Biophysical Tools for Biologists: In Vitro Techniques
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Table of contents
- Contentsv
- Contributorsxv
- Prefacexix
- Dedication Pagexxiii
- Section 1: Solution Methods1
- Chapter 1: Binding: A Polemic and Rough Guide3
- I. Introduction4
- II. Binding Constants Provide an Entry into Thermodynamics5
- III. General Properties of Binding Isotherms7
- IV. Thermodynamics from Thermal Denaturation Methods10
- V. Completing the Thermodynamic Profile13
- VI. Thermodynamics in the Real World: Some Useful Strategies14
- VII. Ligand-Receptor Binding in the Absence of an Optical Signal16
- VIII. Toward High-Throughput Thermodynamics18
- IX. Summary20
- Acknowledgments21
- References21
- Suggested Reading23
- Chapter 2: Linked Equilibria in Regulation of Transcription Initiation25
- I. Introduction26
- II. Multiple Levels of Linkage in Transcription Regulation27
- III. A Road Map for Quantitative Studies of Assembly of Gene Regulatory Complexes30
- IV. Measurements of Binding Interactions in Transcription Regulation30
- A. Determine the Assembly State of the Protein(s) of Interest30
- B. Small Ligand Binding31
- C. Analysis of Binding Data34
- D. Tools for Measuring Protein-Protein Interactions35
- E. Protein-DNA Interactions40
- V. Case Studies of Multiple Linked Equilibria in Transcription Regulatory Systems44
- A. CRP and CytR: Combinatorial Control of Transcription in E. coli45
- B. The Progesterone Receptor: A Nuclear Hormone Receptor48
- References50
- Chapter 3: Biosensor-Surface Plasmon Resonance Methods for Quantitative Analysis of Biomolecular Int53
- I. Introduction54
- II. Rationale: Biomolecular Interactions with SPR Detection55
- III. Materials and Methods58
- A. Instrument Preparation58
- B. Sensor-Chip Surface Preparation61
- C. Sample Preparations64
- D. Data Collection and Processing65
- IV. Results and Data Analysis68
- A. Equilibrium Analysis69
- B. Kinetic Analysis72
- V. Summary74
- Acknowledgments75
- References75
- Chapter 4: Isothermal Titration Calorimetry: Experimental Design, Data Analysis, and Probing Macromo79
- I. Introduction80
- II. Calorimetry Theory and Operation82
- A. Heat Change Measurement and Theory82
- B. Variations in Ligand/Macromolecule Mixing Techniques83
- C. Commercial Availability85
- III. Thermodynamic ITC Experiments85
- A. Preface and Review of Basic Thermodynamics85
- B. Planning the Thermodynamic ITC Experiment88
- C. Running the Thermodynamic ITC Experiment88
- D. Analyzing Thermodynamic Data92
- E. Models95
- F. Error Analysis/Monte Carlo96
- G. Summary99
- IV. Kinetic ITC Experiments100
- A. Reaction Rate Versus Heat Rate100
- B. Planning the Experiment103
- C. Running the Kinetic ITC Experiment105
- D. Analyzing the Kinetic ITC Data107
- E. Models109
- F. Summary109
- V. Conclusions110
- Acknowledgments111
- References111
- Chapter 5: Differential Scanning Calorimetry115
- I. Introduction116
- II. DSC Instrumentation117
- III. Experimental Protocols and Preliminary Data Treatment119
- IV. Modeling DNA Unfolding126
- A. Freire-Biltonen Deconvolution126
- B. Cooperative DNA Unfolding129
- C. Melting Oligomeric Hairpins135
- V. Summary139
- Acknowledgments140
- References140
- Chapter 6: Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium143
- I. Introduction144
- A. Types of Problems That Can be Addressed145
- II. Basic Theory146
- A. Sedimentation Velocity146
- B. Sedimentation Equilibrium147
- III. Dilute Solution Measurements147
- IV. Concentrated and Complex Solutions149
- V. Instrumentation and Optical Systems150
- A. Absorbance152
- B. Interference153
- C. Fluorescence155
- D. High Concentrations and High Concentration Gradients157
- VI. Sample Requirements158
- VII. Sample Preparation160
- VIII. Sedimentation Velocity161
- A. Instrument Operation and Data Collection161
- B. Data Analysis165
- IX. Sedimentation Equilibrium168
- A. Instrument Operation and Data Collection168
- B. Monitoring Approach to Equilibrium170
- C. Data Analysis171
- X. Discussion and Summary174
- Acknowledgment175
- References176
- Chapter 7: Determination of Membrane Protein Molecular Weights and Association Equilibrium Constants181
- I. Introduction182
- II. Rationale183
- A. Why Use AUC?183
- B. General Considerations for Sedimentation Equilibrium Experiments of Membrane Proteins183
- C. Special Considerations for Sedimentation Equilibrium Experiments in the Presence of Detergent Mic185
- III. Materials and Methods190
- A. Expression and Purification of Membrane Proteins Used in This Study190
- B. Determination of the Density-Matching Point for C14SB191
- C. Sedimentation Equilibrium Experiments on Membrane Protein Samples Dispersed in C14SB193
- D. Sedimentation Velocity Experiments on OmpF194
- E. Viscosity Measurements195
- F. Density Measurements195
- IV. Results195
- A. Analysis of OMPLA Dimerization Energetics195
- B. Analysis of OmpF Trimer Stability in C14SB200
- C. Sedimentation Velocity Experiments on OmpF202
- V. Discussion206
- VI. Summary208
- Acknowledgments208
- References209
- Chapter 8: Basic Aspects of Absorption and Fluorescence Spectroscopy and Resonance Energy Transfer M213
- I. Introduction214
- II. Absorption Spectroscopy214
- A. Absorption Spectrophotometers215
- B. Measuring Absorption Spectrum216
- C. Common Applications217
- D. Microplate Reader Spectrophotometers222
- III. Fluorescence Spectroscopy223
- A. Introduction to Fluorescence223
- B. Fluorophores225
- C. Fluorescence Instrumentation228
- D. Absorption Versus Fluorescence229
- E. Measuring Emission and Excitation Spectra229
- F. Common Experimental Problems and Their Solutions230
- G. Fluorescence Quenching233
- H. Environmental Effects on Fluorescence234
- I. Fluorescence Resonance Energy Transfer236
- IV. Summary239
- Acknowledgments239
- References239
- Suggested Reading241
- Chapter 9: Applications of Fluorescence Anisotropy to the Study of Protein-DNA Interactions243
- I. Introduction and General Background244
- A. Fluorescence Anisotropy in a Nutshell244
- B. Anisotropy and Polarization245
- II. Advantages and Disadvantages of Anisotropy in Monitoring DNA Binding247
- III. Equipment248
- IV. Experimental Design and Performance251
- A. Reagents251
- B. Polarizer Calibration and G-Factor253
- C. Sample Compartment Control254
- D. Excitation and Emission Parameters254
- E. Data Collection255
- F. Data Analysis256
- G. Other Controls258
- H. Competition Experiments259
- V. Other Applications of Fluorescence Anisotropy to the Study of Protein-DNA Interactions260
- References260
- Chapter 10: Circular Dichroism and Its Application to the Study of Biomolecules263
- I. Introduction264
- II. Instrumentation and Sample Preparation266
- A. Instrumentation266
- B. Instrument Care and Calibration266
- C. Sample Preparation267
- D. Determination of Sample Concentration268
- III. Data Collection269
- A. Wavelength Range269
- B. Scanning Speed and Time Constant (or Response Time)270
- C. Spectral Bandwidth270
- D. Temperature Control270
- IV. Data Processing and Spectral Characteristics271
- A. Data Processing271
- B. Spectral Characteristics272
- V. Applications276
- A. Secondary Structure Content of Proteins276
- B. Detecting Altered Conformation278
- C. CD in the Study of Protein Stability282
- D. Determination of Equilibrium Dissociation Constants286
- VI. Summary290
- Acknowledgments290
- References290
- Chapter 11: Protein Folding and Stability Using Denaturants295
- I. Introduction296
- II. Rationale297
- III. Methods298
- A. General Features of Chemical Denaturation298
- B. Equilibration and Reversibility300
- C. Automated Titrations301
- D. Modeling the Unfolding Reaction302
- E. Calculating Folding Energies Within Transition Regions303
- F. Modeling the Effects of Denaturant on Folding Energies304
- G. Analyzing Denaturation Curves to Extract Thermodynamic Parameters307
- H. Spectroscopic Tests of the Two-State Model310
- IV. Materials310
- A. Preparation of Denaturant Solutions298
- B. Determination of Concentrations of Denaturant Solutions300
- V. Discussion313
- A. Baselines, Parameter Correlation, and Influence on Stability Estimates314
- B. The Physical Interpretation of m-Values317
- C. Identification of Domain Boundaries319
- D. Assessing the Effects of Missense Mutations on Structure and Stability319
- E. Identification of Interdomain Interactions321
- VI. Summary322
- Acknowledgments323
- References323
- Chapter 12: Hydrodynamic Modeling: The Solution Conformation of Macromolecules and Their Complexes327
- I. Introduction328
- II. Background to HBM329
- II. Model Construction332
- A. AtoB332
- B. PDB2AT, PDB2AM and MAP2GRID339
- C. MAKEPIXB (and HYDROPIX)340
- D. SOMO and ASAB1342
- IV. Model Visualization348
- A. RasMol and RAZ348
- B. VisualBeads349
- V. Hydration349
- VI. Hydrodynamic Calculations353
- A. HYDRO353
- B. HYDROPRO357
- C. SOLPRO361
- D. HYDROSUB361
- E. HYDROMIC362
- F. SUPCW/SUPCWIN362
- G. HYDRONMR (and Fast-HYDRONMR)365
- H. How Do Differently Constructed Models and Different Computations Compare?366
- VII. Advanced Hydrodynamic Calculations366
- A. MULTIHYDRO, MONTEHYDRO and Rayuela367
- B. BROWNRIG and BROWNFLEX369
- Concluding Comments370
- Acknowledgments370
- References370
- Chapter 13: X-Ray and Neutron Scattering Data and Their Constrained Molecular Modeling375
- I. Introduction376
- II. Rationale380
- A. Complementary Structural Approaches380
- B. Properties of X-Ray Scattering380
- C. Properties of Neutron Scattering381
- III. X-R.ay and Neutron Facilities382
- A. High-Flux Sources382
- B. X-Ray Instrumentation384
- C. Neutron Reactor Instrumentation386
- D. Spallation Neutron Instrumentation388
- IV. Experimental Methods390
- A. Applications for X-Ray and Neutron Beam Time390
- B. Sample Preparation for Scattering391
- C. Data Collection Strategies393
- D. Guinier Analyses396
- E. Distance Distribution Function Analyses398
- V. Constrained Scattering Modeling400
- A. Modeling Using Atomic Structures400
- B. Scattering Modeling by Other Approaches404
- C. Comparison with Sedimentation Coefficients406
- VI. Examples407
- A. Type 1: Self-Association of Complement C3d and Other Proteins407
- B. Type 1: Dimeric IgA410
- C. Type 2: SCR-1 and SCR-2 in Complement Receptor Type 2410
- D. Type 3: Antibody Structural Modeling of IgD411
- E. Type 3: Factor HSCR-6/8413
- F. Type N: Secretory Component of IgA and Other Multidomain Proteins414
- VII. Discussion416
- A. Summary and Future Considerations416
- B. Comparison of Scattering with Crystallography and NMR418
- C. Biological Relevance of Scattering Structures419
- Acknowledgments420
- References420
- Chapter 14: Structural Investigations into Microtubule-MAP Complexes425
- I. Introduction426
- II. Rationale428
- III. Methods430
- A. Helical Reconstructions of MAP-Microtubule Complexes430
- B. High-Resolution Surface Shadowing431
- C. 3-D Analysis of Microtubules Complexed with Tau433
- D. 3-D Analysis of a Novel Tubulin Conformation Induced by HURP437
- E. 3-D Analysis of a MAP That Locates to a Highly Specific Structure on the Microtubule Surface439
- IV. Discussion439
- Acknowledgments441
- References441
- Chapter 15: Rapid Kinetic Techniques445
- I. Introduction446
- II. Basic Theory448
- A. First-Order Reactions448
- B. Second-Order Reactions451
- III. Techniques452
- A. Flow Techniques452
- B. Relaxation Techniques454
- C. Flash Photolysis456
- IV. Instrumentation456
- A. Instrument Characteristics457
- B. Instrument Settings458
- V. Probes459
- A. Intrinsic Probes460
- B. Extrinsic Probes460
- C. Indicators, Linked Assays, and Biosensors461
- D. Fluorescence Anisotropy462
- VI. Experimental Design and Data Analysis463
- A. General Considerations463
- B. Displacement Experiments467
- C. Competition Experiments468
- D. Analysis of Stopped-Flow Anisotropy Data469
- VII. Complex Reactions470
- A. Two-Step Mechanisms470
- B. Multistep Mechanisms474
- VIII. Data Analysis in Practice474
- References476
- Chapter 16: Mutagenic Analysis of Membrane Protein Functional Mechanisms: Bacteriorhodopsin as a Mod479
- I. Introduction480
- II. Rationale481
- A. bR as a Model System481
- B. Mutagenic Rationale481
- C. Analytical Rationale481
- III. Materials and Methods486
- A. Prokaryotic Strains486
- B. Media and Growth Conditions486
- C. Reagents486
- D. Site-Directed Mutagenesis487
- E. Scanning Mutagenesis488
- F. Purification of bR Mutants488
- G. Cellular Screen488
- H. Assignment of Phenotypes and Mapping490
- I. Spectra of Purified D85N:Cysteine Mutants490
- IV. Results491
- A. In Vivo Random Mutagenesis of bR481
- B. Site-Directed Mutagenesis of bR481
- C. Scanning Mutagenesis of bR481
- V. Conclusions509
- References511
- Chapter 17: Quantifying DNA-Protein Interactions by Single Molecule Stretching517
- I. Introduction518
- II. Stretching Single DNA Molecules with Optical Tweezers519
- III. Force-Induced Melting of Single DNA Molecules521
- IV. T4 gp32 Interactions with DNA522
- A. Background522
- B. T4 gp32 Destabilizes Single DNA Molecules524
- C. Quantifying Equilibrium Protein Interactions with DNA525
- D. Salt Dependence of T4 Gene 32 Binding to ssDNA529
- V. Model for Salt-Dependent Regulation of T4 Gene 32 Binding to DNA531
- VI. Conclusions537
- Acknowledgments538
- References538
- Chapter 18: Isotopomer-Based Metabolomic Analysis by NMR and Mass Spectrometry541
- I. Introduction542
- II. Rationale543
- III. Materials544
- A. Extractions544
- B. Sample Preparation545
- IV. Methods547
- A. Application of NMR to Isotopomer Analysis547
- B. 2-D NMR and Isotope Editing552
- C. Application of MS to Isotopomer Analysis566
- V. Discussion577
- Short Glossary of Terms578
- Acknowledgments581
- References581
- Chapter 19: Following Molecular Transitions with Single Residue Spatial and Millisecond Time Resolut589
- I. Introduction590
- A. What Is Footprinting?590
- B. Quantitative Footprinting592
- C. The Hydroxyl Radical (bullOH) as a Footprinting Probe592
- D. Nucleic Acid OH Footprinting593
- E. Protein bullOH Footprinting593
- F. Generation of Hydroxyl Radicals for Millisecond Resolution Time-Resolved Studies594
- II. Acquisition of bullOH Footprinting Time-Progress Curves595
- A. Reagents and Solutions595
- B. Minimizing RNase Contamination When Working With RNA596
- C. Determine the Correct Amount of bullOH Production597
- D. A.General Protocol for Fast Fenton Footprinting598
- III. Data Reduction and Production of Time-Progress Curves602
- A. Quantitation of the bullOH Footprinting Reaction Products602
- B. Assembling Nucleotides into Sites of bullOH Protection or Hypersensitivity604
- C. Generation of Individual-Site Time-Progress Curves605
- IV. Interpretation of Individual Nucleotide Time-Progress Curves605
- A. Analysis by Inspection606
- B. Time-Progress Curve Clustering606
- C. Kinetic Modeling608
- Acknowledgments611
- References611
- Chapter 20: Methods and Applications of Site-Directed Spin Labeling EPR Spectroscopy617
- I. Background and Methods618
- A. The Technique618
- B. Motion622
- C. Accessibility628
- D. Distances633
- II. Examples636
- A. Secondary Structure636
- B. Membrane Depth639
- C. Protein Interactions641
- D. Peptides644
- E. Unfolding and Kinetics648
- F. Distances to Determine Structural Arrangements and Monitor Dynamics650
- III. Conclusion653
- References653
- Chapter 21: Fluorescence Correlation Spectroscopy and Its Application to the Characterization of Mol659
- I. Introduction660
- II. Fluorescence Correlation Spectroscopy663
- A. Translational Diffusion664
- III. FCS Experimental Setups666
- IV. Sample Preparation and Some Practical Considerations668
- A. Fluorophores668
- B. Sample Labeling and Practical Tips669
- V. Fluorescence Cross-Correlation Spectroscopy671
- VI. Illustrative Examples of FCS Applications672
- A. Use of FCS to Quantify Translational Diffusion with Varying Solution Conditions672
- B. FCS in Binding Assays673
- C. Use of FCS to Detect Conformational Changes674
- D. Use of FCS to Monitor Polymerization675
- E. FCS as a Diagnostic or Quality Control Tool676
- F. Use of FCCS676
- VII. Conclusions676
- Acknowledgments677
- References677
- Suggested Reading678
- Chapter 22: A Practical Guide on How Osmolytes Modulate Macromolecular Properties679
- I. Introduction680
- A. Overview680
- B. Organic Osmolytes Versus Salts681
- C. Osmolytes: How Are They Useful?682
- D. Notation682
- II. Experimental Systems683
- A. Protein Folding683
- B. Ligand Binding and Molecular Association687
- C. Protein Solubility693
- D. Stressing Assemblies: DNA, Lipids, and Other Macromolecules695
- III. Experimental Methods698
- A. Osmometry698
- B. Calorimetry701
- C. Spectroscopy705
- D. Ion Channels as Probes707
- E. Scattering Techniques708
- F. Phase Diagram Method710
- IV. Solvation Information714
- A. Activity Coefficients714
- B. Kirkwood-Buff Approach716
- C. Charged Osmolytes721
- V. Prospects724
- Acknowledgments725
- References726
- Section 2: Computational Methods737
- Chapter 23: Stupid Statistics!739
- I. Introduction740
- II. Statistics of Data: The 10-Min Review742
- A. Sampling: Location and Dispersion Indices742
- B. Probability Distributions742
- C. A.Simple MC Experiment744
- D. The Central Limit Theorem745
- E. The Importance of Finite Variance747
- III. Linear Least Squares„Theory749
- A. Tenets: Demands and Payback749
- B. The LLS Solution: Matrix Notation750
- C. Parameter Uncertainties: The Variance-Covariance Matrix751
- D. Uncertainties in Variances: The Chi-Square Distribution753
- E. Confidence Limits on Estimated Parameters: The t-Distribution753
- F. Correlation754
- G. Correlated Data754
- H. Fitting with Constraints755
- I. Error Propagation756
- J. Systematic Error756
- IV. Linear Least Squares„Monte Carlo Illustrations757
- A. The Straight Line with Constant Error757
- B. The Straight Line with Proportional Error: Neglect of Weights761
- V. Nonlinear Least Squares763
- A. When Is It Nonlinear and What Then?763
- B. Computational Methods763
- C. The 10% Rule of Thumb765
- VI. Applications and Illustrations766
- A. Univariate Calibration: Beyond the Straight Line766
- B. "Going Straight" ... by Shunning the Straight Line767
- C. Error Propagation: How Good Is a vH Analysis?769
- D. Correlated Fitting: The Guggenheim Method770
- E. Experiment Design Using the V Matrix: ITC772
- F. Wrapping Up: Assessing and Presenting Results776
- VII. Summary778
- Acknowledgment778
- References779
- Chapter 24: Nonlinear Least-Squares Fitting Methods781
- I. Introduction781
- II. Formulate a Hypothesis-Based Mathematical Model785
- III. Determining the Optimal Parameters of the Model787
- IV. Distinguishing Between Multiple Mathematical Models791
- V. Estimate the Precision of the Model Parameters796
- VI. Cross-Correlation of the Estimated Parameters800
- VII. Uniqueness of the Parameters803
- VIII. Conclusions804
- Acknowledgments805
- References805
- Chapter 25: Methods for Simulating the Dynamics of Complex Biological Processes807
- I. Introduction808
- II. Rationale809
- III. Modeling810
- A. Running Example810
- B. From Cartoons to Dynamic Model Diagrams811
- C. Compartments814
- D. Events and State Transitions814
- E. Reaction Kinetics815
- F. Timing of Events in Chemical Reactions821
- IV. Simulation823
- A. Stochastic Methods: The Behavior of Individual Entities823
- B. Deterministic Methods: The Average Behavior of Many Entities831
- C. Comparison of Stochastic and Deterministic Simulation834
- V. Modeling and Simulation in Practice835
- A. Modeling835
- B. Simulation838
- VI. Concluding Remarks840
- Acknowledgments840
- References840
- Chapter 26: Computational Methods for Biomolecular Electrostatics843
- I. Introduction844
- II. Electrostatics in Cellular Systems844
- A. Biomolecule-Ion Interactions845
- B. Biomolecule-Ligand and -Biomolecule Interactions846
- III. Models for Biomolecular Solvation and Electrostatics847
- A. Explicit Solvent Methods847
- B. Implicit Solvent Methods848
- C. Poisson-Boltzmann Methods851
- D. Simpler Models854
- E. Limitations of Implicit Solvent Methods855
- IV. Applications856
- A. Solvation Free Energy856
- B. Electrostatic Free Energy857
- C. Folding Free Energies857
- D. Binding Free Energies858
- E. pKa Calculations860
- F. Biomolecular Association Rates861
- V. Conclusion and Future Directions861
- Acknowledgments862
- References862
- Chapter 27: Ligand Effects on the Protein Ensemble: Unifying the Descriptions of Ligand Binding, Loc871
- I. Introduction872
- II. The Effect of pH on the Conformational Ensemble875
- A. Thermodynamic Model of the Conformational Ensemble875
- B. pH Modulation of the Conformational Ensemble878
- III. Results and Discussion881
- A. pH Dependence of the Ensemble of SNase881
- B. Microscopic Origins of the pH Dependence of Stability884
- C. Application to a Hyperstable Variant of Nuclease885
- IV. Summary and Conclusions886
- References889
- Chapter 28: Molecular Modeling of the Cytoskeleton893
- I. Introduction894
- II. Simulation Methods894
- A. Molecular Dynamics894
- B. Brownian Dynamics896
- C. Molecular Docking897
- III. Applications of Molecular Modeling898
- A. Molecular Dynamics Applications898
- B. Brownian Dynamics Applications901
- C. Ligand-Protein and Protein-Protein Docking902
- IV. Related Methodologies904
- A. QSAR and CoMFA904
- B. Electrostatics Based Approaches905
- C. Normal Mode Analysis906
- V. Conclusions907
- Acknowledgments907
- References907
- Chapter 29: Mathematical Modeling of Cell Migration911
- I. Introduction912
- II. Cell Protrusion912
- A. Single-Filament Modeling912
- B. Many-Filament Modeling917
- C. Continuum Modeling920
- III. Cell Adhesion and Retraction926
- A. Myosin-Based Retraction927
- B. Non-Myosin-Based Contractility929
- IV. Whole-Cell Models930
- V. Summary and Future Outlook933
- Acknowledgments934
- References934
- Index939
- Volumes In series965
Driven in part by the development of genomics, proteomics, and bioinformatics as new disciplines, there has been a tremendous resurgence of interest in physical methods to investigate macromolecular structure and function in the context of living cells. This volume in Methods in Cell Biology is devoted to biophysical techniques in vitro and their applications to cellular biology. The volume covers methods-oriented chapters on fundamental as well as cutting-edge techniques in molecular and cellular biophysics. This book is directed toward the broad audience of cell biologists, biophysicists, pharmacologists, and molecular biologists who employ classical and modern biophysical technologies or wish to expand their expertise to include such approaches. It will also interest the biomedical and biotechnology communities for biophysical characterization of drug formulations prior to FDA approval.
* Describes techniques in the context of important biological problems
* Delineates critical steps and potential pitfalls for each method
* Includes full-color plates to illustrate techniques
* Describes techniques in the context of important biological problems
* Delineates critical steps and potential pitfalls for each method
* Includes full-color plates to illustrate techniques