulation of actin gels......Page 271 3.2 Control of organization and function of actin cortex by cell signalling......Page 276 4 Micromechanics and microrheometry of cells......Page 278 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways......Page 282 6 On cells as adaptive viscoplastic bodies......Page 285 7 Controll of cellular protrusions controlled by actin/myosin cortex......Page 289 References......Page 293 Contents......Page 297 1 Introduction......Page 298 2 Mimicking cell adhesion......Page 303 4 Soft shell adhesion is controlled by a double well interfacial potential......Page 305 5 How is adhesion controlled by membrane elasticity?......Page 308 6 Measurement of adhesion strength by interferometric contour analysis......Page 310 8 Measurement of unbinding forces, receptor-ligand leverage and a new role for stress fibers......Page 311 10 Conclusions......Page 314 A Appendix: Generic interfacial forces......Page 315 References......Page 319 Contents......Page 322 1 Why micro/nanofabrication?......Page 324 1 Introduction: The need to control flows in 2 1/2 D......Page 328 2 Somewhat simple hydrodynamics in 2 1/2 D......Page 330 3 The N-port injector idea......Page 337 References......Page 342 1 Introduction......Page 344 2.1 Fabrication......Page 346 2.4 DNA samples......Page 347 3.1 Basic results and dielectrophoretic force extraction......Page 348 4 Data and analysis......Page 352 5 Origin of the low frequency dielectrophoretic force in DNA......Page 356 6 Conclusion......Page 362 References......Page 363 1 Introduction......Page 365 2 Experimental approach......Page 369 3 Conclusions......Page 373 References......Page 374 2 Design......Page 375 3 Results......Page 376 4 Conclusions......Page 381 References......Page 382 1 The problems with insulators in rachets......Page 383 2 An experimental test......Page 384 References......Page 390 1 Introduction......Page 391 2 The nearfield scanner......Page 392 3 The chip......Page 393 4 Experiments with molecules......Page 396 References......Page 400 2 Blood specifics......Page 401 3 Magnetic separation......Page 406 4 Microfabrication......Page 407 5 Magnetic field gradients......Page 408 6 Device interface......Page 410 7 A preliminary blood cell run......Page 415 8 Conclusions......Page 418 References......Page 419 1 Introduction......Page 420 2 Technology......Page 421 3 Experiments......Page 424 References......Page 427 Contents......Page 430 1 Introduction......Page 431 2 New technologies......Page 433 3 Sequence comparison......Page 435 4 Clustering......Page 438 5 Gene regulation......Page 440 References......Page 441 Contents......Page 444 1 Enzymatic networks. Proofreading knots: How DNA topoisomerases disentangle DNA......Page 446 1.1 Length scales and energy scales......Page 447 1.2 DNA topology......Page 448 1.3 Topoisomerases......Page 449 1.4 Knots and supercoils......Page 452 1.5 Topological equilibrium......Page 454 1.6 Can topoisomerases recognize topology?......Page 455 1.7 Proposal: Kinetic proofreading......Page 456 1.8 How to do it twice......Page 457 1.9 The care and proofreading of knots......Page 459 1.10 Suppression of supercoils......Page 461 1.11 Problems and outlook......Page 463 2.1 The regulation of gene expression......Page 465 2.2 Gene expression arrays......Page 468 2.3 Analysis of array data......Page 471 2.4 Some simplifying assumptions......Page 472 2.5 Probeset analysis......Page 474 2.6 Discussion......Page 478 3 Neural and gene expression networks: Song-induced gene expression in the canary brain......Page 479 3.1 The study of songbirds......Page 480 3.2 Canary song......Page 481 3.3 ZENK......Page 482 3.5 Histological analysis......Page 484 3.6 Natural vs. artificial......Page 487 3.7 The Blush II: gAP......Page 488 3.8 Meditation......Page 489 References......Page 490 Contents......Page 493 1 Introduction......Page 494 2 Photon counting......Page 498 3 Optimal performance at more complex tasks......Page 508 4 Toward a general principle?......Page 525 5 Learning and complexity......Page 545 6 A little bit about molecules......Page 559 7 Speculative thoughts about the hard problems......Page 571 References......Page 580