Handbook of Single-Molecule Biophysics (eBook)

Preface 5
Contents 8
List of Contributors 18
1 Single-Molecule Fluorescent Particle Tracking 21
1.1 The History of Single-Particle Tracking 21
1.2 Localization in Fluorescence Microscopy 23
1.3 Higher Signal, Lower Noise 24
1.4 Fluorescence Imaging with One-Nanometer Accuracy 25
1.5 Tracking the Movement of Molecular Motors 26
1.6 Multicolor Fluorescent Tracking 27
1.7 Tracking Fluorophores inside Living Cells 28
1.8 Rotational Movement 31
1.9 Future Directions 34
1.9.1 Probe Development 34
1.9.2 Instrumentation 35
1.9.3 Beyond the Diffraction Limit 35
References 36
2 Single-Molecule Analysis of Biomembranes 39
2.1 Introduction 39
2.2 Superresolution 40
2.3 Detection and Tracking 43
2.4 Learning from Trajectories 46
2.5 Application 1Synthetic Lipid Bilayers 50
2.6 Application 2Live Cell Plasma Membrane 52
2.7 Acknowledgments 56
References 56
3 Single-Molecule Imaging in Live Cells 63
3.1 Introduction 63
3.2 Fluorescent Labels 64
3.3 Green Fluorescent Protein 65
3.3.1 Discovery of GFP 66
3.3.2 Structure of GFP and the Fluorophore 66
3.4 Properties of Fluorescent Proteins 68
3.4.1 Brightness 68
3.4.2 Fluorescence Lifetime 69
3.4.3 Photobleaching Quantum Yield 70
3.4.4 Fluorescence Blinking 71
3.4.5 Maturation Time 72
3.4.6 Construction and Expression of Fusion Proteins 73
3.4.7 General Guidelines 73
3.5 Derivatives of avGFP and Other GFP-Like Proteins 74
3.5.1 Derivatives of avGFP 74
3.5.1.0 EYFP 74
3.5.1.0 Citrine 79
3.5.1.0 Venus 79
3.5.1.0 Yellow-Fluorescent Protein for Energy Transfer 80
3.5.2 Orange- and Red-Fluorescent Proteins 80
3.5.2.0 Monomeric Orange-Fluorescent Protein Kusabira-Orange 81
3.5.2.0 mOrange and mOrange2 82
3.5.2.0 tdTomato 82
3.5.2.0 TagRFP and TagRFP-T 82
3.5.2.0 mKate 83
3.5.2.0 mCherry 83
3.5.3 Photoinducible Fluorescent Proteins (PI-FPs) 83
3.5.3.0 Dronpa and Its Derivative rsFastLime 84
3.5.3.0 rsCherry and rsCherryRev 86
3.5.3.0 EosFP 87
3.5.3.0 Dendra2 88
3.6 Special Considerations for Live-Cell Imaging 89
3.6.1 Autofluorescence 89
3.6.2 Fluorescence Signal Enhancement 90
3.6.3 Laser-Induced Photodamage of Cells 91
3.7 Instrumentation 93
3.7.1 Illumination Source 93
3.7.2 Illumination Mode 94
3.7.2.0 Wide-Field Illumination 94
3.7.2.0 Confocal Illumination 95
3.7.2.0 Total Internal Reflection Illumination (TIR) 96
3.7.3 Camera-Based Detectors 97
3.7.4 Live-Cell Sample Preparation 98
3.8 Applications 99
3.8.1 Gene Expression 100
3.8.2 Transcription Factor Dynamics 100
3.8.3 Cell Signaling 103
3.8.4 Protein Complex Composition 104
3.9 Outlook 105
References 105
4 Fluorescence Imaging at Sub-Diffraction-Limit Resolution with Stochastic Optical Reconstruction Microscopy 114
4.1 Introduction 114
4.1.1 Basic Principle of STORM 115
4.1.2 Multicolor STORM 115
4.1.3 3D STORM 116
4.1.4 Applications 118
4.2 Labeling Cellular Targets with Photoswitchable Fluorescent Probes 118
4.2.1 Labeling Proteins with Photoswitchable Dyes 119
4.2.2 Immunofluorescence Staining of Cells 120
4.2.3 Labeling Cellular Structures with Photoswitchable Proteins 120
4.3 Instrumentation for Storm Imaging 120
4.3.1 Excitation Pathway 121
4.3.1.0 Light Sources Used for Various Multicolor STORM Imaging Schemes 121
4.3.1.0 Power Control and Temporal Modulation of Excitation Light 123
4.3.1.0 Excitation Light Path 123
4.3.2 Emission Pathway 124
4.3.2.0 Emission Filters 124
4.3.2.0 3D Imaging 124
4.3.2.0 Detection of Fluorescence Emission 125
4.3.3 Focus Lock for Axial Stability 126
4.4 Performing a Storm Experiment 126
4.4.1 Preparation of Cells 127
4.4.2 Calibration of z Position for 3D Imaging 127
4.4.3 Choosing a Method for Drift Correction 128
4.4.4 Imaging a Sample 129
4.5 Data Analysis 129
4.5.1 Peak Finding 129
4.5.2 Localizing Molecules in x , y , and z by Fitting 130
4.5.3 Color Identification 132
4.5.4 Drift Correction 132
4.5.5 Cross-Talk Subtraction 133
4.5.6 Displaying the Image 133
4.5.7 Additional Filtering of the Image 134
4.6 Example Applications 134
4.7 Protocol 1 136
4.7.1 Labeling Antibodies or Other Proteins with Organic Dyes 136
4.7.1.0 Materials 137
4.7.1.0 Methods 138
4.7.1.0 Troubleshooting 138
4.8 Protocol 2 138
4.8.1 Cell Fixation and Staining 138
4.8.1.0 Materials 138
4.8.1.0 Methods 139
4.9 Protocol 3 139
4.9.1 Transient Transfection of Cells for Expression of Fusion Constructs of PA-FPs and Proteins of Interest 140
4.9.1.0 Materials 140
4.9.1.0 Methods 140
4.10 Protocol 4 140
4.10.1 Using STORM to Image a Sample 141
4.10.1.0 Materials 141
4.10.1.0 Methods 142
4.10.1.0 Troubleshooting 143
4.11 Acknowledgments 143
References 143
5 Single-Molecule FRET: Methods and Biological Applications 147
5.1 Introduction 147
5.2 FRET Fundamentals and Ensemble FRET 148
5.3 Single-Molecule FRET Methods Based on Single-Laser Excitation 150
5.4 Single-Molecule FRET Methods Based on Alternating Laser Excitation 154
5.5 Quantitative Single-Molecule FRET 155
5.5.1 Measuring Accurate FRET 157
5.5.2 Obtaining Distances from Single-Molecule FRET Data 157
5.5.3 Triangulation Methods 158
5.6 Current Developments in Single-Molecule FRET 158
5.6.1 Multiple FRET Pair Methods 158
5.6.2 Combinations of Single-Molecule FRET with Other Single-Molecule Methods 159
5.6.2.0 Single-Molecule FRET in Living Cells 161
5.7 Applications of Single-Molecule FRET to Biomolecular Systems 161
5.7.1 Applications to Nucleic Acids 163
5.7.1.0 Holliday Junctions 163
5.7.1.0 RNA Folding 164
5.7.1.0 DNA Nanomachines 164
5.7.2 Applications to Nucleic Acid Machines 165
5.7.2.0 DNA and RNA Helicases 165
5.7.2.0 DNA Polymerase 168
5.7.2.0 RNA Polymerase 168
5.7.2.0 Reverse Transcriptase 170
5.7.3 Applications to Molecular Motors 170
5.7.3.0 ATP Synthase 171
5.7.3.0 Kinesin 172
5.7.4 Applications to Protein Folding and Dynamics 175
5.8 Conclusion and Future Prospects 176
References 177
6 Single-Molecule Enzymology 182
6.1 Introduction 182
6.2 Enzyme Kinetics 183
6.3 Conformational Fluctuations and Dynamics 189
6.4 Enzymology of Multiprotein Complexes 194
References 197
7 Single-Molecule Studies of Rotary Molecular Motors 200
7.1 Introduction 200
7.2 Structure 201
7.2.1 ATP-Synthase 202
7.2.1.0 F1 203
7.2.1.0 Fo 203
7.2.2 Bacterial Flagellar Motor 205
7.3 Single-Molecule Methods for Measuring Rotation 207
7.3.1 ATP-Synthase 208
7.3.2 Bacterial Flagellar Motor 210
7.4 Energy Transduction 212
7.4.1 ATP-Synthase 212
7.4.1.0 Fo 212
7.4.1.0 F1 213
7.4.2 Bacterial Flagellar Motor 215
7.4.2.0 Input 215
7.4.2.0 Output 217
7.5 Mechanism 219
7.5.1 ATP-Synthase 219
7.5.1.0 F1Fo 219
7.5.1.0 F1 221
7.5.2 Bacterial Flagellar Motor 224
7.5.2.0 Independent Torque-Generating Units 224
7.5.2.0 Interactions between Rotor and Stator 225
7.5.2.0 Stepping Rotation 226
References 226
8 Fluorescence Correlation Spectroscopy in Living Cells 234
8.1 Introduction 234
8.2 Measurement Principle 235
8.3 Theoretical Framework 237
8.3.1 Diffusion 237
8.3.2 Chromophore Dynamics 242
8.3.3 Concentrations 244
8.3.4 Interactions 245
8.4 Instrumentation 247
8.4.1 Position-Sensitive Single-Point FCS 248
8.4.2 Scanning FCS 248
8.4.3 Alternative Excitation Modes 250
8.5 Biological Implications 251
8.6 Acknowledgments 254
References 254
9 Precise Measurements of Diffusion in Solutionby Fluorescence Correlations Spectroscopy 259
9.1 Introduction 259
9.2 Optical Setup 261
9.3 Data Acquisition and Evaluation 263
9.4 Measuring Diffusion and Concentration 264
9.4.1 One-Focus FCS 264
9.4.2 Dual-Focus FCS 270
9.5 Conclusion and Outlook 277
References 278
10 Single-Molecule Studies of Nucleic Acid InteractionsUsing Nanopores 280
10.1 Introduction 280
10.2 Nanopore Basics 281
10.3 Biophysical Studies Using Protein Pores 283
10.3.1 -Hemolysin 283
10.3.2 DNA Translocation Dynamics: The Role of Biopolymer--Pore Interactions and DNA Structure inside a Nanometer Confinement 285
10.3.2.0 Polynucleotide Translocation Dynamics 285
10.3.2.0 Orientation Dependence of Polynucleotide Entry and Dynamics 287
10.3.3 Probing Secondary Structure: DNA End-Fraying and DNA Unzipping Kinetics 289
10.3.4 Probing DNA--Protein Interactions: DNA Exonucleases and Polymerases 291
10.3.4.0 Exonuclease I--DNA Interactions 292
10.3.4.0 Nanopore Probing of Deoxyribonucleotide Triphosphate Incorporation by a Klenow Fragment DNA Polymerase 293
10.3.4.0 Probing DNA Polymerase Activity Using a Nanopore 293
10.4 Biophysical Studies Using Solid-State Nanopores 295
10.4.1 Nanopore Fabrication 296
10.4.2 Experimental Considerations 296
10.4.3 DNA Translocation through Solid-State Nanopores 298
10.5 Summary and Future Prospects 301
10.5.1 DNA Sequencing by Ionic Blockade Measurement 301
10.5.2 DNA Sequencing by Transverse Electronic Measurement 302
10.5.3 DNA Sequencing by Optical Readout of DNA Bases 303
10.6 Acknowledgments 303
References 303
11 Nanopores: Generation, Engineering, and Single-Molecule Applications 307
11.1 Introduction 307
11.2 Principles of Nanopore Analytics 308
11.3 Pores 310
11.3.1 Biological and Chemical Pores 310
11.3.2 Engineering of Biological and Chemical Nanopores 312
11.3.3 Biological Nanopores in Lipid Membranes 313
11.3.4 Solid-State and Polymer Nanopores 314
11.3.4.0 Track-Etching Technique 315
11.3.4.0 Pores in Silicon Obtained by Asymmetric Etching 316
11.3.4.0 Ion-Beam Sculpting 316
11.3.4.0 Electron Beam as a Nanofabrication Tool 317
11.3.4.0 Glass Nanopipettes 318
11.3.4.0 Submicrometer Pores with Diameters Larger Than 200 nm 318
11.3.5 Chemical Engineering of Solid-State Nanopores 318
11.3.5.0 Chemical Modification of Nanopores Obtained by the Track-Etching Technique 319
11.3.5.0 Chemical Modification of Silicon-Based Nanopores 321
11.3.5.0 Chemical Modification of Glass Nanopipettes 322
11.4 Applications of Nanopores 322
11.4.1 Sensing and Examining Individual Molecules 322
11.4.1.0 Coulter Counter Method 324
11.4.1.0 Sensing Aided by Pore-Tethered Molecular Recognition Sites 330
11.4.1.0 Sensing of Covalently Attached Analytes 332
11.4.1.0 Controlling the Movement and Position of Molecules within Nanopores 333
11.4.1.0 Theoretical Modeling 334
11.4.2 Separation and Molecular Filtration 335
11.4.3 Nanofluidics 339
11.5 Outlook 340
11.6 Acknowledgments 343
References 343
12 Single-Molecule Manipulation Using Optical Traps 354
12.1 Theory and Design of Optical Traps 354
12.1.1 Theory 354
12.1.2 Design of Optical Traps 356
12.1.2.0 Laser Beams 356
12.1.2.0 Sample Manipulation 357
12.1.2.0 Focusing Optics 357
12.1.2.0 Detection Optics 358
12.1.3 Calibration of Optical Traps 358
12.1.4 Implementation of Single-Molecule Optical Trapping Assays 360
12.1.5 Technical Capabilities 360
12.2 Applications to Studying Single Molecules 361
12.2.1 Studies of Structural and Mechanical Properties 362
12.2.1.0 Elastic Properties of DNA 362
12.2.1.0 Folding Studies 363
12.2.1.0 Binding Reactions 369
12.2.2 Studies of Molecular Motors 369
12.2.2.0 Processive Mechanoenzymes 370
12.2.2.0 Three-Bead Optical Trapping for Nonprocessive Motors 373
12.3 Practical Experimental Considerations 375
12.3.1 Ensuring ''Real'' Signals 375
12.3.1.0 Ensuring the Quality of the Molecules Measured 375
12.3.1.0 Working in the Single-Molecule Regime 375
12.3.1.0 Identifying Signals 376
12.3.2 Sources of Error 377
12.3.2.0 Errors from Bead-Size Polydispersity 377
12.3.2.0 Errors in Stiffness Calibration 377
12.3.2.0 Errors from Trap Potential Anharmonicity 378
12.3.2.0 Errors in Determining the Bead Height above a Surface 378
12.3.3 Comparing Optical Trapping Results to Results from Other Methods 379
12.4 Extending the Capabilities of Optical Traps 379
12.5 Acknowledgments 380
References 380
13 Magnetic Tweezers for Single-Molecule Experiments 384
13.1 Introduction 384
13.2 Experimental Design of the Magnetic Tweezers 385
13.3 Image Analysis 386
13.4 Determination of the Applied Force 387
13.4.1 Calculation of the Applied Force---Analysis of the Brownian Motion of the Bead in Real and Fourier Space 389
13.4.2 Correction for the Camera Integration Time 391
13.5 Nucleic Acids under Force and Torque 393
13.6 Current Capabilities in Terms of Temporal and Spatial Resolution: Practical Limitations 395
13.7 Optimization of the Magnet Geometry 396
13.8 Flow Cells for Magnetic Tweezers 398
13.8.1 Strategies for Tethering Nucleic Acids to the Flow Cell and the Bead 399
13.8.2 Inner Surface Passivation Techniques 399
13.8.3 Considerations When Working with RNA 400
13.9 Use of Magnetic Tweezers in Biological Experiments: Examples 400
13.9.1 Example 1: Supercoils Dynamics, and Supercoil Removal 401
13.9.2 Example 2: DNA Scrunching by RNA Polymerase 403
13.9.3 Example 3: DNA Helicase Activity 404
13.9.4 Example 4: MT Applications in Protein Science 404
13.10 Outlook 405
13.11 Acknowledgments 405
References 405
14 Folding of Proteins under Mechanical Force 409
14.1 A Model for Protein Folding under Force 409
14.2 Protein Refolding at Constant Pulling Velocity 412
14.3 Comparing AFM and Optical Tweezers Experiments 415
14.4 Comparison to Experimental Data and Conclusion 417
References 417
15 Probing the Energy Landscape of Protein-Binding Reactions by Dynamic Force Spectroscopy 419
15.1 Introduction 419
15.2 Dynamic Force Spectroscopy: Principles and Theory 421
15.2.1 Tip and Surface Immobilization 421
15.2.2 The Force--Distance Cycle 427
15.2.3 Spring Constant Determination 428
15.2.4 Force Distributions 430
15.2.5 Theory of Force Spectroscopy 431
15.2.6 The Effect of Hidden Barriers on Kinetic Parameters 437
15.2.7 Free Energy Surface Reconstruction from Nonequilibrium Single-Molecule Pulling Experiments 439
15.3 Applications of Dynamic Force Spectroscopy to Protein Interactions 442
15.3.1 Load-Dependent Dynamics of Protein Interactions 442
15.3.2 Energy Landscape Roughness of Protein Binding Reactions 447
15.3.3 Discrimination between Modes of Protein Activation 450
References 452
16 Probing Single Membrane Proteins by Atomic Force Microscopy 460
16.1 Introduction 460
16.1.1 A Short Synopsis on Membrane Proteins 460
16.1.2 Atomic Force Microscope as a Multifunctional Tool for Characterizing Membrane Protein Structure and Function 462
16.2 The Atomic Force Microscope 463
16.2.1 Principle and Setup 463
16.3 High-Resolution Imaging of Single Native Proteins 464
16.3.1 High-Resolution Imaging of Protein Assemblies 465
16.3.2 High-Resolution Imaging of Membrane Protein Diffusion 469
16.3.3 High-Resolution Imaging of Proteins at Work 471
16.4 Single-Molecule Force Spectroscopy of Membrane Proteins 471
16.4.1 Detecting Unfolding Intermediates and Pathways 472
16.4.2 Importance of Studying Membrane Protein (Un)Folding 473
16.4.3 Why Study Membrane Protein (Un)Folding Under a Mechanical Load? 475
16.4.4 Unfolding Forces Reflect Interactions That Stabilize Structural Regions 475
16.4.5 Origin of Unfolding Forces 475
16.4.6 Elucidating the Unfolding Routes of Membrane Proteins 478
16.4.7 Molecular Nature of Unfolding Intermediates 478
16.4.8 Membrane Proteins of Similar Structures Show Similar Unfolding Patterns 479
16.4.9 Detecting Intermediates and Pathways during Refolding of Membrane Proteins 479
16.4.10 Dynamic Energy Landscape 481
16.4.11 Following the Unfolding Contours of Mutant Proteins in an Energy Landscape 482
16.4.12 Protein Rigidity, Function, and Energy Landscape 485
16.4.13 Screening Membrane Proteins for Small-Molecule Binding 487
16.5 Outlook 488
16.5.1 Characterizing Factors That Sculpt the Energy Landscape 488
16.5.2 Approaches to Screening Drug Targets with Molecular Compounds 489
References 489
17 High-Speed Atomic Force Microscopy 497
17.1 Introduction 497
17.2 Basic Principle of AFM and Various Imaging Modes 498
17.3 Imaging Rate and Feedback Bandwidth 499
17.3.1 Image Acquisition Time and Feedback Bandwidth 500
17.3.2 Feedback Bandwidth as a Function of Various Factors 500
17.3.3 Feedback Operation and Parachuting 501
17.3.4 Refinement of Analytical Expressions for 0p and 0I 503
17.4 Devices for High-Speed AFM 504
17.4.1 Small Cantilevers and Related Devices 504
17.4.2 Tip--Sample Interaction Detection 506
17.4.2.0 Amplitude Detectors 506
17.4.2.0 Force Detectors 508
17.4.3 High-Speed Scanner 509
17.4.3.0 Counterbalance 509
17.4.3.0 Mechanical Scanner Design 510
17.4.4 Active Damping 510
17.4.4.0 Feedback Q Control 511
17.4.4.0 Feedforward Active Damping 512
17.4.4.0 Practice of Active Damping of the Scanner 512
17.4.5 Dynamic PID Control 514
17.4.5.0 Dynamic PID Controller 514
17.4.5.0 Performance of Dynamic PID Control 516
17.4.6 Drift Compensator 518
17.4.7 High-Speed Phase Detector 518
17.5 High-Speed Bioimaging 520
17.5.1 Chaperonin GroEL 520
17.5.2 Lattice Defect Diffusion in Two-Dimensional Protein Crystals 521
17.5.3 Myosin V 523
17.5.4 Intrinsically Disordered Regions of Proteins 524
17.5.5 High-Speed Phase-Contrast Imaging 526
17.5.5.0 Compositional Mapping on Blended Polymers 526
17.5.5.0 Phase-Contrast Imaging of Myosin Filaments 527
17.5.5.0 Phase-Contrast Imaging of GroEL 529
17.6 Substrata for Observing Dynamic Biomolecular Processes 529
References 530
18 Recognition Imaging Using Atomic Force Microscopy 534
18.1 Introduction 534
18.2 Chemical Force Microscopy 535
18.2.1 Methods 536
18.2.2 Chemical Imaging of Live Cells 538
18.3 Recognition Imaging Using Force Spectroscopy 540
18.3.1 Methods 540
18.3.2 Measuring Molecular Recognition Forces 541
18.3.3 Molecular Recognition Imaging Using SMFS 543
18.4 Topography and Recognition Imaging 545
18.4.1 Methods 546
18.4.2 Applications of TREC Imaging 549
18.4.2.0 Chromatin 549
18.4.2.0 Bacterial Surface Layers 550
18.4.2.0 Membranes 553
18.4.2.0 Cells 555
18.5 Immunogold Imaging 557
18.6 Acknowledgments 560
References 560
19 Atomic Force Microscopy of Protein0Protein Interactions 564
19.1 Introduction 564
19.2 AFM Experimentation 565
19.2.1 AFM Principles 565
19.2.2 AFM Measurement of Single-Molecule Interactions 566
19.2.3 Tip and Sample Preparation 567
19.3 Determination of the Energy Landscape from the AFM Measurements 569
19.4 Recent Applications 572
19.4.1 Molecular Basis for Multiple Energy Barriers along with Protein--Protein Dissociation 572
19.4.2 AFM Measurements on Living Cells 573
19.4.3 Energy Landscape Roughness of Protein--Ligand Interaction 575
19.5 Concluding Remarks 575
19.6 Acknowledgments 576
References 576
20 A New Approach to Analysis of Single-Molecule ForceMeasurements 580
20.1 Introduction 580
20.2 Force Probe Design and the Quest for Single-Molecule Statistics 581
20.3 Identifying Events Arising from Nonspecific and Multiple-Specific Attachments 582
20.3.1 Dealing with Nonspecific Events 584
20.3.2 Dealing with Multiple-Specific Events 584
20.4 Establishing Estimators for Initial State Probability and Distribution of Transitions 586
20.5 Two-State Transitions and the Direct Experimental Assay for Kinetic Rates 587
20.6 Experimental Example: Dissociating ICAM-1 from 2 -Integrin with Force Ramps 589
20.6.1 Microsphere Targets 589
20.6.2 PMN Targets 591
20.7 Experimental Example: Unfolding/Refolding of a Polyprotein with Force Ramps 592
20.7.1 Unfolding Kinetics 593
20.7.2 Refolding Kinetics 595
20.8 Acknowledgment 598
References 598
21 Single-Molecule Recognition: Extracting Informationfrom Individual Binding Events and Their Correlation 599
21.1 Introduction 599
21.2 Adhesion Frequency Assay 600
21.3 Thermal Fluctuation Assay 602
21.4 Analysis of Correlation among Outcomes from Sequential Tests 612
21.5 Acknowledgments 617
References 617
Subject Index 619