Analytical Ultracentrifugation (eBook)

Susumu Uchiyama, Ph. D., Associate Professor, Department of Biotechnology, Graduate School of Engineering, Osaka University, 2‐1 Yamadaoka, Suita, Osaka 565‐0871, JapanFumio Arisaka, Ph. D., Professor, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori‐ku, Yokohama 226‐8501, JapanWalter F. Stafford, III, Ph.D., Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USATom Laue, Ph.D., Professor, Biomolecular Interaction Technologies Center (BITC), Center to Advance Molecular Interaction Science (CAMIS), University of New Hampshire, Rudman Hall, Room 379, 46 CollegeRoad, Durham, NH 03824, USA

Preface 6
Contents 8
Part I Introduction 11
1 Important and Essential Theoretical Aspects of AUC 12
1.1 Introduction 12
1.2 Principle for Sedimentation and Diffusion 13
1.2.1 Sedimentation 13
1.2.2 Diffusion 16
1.3 Sedimentation Velocity 18
1.3.1 Lamm Equation 18
1.3.2 Relationship Between s and M 19
1.3.3 Molecular Shape and f/f0 19
1.3.4 Sedimentation Coefficients Estimated from the X-ray Structure of the Proteins 20
1.4 Sedimentation Equilibrium 20
1.5 Conclusions 22
References 23
2 Experimental Design and Practical Aspects 24
2.1 Optical Systems in XL-A and XL-I 24
2.2 Rotors and Centerpieces 26
2.3 Determination of the Appropriate Temperature and Rotor Speed for Measurements 27
2.4 Data Analysis of Sedimentation Velocity and Sedimentation Equilibrium 28
2.5 Important and Useful Websites Related to Analytical Ultracentrifugation 29
References 30
Part II AUC Instrumentation and Analysis 31
3 The CFA Analytical Ultracentrifuge Architecture 32
3.1 Introduction 32
3.2 Hardware Description 33
3.2.1 Hardware Design Rationale 33
3.2.1.1 Data Acquisition Stacks 34
3.2.2 Parallel Digital Bus 34
3.2.3 Serial Bus 34
3.2.4 Motion Control Bus 35
3.2.5 Power Distribution 35
3.2.6 Stack Electronics 35
3.2.6.1 Master Clock 35
3.2.6.2 Synchronizer 36
3.2.6.3 Analog to Digital Converter (ADC) 38
3.2.6.4 Memory 38
3.2.6.5 Source/Detector Control 38
3.3 Software Description 39
3.3.1 Software Design Rationale 39
3.3.1.1 Inter-process Communications 39
3.3.2 Parallel Digital 40
3.3.3 Motion Control 40
3.3.4 Centrifuge Hardware 40
3.3.5 Experiment 41
3.3.6 Hardware Inventory 41
3.3.7 Master Daemon 42
3.4 Databases 42
3.4.1 Database Rationale 42
References 44
4 Fluorescence Detection System 45
4.1 Development of the Fluorescence Detection System 45
4.2 FDS Optical Setup 46
4.3 Advantages and Limitations 47
4.4 Pre-experimental Requirements 48
4.4.1 Sample Labeling 48
4.4.2 Meniscus Detection 50
4.4.3 Sample Loading 50
4.5 Experimental Operation Procedures 51
4.5.1 Experiment Initiation 51
4.5.2 Position Calibration 52
4.5.3 Common Signal Attenuation Issues 53
4.5.3.1 Inner Filter Effect 53
4.5.3.2 Light Beam Clipping 54
4.5.3.3 Focal Height Drift 55
4.5.4 Data Acquisition and Analyses 55
4.5.5 Post-experimental Considerations 55
4.6 Applications of AU-FDS 56
4.6.1 High-Affinity Interactions 56
4.6.2 Studies in Complex Backgrounds 58
4.6.2.1 Behavior of Protein Therapeutics in Blood Serum 58
4.6.2.2 Aggregation of Serum Proteins Linked to Systemic Amyloidosis 60
4.6.2.3 Aggregation of Huntingtin in Cell Lysates 62
4.6.3 Identifying Enzyme Complexes 63
4.7 Conclusions 64
References 64
5 The Multiwavelength UV/Vis Detector: New Possibilities with an Added Spectral Dimension 68
5.1 Introduction 68
5.2 The Multiwavelength Detector 69
5.3 History of MWL-AUC Development 70
5.3.1 First-Generation MWL Detectors 71
5.3.2 Second-Generation MWL Detectors 73
5.3.3 Mechanical Parts of Second-Generation MWL-AUC 74
5.3.4 Electronics of MWL-AUC 75
5.3.5 Software of MWL-AUC 75
5.4 MWL-AUC in the Scientific Literature 76
5.5 Conclusions 83
References 84
6 SEDANAL: Model-Dependent and Model-Independent Analysis of Sedimentation Data 86
6.1 Introduction 86
6.2 Modules of SEDANAL 87
6.2.1 Preprocessor 87
6.2.2 Fitter 87
6.2.3 Simulator 88
6.2.4 DCDT and WDA 88
6.2.5 BioSpin 89
6.2.6 Chemical Equilibrium Calculator 89
6.2.7 Model Editor and Equation Editor 89
6.2.7.1 Determination of the Components 95
6.2.7.2 Determining Equations for Equilibrium 97
6.2.7.3 Representation of the Jacobian 101
6.2.7.4 Determination of the Conservation Relations 101
6.2.7.5 Determination of the Jacobian 102
6.2.7.6 Methods of Calculation 103
6.2.8 Wide Distribution Analysis 104
6.2.8.1 Advantages of WDA vs. DCDT 104
6.2.8.2 Multiwavelength Analysis 104
6.2.8.3 Extracting Component Extinction Spectra 105
6.2.9 Summary 106
References 106
7 SEDANAL: Global Analysis of General Hetero- and Self-Associating Systems by Sedimentation Equilibrium 108
7.1 Introduction 109
7.2 Theoretical Background 109
7.2.1 Radially Dependent Baseline Subtraction 110
7.2.2 Conservation of Mass Relations 111
7.2.2.1 One Species 111
7.2.2.2 Monomer-Oligomer 112
7.2.2.3 Hetero-Association 113
7.2.3 Self-Associating System with Incompetent or Adventitious Species 114
7.2.4 Non-ideality 115
7.3 Fitting to a Heterologous Interacting System 118
7.3.1 Fitting Strategy 119
7.3.2 BIOSPIN 121
7.3.3 Error Analysis 121
7.3.4 Summary 121
References 122
8 Analytical Ultracentrifugation Data Analysis with UltraScan-III 123
8.1 Introduction 123
8.2 UltraScan-III Components 125
8.3 UltraScan-III Concepts 126
8.4 The UltraScan Analysis Workflow 127
8.4.1 Overview 127
8.4.2 Importing Experimental Data 128
8.4.3 Editing Experimental Data 130
8.4.4 Data Refinement 131
8.4.5 Remote Supercomputer Analysis 132
8.4.6 Advanced Data Analysis 133
8.4.7 Global Analysis 136
8.4.8 Models 137
8.4.9 Visualization 137
8.4.10 Reports 143
8.5 Simulation Programs 143
8.6 Utilities 144
References 146
Part III Hydrodynamic Modeling 148
9 Introduction: Calculation of Hydrodynamic Parameters 149
9.1 Introduction 149
9.2 Hydrodynamics 101: A Simple Tutorial 153
9.3 Current Approaches 157
9.3.1 Rigid Body Modelling 157
9.3.2 Flexible Body Modelling 162
9.4 Pros and Cons of Current Approaches 163
9.5 Concluding Comments and Outlook 165
References 166
10 Calculation of Hydrodynamic Parameters: US-SOMO 170
10.1 Introduction 170
10.2 Operational Principles of the Bead Modeling Methods 171
10.3 The US-SOMO Main GUI Interface and Option Settings 174
10.3.1 PDB Function Area 174
10.3.2 BEAD Model Function Area 179
10.3.3 Hydrodynamic Calculations Area 182
10.4 The US-SOMO Model Classifier Module 185
10.5 The US-SOMO Batch Mode/Cluster Operation Module 187
10.6 The US-SOMO BEST Interfaces 189
10.7 Conclusions 192
References 193
11 The HYDRO Software Suite for the Prediction of Solution Properties of Rigid and FlexibleMacromolecules and Nanoparticles 195
11.1 Introduction 195
11.1.1 A Broad Overview 195
11.1.2 The Various Kinds of Bead Modeling 196
11.1.3 Coarse-Grained, Mesoscale, and Multiscale Modeling 197
11.1.4 The HYDRO Suite 199
11.2 Rigid Particles 199
11.2.1 HYDRO++ 200
11.2.2 HYDROPRO and HYDRONMR 200
11.2.3 HYDROMIC 202
11.2.4 HYDROSUB 203
11.2.5 HYDROPIX 204
11.3 Flexible Particles 204
11.3.1 Introduction 204
11.3.2 A General Bead-and-Spring Model 205
11.3.3 MONTEHYDRO and SIMUFLEX 207
11.3.4 Examples: Dendrimers and Intrinsically Disordered Proteins 208
11.3.5 Wormlike Chains 210
11.4 Global Fitting for Structural Determination: HYDROFIT 211
11.4.1 HYDFIT 211
11.4.2 Multi-HYDFIT 212
11.5 Conclusions 213
References 214
12 Accurate Hydrodynamic Modeling with the Boundary Element Method 218
12.1 Introduction 219
12.2 The Integral Equations of Stokes Flow 221
12.2.1 Stick Boundary Conditions 222
12.2.2 Slip Boundary Conditions 223
12.3 Hydrodynamic Hydration Reinterpreted 224
12.4 Studies of Globular Proteins 231
12.5 Combination of Molecular Dynamics Simulation and Hydrodynamic Modeling 234
12.5.1 Implicit Water MD of ?-Chymotrypsin 234
12.5.2 Explicit Water MD of Small Proteins 236
12.5.3 Explicit Water MD Simulations of Trastuzumab 240
12.6 Conclusions 242
References 243
Part IV Applications of AUC: Material Science 247
13 Hydrodynamic Analysis of Synthetic Permanently Charged Polyelectrolytes 248
13.1 Introduction 248
13.2 Polyelectrolyte Synthesis 251
13.3 Supporting Methods 252
13.3.1 Solution Preparation 252
13.3.2 Dilution Viscometry 254
13.3.3 Densitometry 255
13.4 Analytical Ultracentrifugation 255
13.4.1 Synthetic Boundary Experiments 255
13.4.2 Sedimentation Velocity Experiments 256
13.4.2.1 The Limiting Sedimentation Coefficient s0 256
13.4.2.2 The Frictional Ratio f/f0 260
13.4.2.3 Molar Masses 261
13.4.3 Scaling Relationships 261
13.5 Conclusions 263
References 263
14 Different Levels of Self-Sufficiency of the Velocity Sedimentation Method in the Study of Linear Macromolecules 266
14.1 Introduction 266
14.2 Essential Concepts of Molecular Hydrodynamics and Parameters Characterizing the Conformation of Linear Polymers 269
14.3 Relationships Between the Experimental Hydrodynamic Values and the Macromolecular Characteristics 270
14.4 Degree of Solution Dilution 272
14.5 Error of ks Determination 273
14.6 Scaling Relation Between ks and s0 273
14.7 Scaling Relations Between Other Hydrodynamic Values and Molar Mass 278
14.8 Another Level of Self-Sufficiency of Velocity Sedimentation Data: Sedimentation Parameter and Hydrodynamic Invariant 280
14.9 Relationship Between Hydrodynamic Values and Conformational Parameters A and d in the Model of Wormlike Chain 284
14.10 Multi-Hydfit Program 288
14.11 Further Steps of the Analysis of the Hydrodynamics Data of Homologous Series of Linear Macromolecules 288
14.12 Examples of Handling of Velocity Sedimentation Data with Sedfit Suite 291
14.12.1 Model-Less Method for Calculating the Apparent Differential Sedimentation Coefficient Distribution g*(s) 291
14.12.2 Continuous c(s) Model in Sedfit Suite 293
14.12.3 Continuous c(s) Model with General Scaling Law in Sedfit Suite 295
14.13 Conclusions 298
References 299
Part V Applications of AUC: Biological Science 305
15 Applications of Analytical Ultracentrifugation to Membrane Proteins 306
15.1 Introduction 306
15.2 Theoretical Considerations for Mass Evaluation Using the Analytical Ultracentrifuge 307
15.3 Sedimentation Equilibrium of Membrane Proteins 308
15.3.1 Density Matching Strategy 308
15.3.2 Procedure for Density Matching the Detergent in a Buffer of Interest 310
15.3.3 Sedimentation Equilibrium Data Collection and Analysis 311
15.3.4 Analysis of a Reversibly Equilibrating Membrane Protein Interaction 312
15.3.5 Forced Cohabitation of Membrane Proteins 315
15.3.6 Non-equilibrating Systems 316
15.4 Sedimentation Velocity Data Collection and Analysis 317
15.5 Approaches to Disentangle Protein and Detergent Contributions to the Sedimentation Coefficient 317
15.6 Sedimentation Velocity Characterization of Surfactants 319
15.7 Examples of Membrane Protein Complexes Analyzed in the Ultracentrifuge 319
References 320
16 Protein-Ligand Interactions 323
16.1 Introduction 323
16.2 Protein-Small Molecule Interactions 324
16.2.1 Biotin Protein Ligase 325
16.2.2 Dihydrodipicolinate Synthase 328
16.3 Protein-Lipid Interactions 333
16.3.1 Lipoprotein Biology and Apolipoprotein E 333
16.3.2 Models of Lipoprotein Particles 333
16.3.3 Flotation Velocity Analysis of Synthetic Lipid Emulsions 335
16.4 AUC Analyses of ApoE-Emulsion Interactions 340
16.5 Conclusions 344
References 344
17 AUC in the High Concentration of Salts/Cosolvent 348
17.1 Introduction 348
17.2 Experimental Considerations 349
17.2.1 The Sample 349
17.2.2 Cell Preparation for Analytical Ultracentrifugation 350
17.2.3 Programs for Analysis of Ultracentrifugation Data 350
17.3 Definition of the Components and Symbols Used 351
17.3.1 Definition of the Components 351
17.3.2 Symbols 351
17.4 Sedimentation in a Complex Solvent 352
17.4.1 Sedimentation Equilibrium in a Complex Solvent 352
17.4.2 Sedimentation Velocity in a Complex Solvent 353
17.4.3 Brief Conclusion Concerning AUC in a Complex Solvent 353
17.5 Considering a Diluted Macromolecule in a Complex Solvent 354
17.5.1 The Density Increments (??/?c2) 354
17.5.1.1 Partial Specific Volumes Are Derived from (?/?c2) 354
17.5.1.2 The Density Increment at Constant Chemical Potential of Solvent Components (?/?c2) 356
17.5.1.3 Illustration of the Differences Between the Two Density Increments 356
17.5.1.4 About the Measurement of Partial Specific Volumes in Solvent of Different Densities in AUC 357
17.5.2 The Preferential Solvent Binding Parameters 357
17.5.2.1 Units and Relations Between These Parameters 357
17.5.2.2 Preferential Binding Parameters Determine the Density Increment in the Ultracentrifuge 358
17.5.2.3 Molecular Description from the Preferential Binding Parameters 359
17.5.2.4 Models for Describing Solvation 360
17.6 Link Between Protein Equilibrium and Interactions with Solvent Components 361
17.6.1 Preferential Binding Parameters are True Thermodynamic Parameters 361
17.6.2 The Wyman Linkage Relationship 362
17.6.3 Implications 363
17.7 Conclusion 364
References 364
18 Aspects of the Analytical Ultracentrifuge Determination of the Molar Mass Distribution of Polysaccharides 367
18.1 Introduction 368
18.2 SEC-MALS 368
18.3 Sedimentation in the Analytical Ultracentrifuge 369
18.4 Sedimentation Velocity: `Extended Fujita' Method 371
18.5 Sedimentation Equilibrium (SE): SEDFIT-MSTAR 373
18.6 Concluding Remarks 377
References 377
Part VI Applications of AUC: Biopharmaceuticals 379
19 Use of Analytical Ultracentrifugation as an Orthogonal Method for Size Exclusion Chromatography: Assuring Quality for Therapeutic Protein Products and Meeting Regulatory Expectations 380
References 385
20 Biopharmaceuticals: Application of AUC-SV for Quantitative Analysis of Protein Size Distributions 387
20.1 Introduction 388
20.2 Applications of AUC-SV for Biopharmaceutical Development 389
20.2.1 The Utility of AUC-SV as an Orthogonal Method for Quantitation of Protein Aggregation 389
20.2.2 Use of AUC-SV to Guide SEC Method Development 390
20.3 Limitations of AUC-SV for Biopharmaceutical Development 393
20.3.1 Precision of the AUC-SV Method 394
20.3.2 Robustness of the AUC-SV Method 395
20.4 Tailoring the AUC-SV Method to Achieve Its Purpose 396
20.4.1 Optimization of AUC Method Parameters 396
20.4.2 Qualification of AUC-SV as a Characterization Method 396
20.4.3 Concurrent Development and Qualification of a SEC Method 401
20.4.4 Validation of a SEC Method for Routine Use (Correlation of SEC to AUC-SV) 402
20.5 Conclusions 406
References 407
21 Biopharmaceutical Evaluation of Intermolecular Interactions by AUC-SE 409
21.1 Introduction 409
21.2 Theory of AUC-SE Analysis to Determine the Intermolecular Interaction 411
21.3 Analysis of Intermolecular Interaction in Biopharmaceuticals 413
21.4 Evaluation of Intermolecular Interaction in Biopharmaceuticals 417
21.5 Predictions of Aggregation Propensity and Viscosity at High Concentrations Based on B2 at Low Concentrations 424
21.6 Conclusions 426
References 427
Part VII AUC of High-Concentration Systems and Non-ideal Solutions 431
22 Johnston-Ogston Effects in Two Simulated Systems of Polystyrene Beads That Are Polydispersewith Respect to Density 432
22.1 Introduction 433
22.2 Common Features of the Two Model Systems 433
22.2.1 The Implicit Solvent Component of the Model Systems 434
22.2.2 Common Characteristics of the PS Beads of the Model Systems 434
22.2.3 Concentration Dependence of Transport 436
22.3 Distinguishing Features of the Two Model Systems 438
22.4 Simulation Parameters 439
22.5 Notation 441
22.6 Results 441
22.6.1 Ka==30.325 ml/g: Two Components Account for Three of the Species Present 445
22.6.2 Ka Undefined: All Species Are Single-Species Components 446
References 451
23 Analysis of Nonideal, Interacting, and Noninteracting Systems by Sedimentation Velocity AnalyticalUltracentrifugation 452
23.1 Background Theory 452
23.1.1 Self-Associations 453
23.1.1.1 Self-Association: Two Species 453
23.1.1.2 Self-Associations: Multispecies 453
23.1.2 Hetero-Associations 454
23.1.2.1 Hetero-Association: Bimolecular, Single Step 454
23.1.2.2 Hetero-Association: Bimolecular, Two Step 454
23.2 General Discussion of Non-ideality 455
23.2.1 Hydrodynamic Non-ideality 455
23.2.1.1 Single Macromolecular Component, Single-Species Systems 455
23.2.2 Thermodynamic Non-ideality 456
23.2.3 Cross Diffusion Coefficients 461
23.2.4 Nonideal, Interacting Systems: Effects of Non-ideality on the Equilibrium Constant 465
23.3 Curve Fitting: Numerical Solutions to the Lamm Equation 467
Appendix 1 Hydrodynamic Non-ideality 469
References 471
24 Techniques for Dissecting the Johnston-Ogston Effect 472
24.1 Introduction 473
24.2 Methods 474
24.3 Self-Nonidealty Measurements 476
24.4 Cross-Term Nonideality Measurements 479
24.5 Summary of the Nonideality Data and Application to SEDANAL 483
References 486
25 Acquisition and Analysis of Data from High ConcentrationSolutions 488
25.1 Introduction 488
25.1.1 Reasons for Interest 489
25.1.2 Why an AUC Approach Could be of Value 489
25.2 Sedimentation Velocity Analysis (SVA) 490
25.2.1 What is SVA and What Can It Tell Us? 490
25.2.2 Fluid Dynamics of Sedimentation at High Solute Concentration 491
25.2.3 Biophysical Studies of Sedimentation Over the Range of Solute Concentration 492
25.2.4 Fluid Dynamics and Biophysical Analysis – A Common Approach? 493
25.2.5 Practical Aspects of SV Analysis of High Concentration Solutions 493
25.3 Sedimentation Equilibrium Analysis (SE) 494
25.3.1 Method of Simulation for Analysis of SE Data 495
25.3.1.1 M_INVEQ – A Revised Formulation for SE Analysis of High Concentration Data 496
25.3.2 Application of M_INVEQ Analysis to Real Systems 501
25.3.2.1 Application of M_INVEQ to BSA Solutions Containing Dimer and Trimer 501
25.4 Discussion 506
References 508
Part VIII New Applications of AUC 510
26 Detection and Quantitative Characterization of Macromolecular Heteroassociations via Composition Gradient Sedimentation Equilibrium 511
26.1 Introduction 511
26.2 Experimental Procedure 513
26.3 Analysis of the Dependence of MS,av* upon wA and wB 514
26.4 Experimental Test 517
26.5 Discussion 518
Appendix: Verification of the Absence of Hyper- or Hypo-chromicity 519
References 519