Hinterdorfer / Oijen Handbook of Single-Molecule Biophysics

1. Auflage 2009
ISBN: 978-0-387-76497-9
Verlag: Springer
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 626 Seiten

This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.

Autoren/Hrsg.

Oijen, Antoine

Hinterdorfer, Peter

Oijen, Antoine van

Inhaltsverzeichnis

1;Preface;5
2;Contents;8
3;List of Contributors;18
4;1 Single-Molecule Fluorescent Particle Tracking;21
4.1;1.1 The History of Single-Particle Tracking;21
4.2;1.2 Localization in Fluorescence Microscopy;23
4.3;1.3 Higher Signal, Lower Noise;24
4.4;1.4 Fluorescence Imaging with One-Nanometer Accuracy;25
4.5;1.5 Tracking the Movement of Molecular Motors;26
4.6;1.6 Multicolor Fluorescent Tracking;27
4.7;1.7 Tracking Fluorophores inside Living Cells;28
4.8;1.8 Rotational Movement;31
4.9;1.9 Future Directions;34
4.9.1;1.9.1 Probe Development;34
4.9.2;1.9.2 Instrumentation;35
4.9.3;1.9.3 Beyond the Diffraction Limit;35
4.10;References;36
5;2 Single-Molecule Analysis of Biomembranes;39
5.1;2.1 Introduction;39
5.2;2.2 Superresolution;40
5.3;2.3 Detection and Tracking;43
5.4;2.4 Learning from Trajectories;46
5.5;2.5 Application 1Synthetic Lipid Bilayers;50
5.6;2.6 Application 2Live Cell Plasma Membrane;52
5.7;2.7 Acknowledgments;56
5.8;References;56
6;3 Single-Molecule Imaging in Live Cells;63
6.1;3.1 Introduction;63
6.2;3.2 Fluorescent Labels;64
6.3;3.3 Green Fluorescent Protein;65
6.3.1;3.3.1 Discovery of GFP;66
6.3.2;3.3.2 Structure of GFP and the Fluorophore;66
6.4;3.4 Properties of Fluorescent Proteins;68
6.4.1;3.4.1 Brightness;68
6.4.2;3.4.2 Fluorescence Lifetime;69
6.4.3;3.4.3 Photobleaching Quantum Yield;70
6.4.4;3.4.4 Fluorescence Blinking;71
6.4.5;3.4.5 Maturation Time;72
6.4.6;3.4.6 Construction and Expression of Fusion Proteins;73
6.4.7;3.4.7 General Guidelines;73
6.5;3.5 Derivatives of avGFP and Other GFP-Like Proteins;74
6.5.1;3.5.1 Derivatives of avGFP;74
6.5.1.1;3.5.1.0 EYFP;74
6.5.1.2;3.5.1.0 Citrine;79
6.5.1.3;3.5.1.0 Venus;79
6.5.1.4;3.5.1.0 Yellow-Fluorescent Protein for Energy Transfer;80
6.5.2;3.5.2 Orange- and Red-Fluorescent Proteins;80
6.5.2.1;3.5.2.0 Monomeric Orange-Fluorescent Protein Kusabira-Orange;81
6.5.2.2;3.5.2.0 mOrange and mOrange2;82
6.5.2.3;3.5.2.0 tdTomato;82
6.5.2.4;3.5.2.0 TagRFP and TagRFP-T;82
6.5.2.5;3.5.2.0 mKate;83
6.5.2.6;3.5.2.0 mCherry;83
6.5.3;3.5.3 Photoinducible Fluorescent Proteins (PI-FPs);83
6.5.3.1;3.5.3.0 Dronpa and Its Derivative rsFastLime;84
6.5.3.2;3.5.3.0 rsCherry and rsCherryRev;86
6.5.3.3;3.5.3.0 EosFP;87
6.5.3.4;3.5.3.0 Dendra2;88
6.6;3.6 Special Considerations for Live-Cell Imaging;89
6.6.1;3.6.1 Autofluorescence;89
6.6.2;3.6.2 Fluorescence Signal Enhancement;90
6.6.3;3.6.3 Laser-Induced Photodamage of Cells;91
6.7;3.7 Instrumentation;93
6.7.1;3.7.1 Illumination Source;93
6.7.2;3.7.2 Illumination Mode;94
6.7.2.1;3.7.2.0 Wide-Field Illumination;94
6.7.2.2;3.7.2.0 Confocal Illumination;95
6.7.2.3;3.7.2.0 Total Internal Reflection Illumination (TIR);96
6.7.3;3.7.3 Camera-Based Detectors;97
6.7.4;3.7.4 Live-Cell Sample Preparation;98
6.8;3.8 Applications;99
6.8.1;3.8.1 Gene Expression;100
6.8.2;3.8.2 Transcription Factor Dynamics;100
6.8.3;3.8.3 Cell Signaling;103
6.8.4;3.8.4 Protein Complex Composition;104
6.9;3.9 Outlook;105
6.10;References;105
7;4 Fluorescence Imaging at Sub-Diffraction-Limit Resolution with Stochastic Optical Reconstruction Microscopy;114
7.1;4.1 Introduction;114
7.1.1;4.1.1 Basic Principle of STORM;115
7.1.2;4.1.2 Multicolor STORM;115
7.1.3;4.1.3 3D STORM;116
7.1.4;4.1.4 Applications;118
7.2;4.2 Labeling Cellular Targets with Photoswitchable Fluorescent Probes;118
7.2.1;4.2.1 Labeling Proteins with Photoswitchable Dyes;119
7.2.2;4.2.2 Immunofluorescence Staining of Cells;120
7.2.3;4.2.3 Labeling Cellular Structures with Photoswitchable Proteins;120
7.3;4.3 Instrumentation for Storm Imaging;120
7.3.1;4.3.1 Excitation Pathway;121
7.3.1.1;4.3.1.0 Light Sources Used for Various Multicolor STORM Imaging Schemes;121
7.3.1.2;4.3.1.0 Power Control and Temporal Modulation of Excitation Light;123
7.3.1.3;4.3.1.0 Excitation Light Path;123
7.3.2;4.3.2 Emission Pathway;124
7.3.2.1;4.3.2.0 Emission Filters;124
7.3.2.2;4.3.2.0 3D Imaging;124
7.3.2.3;4.3.2.0 Detection of Fluorescence Emission;125
7.3.3;4.3.3 Focus Lock for Axial Stability;126
7.4;4.4 Performing a Storm Experiment;126
7.4.1;4.4.1 Preparation of Cells;127
7.4.2;4.4.2 Calibration of z Position for 3D Imaging;127
7.4.3;4.4.3 Choosing a Method for Drift Correction;128
7.4.4;4.4.4 Imaging a Sample;129
7.5;4.5 Data Analysis;129
7.5.1;4.5.1 Peak Finding;129
7.5.2;4.5.2 Localizing Molecules in x , y , and z by Fitting;130
7.5.3;4.5.3 Color Identification;132
7.5.4;4.5.4 Drift Correction;132
7.5.5;4.5.5 Cross-Talk Subtraction;133
7.5.6;4.5.6 Displaying the Image;133
7.5.7;4.5.7 Additional Filtering of the Image;134
7.6;4.6 Example Applications;134
7.7;4.7 Protocol 1;136
7.7.1;4.7.1 Labeling Antibodies or Other Proteins with Organic Dyes;136
7.7.1.1;4.7.1.0 Materials;137
7.7.1.2;4.7.1.0 Methods;138
7.7.1.3;4.7.1.0 Troubleshooting;138
7.8;4.8 Protocol 2;138
7.8.1;4.8.1 Cell Fixation and Staining;138
7.8.1.1;4.8.1.0 Materials;138
7.8.1.2;4.8.1.0 Methods;139
7.9;4.9 Protocol 3;139
7.9.1;4.9.1 Transient Transfection of Cells for Expression of Fusion Constructs of PA-FPs and Proteins of Interest;140
7.9.1.1;4.9.1.0 Materials;140
7.9.1.2;4.9.1.0 Methods;140
7.10;4.10 Protocol 4;140
7.10.1;4.10.1 Using STORM to Image a Sample;141
7.10.1.1;4.10.1.0 Materials;141
7.10.1.2;4.10.1.0 Methods;142
7.10.1.3;4.10.1.0 Troubleshooting;143
7.11;4.11 Acknowledgments;143
7.12;References;143
8;5 Single-Molecule FRET: Methods and Biological Applications;147
8.1;5.1 Introduction;147
8.2;5.2 FRET Fundamentals and Ensemble FRET;148
8.3;5.3 Single-Molecule FRET Methods Based on Single-Laser Excitation;150
8.4;5.4 Single-Molecule FRET Methods Based on Alternating Laser Excitation;154
8.5;5.5 Quantitative Single-Molecule FRET;155
8.5.1;5.5.1 Measuring Accurate FRET;157
8.5.2;5.5.2 Obtaining Distances from Single-Molecule FRET Data;157
8.5.3;5.5.3 Triangulation Methods;158
8.6;5.6 Current Developments in Single-Molecule FRET;158
8.6.1;5.6.1 Multiple FRET Pair Methods;158
8.6.2;5.6.2 Combinations of Single-Molecule FRET with Other Single-Molecule Methods;159
8.6.2.1;5.6.2.0 Single-Molecule FRET in Living Cells;161
8.7;5.7 Applications of Single-Molecule FRET to Biomolecular Systems;161
8.7.1;5.7.1 Applications to Nucleic Acids;163
8.7.1.1;5.7.1.0 Holliday Junctions;163
8.7.1.2;5.7.1.0 RNA Folding;164
8.7.1.3;5.7.1.0 DNA Nanomachines;164
8.7.2;5.7.2 Applications to Nucleic Acid Machines;165
8.7.2.1;5.7.2.0 DNA and RNA Helicases;165
8.7.2.2;5.7.2.0 DNA Polymerase;168
8.7.2.3;5.7.2.0 RNA Polymerase;168
8.7.2.4;5.7.2.0 Reverse Transcriptase;170
8.7.3;5.7.3 Applications to Molecular Motors;170
8.7.3.1;5.7.3.0 ATP Synthase;171
8.7.3.2;5.7.3.0 Kinesin;172
8.7.4;5.7.4 Applications to Protein Folding and Dynamics;175
8.8;5.8 Conclusion and Future Prospects;176
8.9;References;177
9;6 Single-Molecule Enzymology;182
9.1;6.1 Introduction;182
9.2;6.2 Enzyme Kinetics;183
9.3;6.3 Conformational Fluctuations and Dynamics;189
9.4;6.4 Enzymology of Multiprotein Complexes;194
9.5;References;197
10;7 Single-Molecule Studies of Rotary Molecular Motors;200
10.1;7.1 Introduction;200
10.2;7.2 Structure;201
10.2.1;7.2.1 ATP-Synthase;202
10.2.1.1;7.2.1.0 F1 ;203
10.2.1.2;7.2.1.0 Fo;203
10.2.2;7.2.2 Bacterial Flagellar Motor;205
10.3;7.3 Single-Molecule Methods for Measuring Rotation;207
10.3.1;7.3.1 ATP-Synthase;208
10.3.2;7.3.2 Bacterial Flagellar Motor;210
10.4;7.4 Energy Transduction;212
10.4.1;7.4.1 ATP-Synthase;212
10.4.1.1;7.4.1.0 Fo ;212
10.4.1.2;7.4.1.0 F1 ;213
10.4.2;7.4.2 Bacterial Flagellar Motor;215
10.4.2.1;7.4.2.0 Input;215
10.4.2.2;7.4.2.0 Output;217
10.5;7.5 Mechanism;219
10.5.1;7.5.1 ATP-Synthase;219
10.5.1.1;7.5.1.0 F1Fo ;219
10.5.1.2;7.5.1.0 F1 ;221
10.5.2;7.5.2 Bacterial Flagellar Motor;224
10.5.2.1;7.5.2.0 Independent Torque-Generating Units;224
10.5.2.2;7.5.2.0 Interactions between Rotor and Stator;225
10.5.2.3;7.5.2.0 Stepping Rotation;226
10.6;References;226
11;8 Fluorescence Correlation Spectroscopy in Living Cells;234
11.1;8.1 Introduction;234
11.2;8.2 Measurement Principle;235
11.3;8.3 Theoretical Framework;237
11.3.1;8.3.1 Diffusion;237
11.3.2;8.3.2 Chromophore Dynamics;242
11.3.3;8.3.3 Concentrations;244
11.3.4;8.3.4 Interactions;245
11.4;8.4 Instrumentation;247
11.4.1;8.4.1 Position-Sensitive Single-Point FCS;248
11.4.2;8.4.2 Scanning FCS;248
11.4.3;8.4.3 Alternative Excitation Modes;250
11.5;8.5 Biological Implications;251
11.6;8.6 Acknowledgments;254
11.7;References;254
12;9 Precise Measurements of Diffusion in Solutionby Fluorescence Correlations Spectroscopy;259
12.1;9.1 Introduction;259
12.2;9.2 Optical Setup;261
12.3;9.3 Data Acquisition and Evaluation;263
12.4;9.4 Measuring Diffusion and Concentration;264
12.4.1;9.4.1 One-Focus FCS;264
12.4.2;9.4.2 Dual-Focus FCS;270
12.5;9.5 Conclusion and Outlook;277
12.6;References;278
13;10 Single-Molecule Studies of Nucleic Acid InteractionsUsing Nanopores;280
13.1;10.1 Introduction;280
13.2;10.2 Nanopore Basics;281
13.3;10.3 Biophysical Studies Using Protein Pores;283
13.3.1;10.3.1 -Hemolysin;283
13.3.2;10.3.2 DNA Translocation Dynamics: The Role of Biopolymer--Pore Interactions and DNA Structure inside a Nanometer Confinement;285
13.3.2.1;10.3.2.0 Polynucleotide Translocation Dynamics;285
13.3.2.2;10.3.2.0 Orientation Dependence of Polynucleotide Entry and Dynamics;287
13.3.3;10.3.3 Probing Secondary Structure: DNA End-Fraying and DNA Unzipping Kinetics;289
13.3.4;10.3.4 Probing DNA--Protein Interactions: DNA Exonucleases and Polymerases;291
13.3.4.1;10.3.4.0 Exonuclease I--DNA Interactions;292
13.3.4.2;10.3.4.0 Nanopore Probing of Deoxyribonucleotide Triphosphate Incorporation by a Klenow Fragment DNA Polymerase;293
13.3.4.3;10.3.4.0 Probing DNA Polymerase Activity Using a Nanopore;293
13.4;10.4 Biophysical Studies Using Solid-State Nanopores;295
13.4.1;10.4.1 Nanopore Fabrication;296
13.4.2;10.4.2 Experimental Considerations;296
13.4.3;10.4.3 DNA Translocation through Solid-State Nanopores;298
13.5;10.5 Summary and Future Prospects;301
13.5.1;10.5.1 DNA Sequencing by Ionic Blockade Measurement;301
13.5.2;10.5.2 DNA Sequencing by Transverse Electronic Measurement;302
13.5.3;10.5.3 DNA Sequencing by Optical Readout of DNA Bases;303
13.6;10.6 Acknowledgments;303
13.7;References;303
14;11 Nanopores: Generation, Engineering, and Single-Molecule Applications;307
14.1;11.1 Introduction;307
14.2;11.2 Principles of Nanopore Analytics;308
14.3;11.3 Pores;310
14.3.1;11.3.1 Biological and Chemical Pores;310
14.3.2;11.3.2 Engineering of Biological and Chemical Nanopores;312
14.3.3;11.3.3 Biological Nanopores in Lipid Membranes;313
14.3.4;11.3.4 Solid-State and Polymer Nanopores;314
14.3.4.1;11.3.4.0 Track-Etching Technique;315
14.3.4.2;11.3.4.0 Pores in Silicon Obtained by Asymmetric Etching;316
14.3.4.3;11.3.4.0 Ion-Beam Sculpting;316
14.3.4.4;11.3.4.0 Electron Beam as a Nanofabrication Tool;317
14.3.4.5;11.3.4.0 Glass Nanopipettes;318
14.3.4.6;11.3.4.0 Submicrometer Pores with Diameters Larger Than 200 nm;318
14.3.5;11.3.5 Chemical Engineering of Solid-State Nanopores;318
14.3.5.1;11.3.5.0 Chemical Modification of Nanopores Obtained by the Track-Etching Technique;319
14.3.5.2;11.3.5.0 Chemical Modification of Silicon-Based Nanopores;321
14.3.5.3;11.3.5.0 Chemical Modification of Glass Nanopipettes;322
14.4;11.4 Applications of Nanopores;322
14.4.1;11.4.1 Sensing and Examining Individual Molecules;322
14.4.1.1;11.4.1.0 Coulter Counter Method;324
14.4.1.2;11.4.1.0 Sensing Aided by Pore-Tethered Molecular Recognition Sites;330
14.4.1.3;11.4.1.0 Sensing of Covalently Attached Analytes;332
14.4.1.4;11.4.1.0 Controlling the Movement and Position of Molecules within Nanopores;333
14.4.1.5;11.4.1.0 Theoretical Modeling;334
14.4.2;11.4.2 Separation and Molecular Filtration;335
14.4.3;11.4.3 Nanofluidics;339
14.5;11.5 Outlook;340
14.6;11.6 Acknowledgments;343
14.7;References;343
15;12 Single-Molecule Manipulation Using Optical Traps;354
15.1;12.1 Theory and Design of Optical Traps;354
15.1.1;12.1.1 Theory;354
15.1.2;12.1.2 Design of Optical Traps;356
15.1.2.1;12.1.2.0 Laser Beams;356
15.1.2.2;12.1.2.0 Sample Manipulation;357
15.1.2.3;12.1.2.0 Focusing Optics;357
15.1.2.4;12.1.2.0 Detection Optics;358
15.1.3;12.1.3 Calibration of Optical Traps;358
15.1.4;12.1.4 Implementation of Single-Molecule Optical Trapping Assays;360
15.1.5;12.1.5 Technical Capabilities;360
15.2;12.2 Applications to Studying Single Molecules;361
15.2.1;12.2.1 Studies of Structural and Mechanical Properties;362
15.2.1.1;12.2.1.0 Elastic Properties of DNA;362
15.2.1.2;12.2.1.0 Folding Studies;363
15.2.1.3;12.2.1.0 Binding Reactions;369
15.2.2;12.2.2 Studies of Molecular Motors;369
15.2.2.1;12.2.2.0 Processive Mechanoenzymes;370
15.2.2.2;12.2.2.0 Three-Bead Optical Trapping for Nonprocessive Motors;373
15.3;12.3 Practical Experimental Considerations;375
15.3.1;12.3.1 Ensuring ''Real'' Signals;375
15.3.1.1;12.3.1.0 Ensuring the Quality of the Molecules Measured;375
15.3.1.2;12.3.1.0 Working in the Single-Molecule Regime;375
15.3.1.3;12.3.1.0 Identifying Signals;376
15.3.2;12.3.2 Sources of Error;377
15.3.2.1;12.3.2.0 Errors from Bead-Size Polydispersity;377
15.3.2.2;12.3.2.0 Errors in Stiffness Calibration;377
15.3.2.3;12.3.2.0 Errors from Trap Potential Anharmonicity;378
15.3.2.4;12.3.2.0 Errors in Determining the Bead Height above a Surface;378
15.3.3;12.3.3 Comparing Optical Trapping Results to Results from Other Methods;379
15.4;12.4 Extending the Capabilities of Optical Traps;379
15.5;12.5 Acknowledgments;380
15.6;References;380
16;13 Magnetic Tweezers for Single-Molecule Experiments;384
16.1;13.1 Introduction;384
16.2;13.2 Experimental Design of the Magnetic Tweezers;385
16.3;13.3 Image Analysis;386
16.4;13.4 Determination of the Applied Force;387
16.4.1;13.4.1 Calculation of the Applied Force---Analysis of the Brownian Motion of the Bead in Real and Fourier Space;389
16.4.2;13.4.2 Correction for the Camera Integration Time;391
16.5;13.5 Nucleic Acids under Force and Torque;393
16.6;13.6 Current Capabilities in Terms of Temporal and Spatial Resolution: Practical Limitations;395
16.7;13.7 Optimization of the Magnet Geometry;396
16.8;13.8 Flow Cells for Magnetic Tweezers;398
16.8.1;13.8.1 Strategies for Tethering Nucleic Acids to the Flow Cell and the Bead;399
16.8.2;13.8.2 Inner Surface Passivation Techniques;399
16.8.3;13.8.3 Considerations When Working with RNA;400
16.9;13.9 Use of Magnetic Tweezers in Biological Experiments: Examples;400
16.9.1;13.9.1 Example 1: Supercoils Dynamics, and Supercoil Removal;401
16.9.2;13.9.2 Example 2: DNA Scrunching by RNA Polymerase;403
16.9.3;13.9.3 Example 3: DNA Helicase Activity;404
16.9.4;13.9.4 Example 4: MT Applications in Protein Science;404
16.10;13.10 Outlook;405
16.11;13.11 Acknowledgments;405
16.12;References;405
17;14 Folding of Proteins under Mechanical Force;409
17.1;14.1 A Model for Protein Folding under Force;409
17.2;14.2 Protein Refolding at Constant Pulling Velocity;412
17.3;14.3 Comparing AFM and Optical Tweezers Experiments;415
17.4;14.4 Comparison to Experimental Data and Conclusion;417
17.5;References;417
18;15 Probing the Energy Landscape of Protein-Binding Reactions by Dynamic Force Spectroscopy;419
18.1;15.1 Introduction;419
18.2;15.2 Dynamic Force Spectroscopy: Principles and Theory;421
18.2.1;15.2.1 Tip and Surface Immobilization;421
18.2.2;15.2.2 The Force--Distance Cycle;427
18.2.3;15.2.3 Spring Constant Determination;428
18.2.4;15.2.4 Force Distributions;430
18.2.5;15.2.5 Theory of Force Spectroscopy;431
18.2.6;15.2.6 The Effect of Hidden Barriers on Kinetic Parameters;437
18.2.7;15.2.7 Free Energy Surface Reconstruction from Nonequilibrium Single-Molecule Pulling Experiments;439
18.3;15.3 Applications of Dynamic Force Spectroscopy to Protein Interactions;442
18.3.1;15.3.1 Load-Dependent Dynamics of Protein Interactions;442
18.3.2;15.3.2 Energy Landscape Roughness of Protein Binding Reactions;447
18.3.3;15.3.3 Discrimination between Modes of Protein Activation;450
18.4;References;452
19;16 Probing Single Membrane Proteins by Atomic Force Microscopy;460
19.1;16.1 Introduction;460
19.1.1;16.1.1 A Short Synopsis on Membrane Proteins;460
19.1.2;16.1.2 Atomic Force Microscope as a Multifunctional Tool for Characterizing Membrane Protein Structure and Function;462
19.2;16.2 The Atomic Force Microscope;463
19.2.1;16.2.1 Principle and Setup;463
19.3;16.3 High-Resolution Imaging of Single Native Proteins;464
19.3.1;16.3.1 High-Resolution Imaging of Protein Assemblies;465
19.3.2;16.3.2 High-Resolution Imaging of Membrane Protein Diffusion;469
19.3.3;16.3.3 High-Resolution Imaging of Proteins at Work;471
19.4;16.4 Single-Molecule Force Spectroscopy of Membrane Proteins;471
19.4.1;16.4.1 Detecting Unfolding Intermediates and Pathways;472
19.4.2;16.4.2 Importance of Studying Membrane Protein (Un)Folding;473
19.4.3;16.4.3 Why Study Membrane Protein (Un)Folding Under a Mechanical Load?;475
19.4.4;16.4.4 Unfolding Forces Reflect Interactions That Stabilize Structural Regions;475
19.4.5;16.4.5 Origin of Unfolding Forces;475
19.4.6;16.4.6 Elucidating the Unfolding Routes of Membrane Proteins;478
19.4.7;16.4.7 Molecular Nature of Unfolding Intermediates;478
19.4.8;16.4.8 Membrane Proteins of Similar Structures Show Similar Unfolding Patterns;479
19.4.9;16.4.9 Detecting Intermediates and Pathways during Refolding of Membrane Proteins;479
19.4.10;16.4.10 Dynamic Energy Landscape;481
19.4.11;16.4.11 Following the Unfolding Contours of Mutant Proteins in an Energy Landscape;482
19.4.12;16.4.12 Protein Rigidity, Function, and Energy Landscape;485
19.4.13;16.4.13 Screening Membrane Proteins for Small-Molecule Binding;487
19.5;16.5 Outlook;488
19.5.1;16.5.1 Characterizing Factors That Sculpt the Energy Landscape;488
19.5.2;16.5.2 Approaches to Screening Drug Targets with Molecular Compounds;489
19.6;References;489
20;17 High-Speed Atomic Force Microscopy;497
20.1;17.1 Introduction;497
20.2;17.2 Basic Principle of AFM and Various Imaging Modes;498
20.3;17.3 Imaging Rate and Feedback Bandwidth;499
20.3.1;17.3.1 Image Acquisition Time and Feedback Bandwidth;500
20.3.2;17.3.2 Feedback Bandwidth as a Function of Various Factors;500
20.3.3;17.3.3 Feedback Operation and Parachuting;501
20.3.4;17.3.4 Refinement of Analytical Expressions for 0p and 0I ;503
20.4;17.4 Devices for High-Speed AFM;504
20.4.1;17.4.1 Small Cantilevers and Related Devices;504
20.4.2;17.4.2 Tip--Sample Interaction Detection;506
20.4.2.1;17.4.2.0 Amplitude Detectors;506
20.4.2.2;17.4.2.0 Force Detectors;508
20.4.3;17.4.3 High-Speed Scanner;509
20.4.3.1;17.4.3.0 Counterbalance;509
20.4.3.2;17.4.3.0 Mechanical Scanner Design;510
20.4.4;17.4.4 Active Damping;510
20.4.4.1;17.4.4.0 Feedback Q Control;511
20.4.4.2;17.4.4.0 Feedforward Active Damping;512
20.4.4.3;17.4.4.0 Practice of Active Damping of the Scanner;512
20.4.5;17.4.5 Dynamic PID Control;514
20.4.5.1;17.4.5.0 Dynamic PID Controller;514
20.4.5.2;17.4.5.0 Performance of Dynamic PID Control;516
20.4.6;17.4.6 Drift Compensator;518
20.4.7;17.4.7 High-Speed Phase Detector;518
20.5;17.5 High-Speed Bioimaging;520
20.5.1;17.5.1 Chaperonin GroEL;520
20.5.2;17.5.2 Lattice Defect Diffusion in Two-Dimensional Protein Crystals;521
20.5.3;17.5.3 Myosin V;523
20.5.4;17.5.4 Intrinsically Disordered Regions of Proteins;524
20.5.5;17.5.5 High-Speed Phase-Contrast Imaging;526
20.5.5.1;17.5.5.0 Compositional Mapping on Blended Polymers;526
20.5.5.2;17.5.5.0 Phase-Contrast Imaging of Myosin Filaments;527
20.5.5.3;17.5.5.0 Phase-Contrast Imaging of GroEL;529
20.6;17.6 Substrata for Observing Dynamic Biomolecular Processes;529
20.7;References;530
21;18 Recognition Imaging Using Atomic Force Microscopy;534
21.1;18.1 Introduction;534
21.2;18.2 Chemical Force Microscopy;535
21.2.1;18.2.1 Methods;536
21.2.2;18.2.2 Chemical Imaging of Live Cells;538
21.3;18.3 Recognition Imaging Using Force Spectroscopy;540
21.3.1;18.3.1 Methods;540
21.3.2;18.3.2 Measuring Molecular Recognition Forces;541
21.3.3;18.3.3 Molecular Recognition Imaging Using SMFS;543
21.4;18.4 Topography and Recognition Imaging;545
21.4.1;18.4.1 Methods;546
21.4.2;18.4.2 Applications of TREC Imaging;549
21.4.2.1;18.4.2.0 Chromatin;549
21.4.2.2;18.4.2.0 Bacterial Surface Layers;550
21.4.2.3;18.4.2.0 Membranes;553
21.4.2.4;18.4.2.0 Cells;555
21.5;18.5 Immunogold Imaging;557
21.6;18.6 Acknowledgments;560
21.7;References;560
22;19 Atomic Force Microscopy of Protein0Protein Interactions;564
22.1;19.1 Introduction;564
22.2;19.2 AFM Experimentation;565
22.2.1;19.2.1 AFM Principles;565
22.2.2;19.2.2 AFM Measurement of Single-Molecule Interactions;566
22.2.3;19.2.3 Tip and Sample Preparation;567
22.3;19.3 Determination of the Energy Landscape from the AFM Measurements ;569
22.4;19.4 Recent Applications;572
22.4.1;19.4.1 Molecular Basis for Multiple Energy Barriers along with Protein--Protein Dissociation;572
22.4.2;19.4.2 AFM Measurements on Living Cells;573
22.4.3;19.4.3 Energy Landscape Roughness of Protein--Ligand Interaction;575
22.5;19.5 Concluding Remarks;575
22.6;19.6 Acknowledgments;576
22.7;References;576
23;20 A New Approach to Analysis of Single-Molecule ForceMeasurements;580
23.1;20.1 Introduction;580
23.2;20.2 Force Probe Design and the Quest for Single-Molecule Statistics;581
23.3;20.3 Identifying Events Arising from Nonspecific and Multiple-Specific Attachments;582
23.3.1;20.3.1 Dealing with Nonspecific Events;584
23.3.2;20.3.2 Dealing with Multiple-Specific Events;584
23.4;20.4 Establishing Estimators for Initial State Probability and Distribution of Transitions;586
23.5;20.5 Two-State Transitions and the Direct Experimental Assay for Kinetic Rates;587
23.6;20.6 Experimental Example: Dissociating ICAM-1 from 2 -Integrin with Force Ramps;589
23.6.1;20.6.1 Microsphere Targets;589
23.6.2;20.6.2 PMN Targets;591
23.7;20.7 Experimental Example: Unfolding/Refolding of a Polyprotein with Force Ramps;592
23.7.1;20.7.1 Unfolding Kinetics;593
23.7.2;20.7.2 Refolding Kinetics;595
23.8;20.8 Acknowledgment;598
23.9;References;598
24;21 Single-Molecule Recognition: Extracting Informationfrom Individual Binding Events and Their Correlation;599
24.1;21.1 Introduction;599
24.2;21.2 Adhesion Frequency Assay;600
24.3;21.3 Thermal Fluctuation Assay;602
24.4;21.4 Analysis of Correlation among Outcomes from Sequential Tests;612
24.5;21.5 Acknowledgments;617
24.6;References;617
25;Subject Index;619