Methods in molecular biophysics: structure, dynamics, function

Cover......Page 1
Half-title......Page 3
Title......Page 7
Copyright......Page 8
Dedication......Page 9
Contents......Page 11
Foreword 01......Page 13
Foreword 02......Page 15
Preface......Page 17
A brief history and perspectives......Page 19
Languages and tools......Page 23
Length and time scales in biology......Page 24
The structure-function hypothesis......Page 25
Complementarity of physical methods......Page 26
Thermodynamics......Page 27
Hydrodynamics......Page 28
Radiation scattering......Page 29
Spectroscopy......Page 30
Single-molecule detection......Page 31
Part A Biological macromolecules and physical tools......Page 37
A1.1 Historical review......Page 39
A1.2.2 Partial volume......Page 40
A1.2.3 Colligative properties......Page 42
A1.2.4 Chemical potential and activity......Page 43
A1.2.6 Osmotic pressure......Page 44
A1.3 Macromolecules, water and salt......Page 46
A1.3.1 Ionic strength and Debye-Hückel theory......Page 48
A1.3.3 Macromolecule-solvent interactions......Page 49
A1.4 Checklist of key ideas......Page 53
Suggestions for further reading......Page 55
A2.1 Historical review and biological applications......Page 56
A2.2 Biological molecules and the flow of genetic information......Page 58
A2.3 Proteins......Page 61
A2.3.2 Structures of higher order......Page 62
Tertiary structure......Page 65
Quaternary structure......Page 67
A2.4.1 Chemical composition and primary structure......Page 68
DNA......Page 70
RNA......Page 71
A2.5 Carbohydrates......Page 72
A2.5.1 Chemical composition and primary structure......Page 73
A2.5.2 Higher-order structures......Page 75
A2.6.1 Chemical composition......Page 76
A2.6.2 Higher-order structures......Page 78
A2.7 Checklist of key ideas......Page 79
Suggestions for further reading......Page 81
A3.1 Historical review......Page 83
Sines and cosines......Page 85
Complex exponentials......Page 89
Polarization......Page 92
A simple harmonic oscillator......Page 93
Normal modes in one dimension......Page 95
Normal modes in three dimensions......Page 96
Periodic functions, Fourier series and Fourier transforms......Page 97
The Dirac delta function......Page 99
The Fourier integral and continuous Fourier transform......Page 100
Convolution......Page 101
Planck’s constant, energy quanta and photons......Page 104
The wave-particle duality......Page 105
Heisenberg’s uncertainty principle......Page 106
Energy levels......Page 107
A3.2.5 Measurement space, mathematical functions and straight lines......Page 109
A3.3 Dynamics and structure, kinetics, kinematics, relaxation......Page 110
A3.3.1 Macromolecular stabilisation forces......Page 111
A3.3.2 Length and time scales in macromolecular dynamics......Page 112
Conformational substates in the energy landscape and protein specific motions......Page 113
The dynamical transition and mean effective force constants......Page 115
Solvent effects, membrane and protein hydration......Page 118
Motion pictures of intermediate structures......Page 119
A3.4 Checklist of key ideas......Page 123
Part B Mass spectrometry......Page 127
B1.1 Historical review......Page 129
B1.2 Introduction to biological applications......Page 131
B1.3 Ions in electric and magnetic fields......Page 132
B1.4.2 Molecular mass accuracy......Page 133
B1.5.1 From ions in solution to ions in the gas phase......Page 136
B1.5.4 Fast atom bombardment (FAB)......Page 137
B1.5.5 Plasma desorption (PD)......Page 138
B1.5.6 Laser desorption and matrix-assisted laser desorption ionisation......Page 139
B1.6 Instrumentation and innovative techniques......Page 142
B1.6.1 Single- and double-focusing mass spectrometers......Page 143
B1.6.2 Quadrupole mass filter......Page 144
B1.6.3 Quadrupole ion trap......Page 145
B1.6.4 Ion cyclotron resonance mass spectrometry (ICR-MS)......Page 146
B1.6.5 TOF mass spectrometer......Page 149
B1.6.6 Fourier transform mass spectrometry (FT-MS)......Page 150
B1.6.7 Tandem mass spectrometry (MS-MS)......Page 151
B1.7 Checklist of key ideas......Page 152
Instrumentation and innovative techniques......Page 153
B2.1.1 Mass determination......Page 154
B2.1.2 Non-covalent complexes......Page 157
B2.1.3 Protein folding and dynamics......Page 159
Folded and unfolded states......Page 160
Protein folding intermediates......Page 161
B2.1.4 Protein sequencing......Page 164
Computation of protein sequence......Page 165
B2.1.5 Protein identification from two-dimensional electrophoresis......Page 167
B2.2.1 The role of mass spectrometry......Page 169
B2.3 Nucleic acids......Page 171
Computation of nucleotide sequence......Page 172
B2.3.2 Non-covalent complexes......Page 173
B2.3.3 Large and very large nucleic acids......Page 174
B2.3.4 DNA sequencing......Page 175
B2.4 Carbohydrates......Page 176
B2.4.1 Oligosaccharide......Page 177
B2.4.2 Glycopeptides......Page 179
B2.5.1 Ribosomes, ribosomal subunits and ribosomal proteins......Page 181
B2.6 Mass spectrometry in medicine......Page 182
B2.6.1 Clinical oncology......Page 183
B2.7 Imaging mass spectrometry......Page 184
B2.7.2 Mammalian tissue level......Page 185
B2.8 Checklist of key ideas......Page 186
Nucleic acids......Page 187
Imaging mass spectrometry......Page 188
Part C Thermodynamics......Page 189
C1.1 Historical overview and biological applications......Page 191
C1.2 The laws of thermodynamics......Page 193
Adiabatic, isothermal systems and the concept of equilibrium......Page 194
Energy conservation......Page 195
C1.2.3 The second law and entropy......Page 196
Entropy and disorder......Page 197
C1.2.4 The third law and absolute zero......Page 198
Enthalpy......Page 199
Gibbs free energy......Page 200
The partition function......Page 202
The biological standard state......Page 203
Van’t Hoff analysis......Page 204
Binding to many sites......Page 205
Calculating standard enthalpy and entropy changes upon binding......Page 206
Avidity......Page 207
Heat capacity......Page 208
C1.3.4 Activation thermodynamics......Page 209
C1.4 Checklist of key ideas......Page 210
Suggestions for further reading......Page 211
C2.3.1 Instrument specifications......Page 212
C2.3.2 Sensitivity of heat capacity measurements......Page 213
C2.3.3 Sample requirements......Page 214
C2.4.2 Partition function analysis of the heat capacity curve......Page 215
C2.4.3 Two-state transition: calorimetric and van’t Hoff enthalpies are equal......Page 216
C2.4.4 Calorimetric and van’t Hoff enthalpies are not equal: cooperative domains......Page 218
Multidomain proteins......Page 219
Are there folding intermediates in monodomain proteins?......Page 220
Mutant stability......Page 221
C2.4.6 Complex proteins......Page 222
Ion-binding stabilisation......Page 223
Effects of pH......Page 224
Polypeptide chain energies: the primary heat capacity......Page 225
Internal protein interactions: the heat capacity of an anhydrous folded protein......Page 226
Global fit of the heat capacity by a single mathematical function......Page 227
Reliability of the calculations......Page 229
C2.4.9 Protein stabilisation forces......Page 230
Solvent effects in protein folding......Page 231
The hydrophobic effect......Page 232
The enthalpy and entropy of native protein stabilisation: apolar and polar group hydration and the compactness of the protein interior......Page 233
C2.5 Nucleic acids and lipids......Page 235
C2.6 Checklist of key ideas......Page 236
Suggestions for further reading......Page 238
C3.2.1 Measuring protocol and samples......Page 239
C3.2.2 Binding enthalpy and heat capacity......Page 241
C3.2.3 Affinity constants......Page 242
Reactions with high binding affinities......Page 243
C3.3.1 Entropic versus enthalpic optimisation......Page 244
ITC, Mass Spectrometry, analytical ultracentrifugation (AUC) and surface plasmon resonance (SPR)......Page 245
C3.4 Checklist of key ideas......Page 250
Suggestions for further reading......Page 251
C4.1 Historical overview and introduction to biological problems......Page 252
C4.2.1 Lay-out of a biosensor......Page 253
C4.2.2 SPR biosensor......Page 254
C4.2.4 Other types of biosensor......Page 256
C4.3.1 Thermodynamics of surface interactions......Page 257
C4.3.2 Measurement of the equilibrium constant......Page 258
C4.3.3 The determination of the and of an interaction......Page 259
C4.4.1 Scope of analytes......Page 260
C4.4.3 Cell-cell interactions......Page 261
C4.4.4 SPR and mass spectrometry......Page 262
C4.5 Checklist of key ideas......Page 263
Suggestions for further reading......Page 264
Part D Hydrodynamics......Page 265
D1.1 History and introduction to bioliogical problems......Page 269
D1.2.1 Reynolds number......Page 273
D1.2.2 Movement at low Reynolds number......Page 274
D1.3 Hydration......Page 275
D1.4.2 Hydrodynamic experiments......Page 277
Translational friction coefficient......Page 279
Intrinsic viscosity......Page 280
D1.5 Prediction of particle friction properties......Page 281
D1.5.3 Particles of arbitrary shape......Page 282
D1.6 Checklist of key ideas......Page 283
Prediction of particle friction properties......Page 284
D2.1 Historical review......Page 286
Sphere......Page 290
Ellipsoid of revolution......Page 291
Circular cylinder......Page 292
Modelling the entire particle with a set of spheres (‘beads’ method)......Page 293
Modelling the surface of a particle with a set of small panel elements (the ‘platelets’ method)......Page 299
D2.2.3 Translational friction coefficient and electrostatic capacitance......Page 300
Modelling the surface of a protein with a set of panel elements (the ‘platelet’ method)......Page 302
Platelets modelling of DNA from atomic coordinates......Page 304
D2.2.5 Rigid particles with segmental mobility......Page 305
D2.2.6 Experimental methods for measurement of the translational frictional coefficient......Page 306
D2.3.1 Rotational motion in one dimension......Page 308
D2.3.2 Rotational motion in three dimensions......Page 309
D2.3.3 Rotational motion and relaxation times......Page 310
D2.3.4 Regularly shaped rigid particles......Page 311
Ellipsoid of revolution......Page 313
D2.3.5 Arbitrarily shaped particles......Page 314
D2.4.1 Viscosity as a local energy dissipation effect......Page 315
D2.4.2 Relative, specific (reduced) and intrinsic viscosity......Page 317
Spherical particles......Page 318
Ellipsoidal particles......Page 320
Rod-like particles......Page 321
D2.4.4 Arbitrarily shaped particles......Page 322
Rotational frictional coefficient and intrinsic viscosity (the δ-function)......Page 324
Translational frictional coefficient and molecular covolume (the ψ-function)......Page 326
Intrinsic viscosity and molecular covolume (Pi-function)......Page 327
D2.5.3 Tri-axial ellipsoids: volume-independent shape functions......Page 328
D2.5.4 Whole-body approach and bead model......Page 329
D2.6 Homologous series of macromolecules......Page 330
D2.7 Checklist of key ideas......Page 332
Arbitrarily shaped particles......Page 334
Determination of the shape of macromolecules from translational friction: homologous series......Page 335
D3.1 Historical review......Page 336
D3.2 Translational diffusion coefficients......Page 337
D3.3 Microscopic theory of diffusion......Page 338
D3.4.2 Fick’s second equation......Page 340
D3.4.3 Time-dependent solutions of Fick’s equations......Page 341
D3.4.4 Steady-state solutions of Fick’s equations......Page 342
D3.5.2 Fluorescence photobleaching recovery (FPR)......Page 343
D3.6 Prediction of the diffusion coefficients of globular proteins of known structure and comparison with experiment......Page 345
D3.7.1 Einstein-Smoluchowski relation......Page 346
D3.7.2 Diffusion coefficients of biological macromolecules......Page 348
D3.7.3 Dependence of the diffusion coefficient on the molecular mass of globular proteins......Page 349
D3.7.4 Dependence of the diffusion coefficient on the molecular mass of DNA......Page 350
D3.7.5 The limit of application of Stokes’ law. ‘Large’, ‘medium’ and ‘small’ molecules......Page 351
D3.8 Checklist of key ideas......Page 354
Experimental methods of determination diffusion coefficients......Page 355
Translational friction and diffusion coefficients......Page 356
D4.1 Historical review......Page 357
D4.2.1 Rotors and cells......Page 360
D4.2.2 Optical detection systems......Page 362
D4.2.3 Data acquisition......Page 364
D4.3 The Lamm equation......Page 365
No sedimentation......Page 366
D4.4.2 Analytical solutions......Page 367
D4.4.3 Numerical solutions......Page 369
D4.5 Sedimentation velocity......Page 370
D4.5.1 Macromolecules in a strong gravitational field......Page 371
Routine mode AUC: s and D are well defined......Page 374
Complex mode AUC: analysis of the boundary in terms of s and D......Page 375
D4.5.3 Highly heterogeneous systems......Page 377
D4.5.4 Sedimentation coefficients of biological macromolecules......Page 378
D4.5.5 Differential sedimentation for measuring small changes in sedimentation coefficients......Page 382
D4.7.1 Molecular mass......Page 383
D4.7.2 Binding constants......Page 386
D4.8 The partial specific volume......Page 388
D4.9.1 Velocity zonal method......Page 390
D4.9.2 Equilibrium sedimentation in a density gradient......Page 392
D4.10 Molecular shape from sedimentation data......Page 394
D4.10.1 Homologous series of quasi-spherical particles: globular proteins in water......Page 395
D4.10.2 Homologous series of random coils: proteins in guanidine hydrochloride......Page 397
D4.10.3 From slightly flexible rod to nearly perfect random coil: DNA......Page 398
D4.10.4 Ribosomal RNAs, ribosomal particles and RNP complexes......Page 399
D4.11 Checklist of key ideas......Page 403
Instrumentation and innovative techniques......Page 404
Sedimentation equilibrium......Page 405
D5.1 Historical review......Page 406
D5.2 Introduction to biological problems......Page 408
D5.4.1 Charge and electrophoretic mobility......Page 409
D5.4.2 Modelling of electrophoretic mobility......Page 411
D5.5.1 Moving-boundary method......Page 413
D5.5.2 Steady-state electrophoresis (SSE)......Page 414
D5.6 Electrophoretic methods in the presence of a support medium: zonal electrophoresis......Page 415
D5.6.1 Separation according to frictional properties: SDS gel electrophoresis......Page 416
D5.6.2 Separation according to the charge: isoelectric focusing (IEF)......Page 417
D5.6.3 Separation according to the charge and frictional properties simultaneously: two-dimensional gel electrophoresis......Page 419
D5.7.1 One-dimensional electrophoresis......Page 420
D5.7.2 Ultrafast capillary electrophoresis......Page 423
D5.7.3 Electrophoretic mobility of DNA......Page 424
D5.7.4 Two-dimensional capillary electrophoresis......Page 425
D5.7.5 Separation of chiral molecules......Page 426
D5.8 Capillary electrochromatography (CEC)......Page 427
D5.9 Checklist of key ideas......Page 429
Electrophoretic methods in the presence of support medium: zonal electrophoresis......Page 430
Capillary electrochromatography......Page 431
D6.1 Historical review and introduction to biological problems......Page 432
D6.2.1 Dipole moments of biological macromolecules......Page 433
D6.2.2 Electric birefringence and dichroism......Page 434
D6.3 Theoretical background of TEB measurements......Page 435
D6.3.1 Steady-state birefringence......Page 436
D6.3.2 Decay process......Page 437
D6.4.1 Experimental schemes......Page 438
D6.4.2 Interpretation of the birefringence decay profile......Page 440
D6.5.1 Internal structure of ‘motor’ domains from TEB......Page 441
D6.5.2 Hydrodynamic dimensions of globular proteins from TEB......Page 442
D6.6.1 Flexibility of DNA in B- and A-forms......Page 443
D6.6.2 Flexibility of dsRNA......Page 448
D6.6.3 Conformational of non-helical elements in RNA......Page 449
D6.7 Checklist of key ideas......Page 450
Global structure and flexibility of nucleic acids......Page 451
D7.1 Historical review......Page 453
D7.2 Introduction to biological problems......Page 454
D7.3 Steady-state flow birefringence......Page 455
D7.4 Decay of flow dichroism......Page 458
D7.5 Oscillatory flow birefringence......Page 459
D7.6 Orientation of macromolecules in flow as a dynamic phenomenon......Page 460
D7.7 Checklist of key ideas......Page 461
Decay of flow dichroism......Page 462
Orientation of macromolecules in flow as a dynamic phenomenon......Page 463
D8.1 Historical review......Page 464
D8.2 Introduction to biological problems......Page 465
D8.3.1 Fluorescence as a physical phenomenon......Page 466
D8.3.2 Lifetime of fluorophore and rotational correlation time......Page 468
D8.3.3 Steady-state fluorescence depolarisation......Page 469
D8.3.4 Time-resolved fluorescence depolarisation......Page 470
D8.4 Instrumentation......Page 471
D8.5.1 Steady-state or static polarisation......Page 473
D8.5.2 Fluorescence anisotropy decay time......Page 475
D8.5.3 Rotational correlation time of globular proteins......Page 476
D8.6 Depolarised fluorescence and molecular interactions......Page 480
D8.7 Checklist of key ideas......Page 481
Instrumentation......Page 482
Depolarised fluorescence and molecular interactions......Page 483
D9.1 Historical review......Page 484
D9.3.1 Capillary viscometer......Page 485
D9.3.2 Rotor viscometer......Page 487
D9.3.3 Pressure imbalance differential viscometer......Page 488
D9.3.4 The effect of concentration......Page 489
D9.4 Comparison of theory with experiment......Page 490
D9.5.1 Synthetic polypeptides......Page 491
D9.5.2 Proteins in globular conformations......Page 492
D9.5.3 Proteins in coil conformation......Page 493
D9.5.4 DNA......Page 494
D9.6 Monitoring of conformational change in proteins and nucleic acids......Page 495
D9.7 Checklist of key ideas......Page 496
Shapes of macromolecules from their intrinsic viscosities: homologous series......Page 497
Monitoring of conformational change in proteins and nucleic acids......Page 498
D10.1 Historical review......Page 499
D10.2 Introduction to biological problems......Page 501
D10.3 Dynamic light scattering as a spectroscopy of very high resolution......Page 502
Construction of time-dependent correlation functions......Page 504
Field and intensity correlation functions......Page 505
Correlation functions and frequency spectra of fluctuations......Page 506
Filter technique......Page 507
Homodyne and heterodyne techniques......Page 508
Multiple decay analysis of the correlation function......Page 509
D10.4.1 Particles small compared with the wavelength of the light......Page 511
Depolarised component of scattered light......Page 512
D10.4.3 Flexible macromolecules: DNA......Page 513
D10.4.4 Macromolecules in uniform motion: electrophoretic light scattering (ELS)......Page 514
Experimental geometry......Page 516
D10.5.1 Scattering of a small number of particles (number fluctuations)......Page 517
D10.5.2 Cross-correlation (method of two detectors)......Page 519
D10.6 Checklist of key ideas......Page 520
Historical review and introduction to biological problems......Page 521
DLS under non-Gaussian statistics......Page 522
D11.1 Historical review......Page 523
D11.3 General principles of FCS......Page 524
Three- and two-dimensional diffusion......Page 525
Different mobility modes......Page 527
D11.4 Dual-colour fluorescence cross-correlation spectroscopy......Page 530
D11.5 Checklist of key ideas......Page 532
Dual-colour fluorescence cross-correlation spectroscopy......Page 533
Part E Optical spectroscopy......Page 535
E1.1 Brief historical review and biological applications......Page 537
E1.2 Brief theoretical outline......Page 540
E1.3 The UV-visible spectral range......Page 542
E1.3.1 UV-visible spectrophotometers and measurement strategies......Page 543
Difference spectra......Page 544
E1.3.2 UV absorption spectra of proteins......Page 545
E1.3.3 Visible absorption spectra of protein-associated groups......Page 550
Flash photolysis of CO-myoglobin......Page 551
The photocycle of bacteriorhodopsin......Page 554
E1.3.4 UV absorption spectra of nucleic acids......Page 556
E1.4.1 IR spectrometers......Page 558
Fourier transform infrared (FTIR) spectrometers......Page 559
Attenuated total reflection......Page 560
Vibration and polarised light......Page 561
The harmonic approximation......Page 562
Anharmonicity......Page 563
Amide bands of polypeptides and proteins......Page 564
Second derivative spectra......Page 566
Fitting deconvoluted spectra......Page 568
Comparison with known structure......Page 570
E1.4.8 IR difference spectroscopy......Page 572
E1.4.10 DNA conformation......Page 573
E1.5 Checklist of key ideas......Page 576
The UV-visible spectral range......Page 578
E2.1 Historical review and introduction to biological problems......Page 580
E2.3.1 Pump probe experiments......Page 581
E2.3.2 Selection rules for two-dimensional spectroscopy......Page 582
E2.4 From amide bands to protein tertiary structure......Page 584
E2.4.2 Determination of peptide structures......Page 585
E2.5 Checklist of key ideas......Page 588
From amide bands to protein tertiary structure......Page 589
NMR and 2D-IR spectroscopy: similarity and difference......Page 590
E3.2 Classical Raman spectroscopy......Page 591
E3.2.1 Raman spectra......Page 592
E3.2.2 Frequency, intensity and polarisation......Page 593
E3.2.3 Raman spectrometers and Raman microscopes......Page 596
E3.2.4 Protein secondary structure from Raman spectra......Page 597
E3.2.5 Protein conformational dynamics in solution and in crystals......Page 600
E3.3 Resonance Raman spectroscopy (RRS)......Page 603
E3.4 Surface enhanced Raman spectroscopy (SERS)......Page 604
E3.5.1 Vibrational circular dichroism (VCD)......Page 605
Polypeptides and proteins......Page 606
Nucleic acids......Page 609
E3.6 Differential Raman spectroscopy......Page 610
E3.7 Time-resolved resonance Raman spectroscopy......Page 611
E3.7.1 Light-initiated methods......Page 612
Haem proteins......Page 613
E3.7.2 Rapid-mixing methods......Page 615
E3.8 Checklist of key ideas......Page 616
Non-resonance Raman spectroscopy......Page 617
Time-resolved resonance Raman spectroscopy......Page 618
E4.1 Historical review and introduction to biological problems......Page 619
E4.2.1 Plane, circularly and elliptically polarised light......Page 621
E4.2.2 CD, ellipticity and ORD......Page 623
E4.2.3 Electronic transitions, dipole and rotational strengths......Page 624
E4.2.4 Rotational strength and structural organisation......Page 625
E4.4.1 CD of protein secondary structures......Page 627
Fitting spectra with basic sets......Page 629
Membrane proteins......Page 631
E4.4.2 Near-UV CD and protein tertiary structure......Page 633
Molten globules......Page 634
E4.5 Nucleic acids and protein-nucleic acid interactions......Page 635
E4.5.1 RNA......Page 636
E4.5.2 DNA......Page 637
E4.5.3 Protein-nucleic acid interactions......Page 638
E4.6 Carbohydrates......Page 639
E4.8 Checklist of key ideas......Page 641
Suggestion for further reading......Page 642
Part F Optical microscopy......Page 643
F1.1.1 Light microscope......Page 645
F1.1.2 Confocal microscopy......Page 646
F1.2 Light microscopy inside the classical limit......Page 647
One-lens microscope......Page 648
Diffraction limit of resolution......Page 649
Dark-field microscopy......Page 651
Polarization microscopy......Page 652
F1.3.1 Confocal microscopy......Page 653
F1.4 Lensless microscopy......Page 654
Main component of the near-field microscope......Page 655
NSOM in liquids......Page 656
Subwavelength resolution within the restriction of geometrical optics......Page 657
Lensless microscopy......Page 658
F2.1 Historical review......Page 659
F2.3 General principles......Page 660
F2.3.1 The tip, a key element of an SFM......Page 661
Contact (constant force) mode......Page 663
F2.4 Imaging of biological structures......Page 664
F2.4.1 Imaging of DNA structure......Page 665
Two-dimensional crystals......Page 667
RNA polymerase activity......Page 669
F2.4.4 AFM probe as nanoscalpel......Page 670
F2.4.5 Study of crystal growth......Page 671
F2.5 Combination of NSOM and AFM......Page 672
F2.6 Checklist of key ideas......Page 673
Imaging of biological structures......Page 674
Combination of NSOM and AFM......Page 675
F3.1 Historical review......Page 676
F3.3.1 The standard wide-field fluorescence microscope......Page 678
F3.3.2 Two-photon excited microscopy......Page 679
F3.3.3 Total internal reflectance fluorescence microscopy (TIRFM)......Page 682
F3.4.1 4Pi-confocal microscopy......Page 684
F3.4.2 Stimulated emission depletion (STED) microscopy......Page 685
F3.4.3 Standing-wave illumination fluorescence microscopy (SWFM)......Page 687
F3.5 Fluorescence resonance energy transfer (FRET)......Page 688
F3.5.1 FRET as a spectroscopic ruler in static and dynamic regimes......Page 689
F3.6 Green fluorescent protein (GFP)......Page 693
F3.6.1 GFP as a conformational sensor......Page 694
F3.6.2 GFP as a cellular reporter......Page 695
F3.7 Fluorescence lifetime imaging (FLI) microscopy: seeing the machinery of live cells......Page 696
F3.8 Checklist of key ideas......Page 698
FRET......Page 699
FLI microscopy......Page 700
F4.1 Historical review......Page 701
F4.2 Introduction to biological problems......Page 702
F4.3.1 Laser-induced fluorescence......Page 704
F4.3.2 Labelling schemes and observable values......Page 706
F4.4 Optical detection of single molecules in the solid state......Page 708
F4.5.1 Levitated microdroplets......Page 709
F4.5.2 Liquid streams......Page 710
F4.5.3 Trapping with capillaries......Page 711
F4.5.4 Electrical trapping......Page 712
F4.6.1 Near-field scanning optical microscopy (NSOM)......Page 714
F4.6.2 Far-field confocal microscopy......Page 715
F4.6.3 Wide-field epi-illumination......Page 716
F4.6.5 Integrated optical and atomic force microscopy studies......Page 718
F4.6.6 Scanning near-field Raman spectroscopy......Page 719
F4.7 Single-molecule studies tell more than ensemble measurements......Page 720
F4.7.1 Direct observation of enzyme activity......Page 721
F4.7.3 RNA catalysis and folding......Page 723
Fluorescence spectroscopy of single molecules......Page 725
Single-molecule studies tell us more than ensemble measurements......Page 726
F5.1 Historical review and introduction to biological problems......Page 727
F5.2.1 Optical traps (laser tweezers)......Page 728
F5.2.2 Magnetic traps (magnetic tweezers)......Page 731
F5.2.3 Cantilever in the force-measuring mode of the AFM......Page 732
F5.2.4 Glass microneedles......Page 734
F5.3 Macromolecular mechanics: nanometre steps and piconewton forces......Page 735
F5.3.1 Linear molecular motors......Page 736
Kinesin: vesicle transport along microtubules......Page 737
Dynein: eukaryotic flagella motor......Page 740
Myosin: the muscle motor......Page 742
RNA polymerase: a processive DNA transcription machine......Page 745
F5.3.2 Rotary molecular motors......Page 747
F0F1 ATPase motor......Page 748
The bacterial flagella as a propeller screw......Page 750
F5.3.3 The bacteriophage φ29 DNA packaging motor......Page 752
F5.3.4 Molecular motors and Brownian motion......Page 754
F5.3.5 Molecular motors and the second law of thermodynamics......Page 755
Molecular yoga of DNA......Page 756
Topoisomerases and uncoiling of DNA......Page 760
Unzipping the DNA double helix......Page 762
Unfolding of single chromatin fibres and individual nucleosomes......Page 763
F5.3.7 RNA mechanics......Page 766
Polyproteins......Page 768
Models of elasticity......Page 770
Protein folds and mechanical stability......Page 772
F5.3.9 Deformation of polysaccharides......Page 776
F5.4 Checklist of key ideas......Page 778
Historical review and introduction to biological problems......Page 779
DNA and RNA mechanics......Page 780
How strong is a covalent bond?......Page 781
Part G X-ray and neutron diffraction......Page 783
G1.1 Historical review and introduction to biological applications......Page 785
G1.2 Radiation and matter......Page 788
X-rays......Page 789
G1.2.3 Energy momentum and wavelength......Page 790
G1.3 Scattering by a single atom (the geometric view)......Page 791
G1.3.1 Point scattering. Scattering length......Page 792
X-ray scattering amplitudes......Page 793
G1.3.2 Cross-sections and sample size......Page 794
G1.4 Scattering vector and resolution......Page 795
G1.5.1 Coherent and incoherent scattering......Page 797
G1.5.2 Elastic and inelastic scattering......Page 798
G1.5.3 Summing waves, Fourier transformation and reciprocal space......Page 799
G1.6 Solutions and crystals......Page 800
G1.6.1 One-dimensional crystals......Page 801
G1.6.2 Two-and three-dimensional crystals......Page 802
G1.6.3 Disordered systems......Page 803
G1.7 Resolution and contrast......Page 804
X-rays......Page 806
Large-scale facilities......Page 807
Diffractometers......Page 808
G1.9 Checklist of key ideas......Page 809
G2.1.1 Dilute solutions of identical particles......Page 812
G2.1.2 The scattering curve at small Q values. The Guinier approximation, the forward scattered intensity and radius of gyration......Page 815
G2.1.3 Asymptotic behaviour of the scattering curve at large Q values. The Porod relation......Page 819
G2.1.4 The full scattering curve. The distance distribution function......Page 820
G2.1.5 The information content in p(r) and I(Q) for a monodisperse solution of a particle with a well-defined envelope......Page 823
G2.1.6 Polydisperse solutions......Page 824
G2.1.7 Interactions between the particles......Page 825
Spheres and ellipsoids......Page 827
Rods and sheets......Page 829
Atomic coordinates......Page 831
Monte Carlo and simulated annealing algorithms......Page 833
Genetic algorithms......Page 834
G2.3 General contrast variation. Particles in different solvents ‘seen’ by X-rays and ‘seen’ by neutrons......Page 835
G2.3.1 Two-component particles and the parallel axes theorem......Page 836
G2.3.2 The Stuhrmann analysis of contrast variation......Page 838
G2.3.3 Deuterium labelling and triangulation......Page 841
G2.4.1 Fluctuations in hydrodynamics and scattering......Page 842
Neutron scattering......Page 844
G2.4.2 Relating the thermodynamics and particle approaches......Page 846
G2.5.1 Aminoacyl tRNA interactions with tRNA......Page 847
G2.5.2 ATP, solvent- and temperature-induced structural changes of the thermosome......Page 849
G2.5.3 Membrane proteins......Page 850
G2.6 Multiangle laser light scattering (MALLS)......Page 852
G2.7 Checklist of key ideas......Page 854
Suggestions for further reading......Page 855
G3.1 Historical review......Page 856
G3.2 From crystal to model......Page 858
G3.2.1 Reciprocal lattice, Ewald sphere and structure factors......Page 859
G3.2.2 Space group symmetry......Page 861
G3.2.4 Technical challenges and the crystallographic model......Page 862
G3.3 Crystal growth: general principles involved in the transfer of a macromolecule from solution to a crystal form......Page 863
G3.3.2 Crystallization screens......Page 864
Seeding......Page 865
G3.3.4 Identifying crystals and precipitates – crystal shapes and sizes......Page 866
Goniometer......Page 867
Heavy atom derivatives......Page 868
Biosynthetic labelling for anomalous dispersion......Page 869
G3.4.1 Data collection and processing......Page 870
G3.4.2 Indexing Bragg reflections......Page 871
G3.4.4 Twinning......Page 872
G3.4.5 Radiation damage......Page 873
G3.4.8 Redundancy and statistics......Page 874
G3.4.9 Molecular packing in the unit cell and the Patterson function......Page 875
Patterson function......Page 876
Non-crystallographic symmetry......Page 877
G3.5.2 Assessing agreement between the model and the data......Page 878
G3.5.3 Assessing agreement between the model and chemistry......Page 880
G3.6.1 Argand diagram......Page 881
G3.6.2 Molecular replacement......Page 882
Phase relationships between structure factors......Page 883
G3.6.4 Single and multiple isomorphous replacement (SIR, MIR)......Page 884
G3.6.5 Single and multiple anomalous dispersion (SAD, MAD)......Page 885
G3.7 From the electron density to the atomic model – refinement of the model – phase improvement......Page 887
G3.7.2 Minimisation of a target function (maximum likelihood)......Page 888
Bulk solvent correction......Page 889
Phase extension......Page 890
Simulated annealing......Page 891
Publishing a structure......Page 892
G3.8.1 Trapping of intermediate states......Page 893
G3.8.2 Laue diffraction and time-resolved crystallography......Page 894
G3.9 Neutron crystallography......Page 896
G3.10 Checklist of key ideas......Page 897
Suggestions for further reading......Page 899
Part H Electron diffraction......Page 901
H1.1 Historical review......Page 903
H1.2.2 Applications of EM......Page 904
Effect of the electron charge......Page 905
H1.3.2 Electromagnetic lens......Page 906
Noise and radiation damage......Page 907
Effect of the microscope on the path of the electron......Page 908
Contrast transfer function and defocus......Page 909
H1.4.1 Electron beam generation......Page 911
H1.4.2 Transmission and scanning electron microscopes......Page 912
Imaging phase objects......Page 913
Pixelisation......Page 914
Method for negative staining......Page 915
Reasons for electron cryo-microscopy......Page 916
Sample concentration......Page 917
H1.6.3 Imaging crystals and helical molecules......Page 918
H1.6.4 Tomography......Page 919
H1.8 Checklist of key ideas......Page 920
Suggestion for further reading......Page 921
H2.1.2 Examples of electron cryo-microscopy reconstructions......Page 922
H2.2.1 Preliminary analysis of the image......Page 923
H2.2.3 Correction for the Contrast Transfer Function......Page 924
H2.3.1 Coordinate system......Page 925
H2.3.2 Reconstruction from projections......Page 926
H2.3.4 Common lines reconstruction procedure......Page 927
H2.3.5 Polar Fourier transform reconstruction......Page 928
H2.3.7 Focal pairs......Page 929
H2.4.1 Helical reconstruction......Page 930
H2.4.2 Electron crystallography......Page 932
H2.5.2 Multivariate statistical analysis......Page 933
H2.6.1 Number of images required for a reconstruction......Page 934
Phase residual......Page 935
H2.7.2 Weighting......Page 937
H2.8.2 Icosohedral viruses......Page 938
H2.8.3 Microtubules......Page 941
H2.8.4 Integral membrane proteins......Page 942
H2.9 Checklist of key ideas......Page 944
Suggestions for further reading......Page 945
Part I Molecular dynamics......Page 947
I1.1 Historical review of biological applications......Page 949
I1.2 Dynamics, kinetics, kinematics, and molecular stabilisation forces......Page 950
I1.4 Normal mode analysis......Page 951
I1.5.1 Force field......Page 952
I1.5.3 Potential energy surface and energy minimisation......Page 954
I1.5.4 Modelling the solvent......Page 955
Radius of gyration......Page 956
Diffusion coefficients......Page 957
I1.6.2 Protein folding......Page 958
I1.6.4 ATP synthase, a molecular machine......Page 959
I1.7 Checklist of key ideas......Page 963
Suggestions for further reading......Page 965
I2.1 Historical overview and introduction to biological applications......Page 966
I2.2.1 Momentum and energy, distance travelled and time......Page 968
I2.2.2 The dynamic structure factor, intermediate scattering function and correlation function......Page 969
I2.3 Applications......Page 971
I2.3.2 Space-time window......Page 972
I2.3.3 Q dependence of the elastic intensity......Page 974
Mean square fluctuations and effective force constants......Page 975
I2.3.4 Quasi-elastic scattering and diffusion......Page 977
I2.3.5 Inelastic scattering and vibrations......Page 978
The triple-axis spectrometer......Page 980
Time of flight......Page 981
Spin echo......Page 982
I2.5 Checklist of key ideas......Page 984
Suggestions for further reading......Page 985
Part J Nuclear magnetic resonance......Page 987
J1.1 Historical review......Page 989
Magnetic properties of nuclei: nuclear spin......Page 993
Nuclear magnetization......Page 995
Distribution of nuclei between magnetic quantum states......Page 996
J1.2.2 Classical mechanical description......Page 998
Laboratory frame and rotating frame......Page 1000
The effect of the radio-frequency field......Page 1001
Relaxation processes......Page 1002
J1.2.3 Nuclear environment effects on NMR......Page 1003
Chemical shift......Page 1004
Spin-spin coupling......Page 1009
Three-bond couplings......Page 1011
Weakly and strongly coupled spins......Page 1012
J1.3 Checklist of key ideas......Page 1015
Historical review......Page 1016
Fundamental concepts......Page 1017
J2.1.1 Principles......Page 1018
J2.1.2 The Fourier transform NMR spectrometer......Page 1021
J2.2.1 Data acquisition and processing......Page 1022
J2.2.2 Free induction decay (FID)......Page 1024
J2.3 Multiple-pulse experiments......Page 1026
J2.3.1 The inversion recovery method to measure spin-lattice relaxation time T1......Page 1028
J2.3.2 The spin-echo effect to measure T2......Page 1029
J2.3.3 Polarization transfer......Page 1030
J2.4 Nuclear Overhauser Enhancement (NOE)......Page 1032
J2.5 Two-dimensional NMR......Page 1035
J2.5.1 Correlation spectroscopy (COSY)......Page 1037
J2.5.2 Nuclear Overhauser enhanced spectroscopy (NOESY)......Page 1040
J2.5.3 Transverse relaxation-optimised spectroscopy (TROSY)......Page 1042
J2.6 Multi-dimensional, homo- and hetero-nuclear NMR......Page 1044
J2.7 Sterically induced alignment......Page 1045
J2.7.1 Residual dipolar couplings......Page 1046
J2.7.2 Chemical shift anisotropy (CSA)......Page 1049
J2.8.1 Labelling strategies for proteins......Page 1050
J2.8.2 Labelling strategies for RNA......Page 1051
J2.9 Encapsulated proteins in low-viscosity fluids......Page 1052
Single-pulse experiments......Page 1054
Isotope labelling of proteins and nucleic acids......Page 1055
Encapsulated proteins in low viscosity......Page 1056
J3.1 Structure calculation strategies from NMR data......Page 1057
Proteins of medium size (20-60 kDa)......Page 1060
Protein-protein interactions......Page 1062
J3.2.2 Nucleic acids......Page 1064
J3.2.3 Carbohydrates......Page 1066
J3.3 Dynamics of biological macromolecules......Page 1068
J3.3.1 Protein dynamics from relaxation measurements......Page 1069
J3.3.3 Dynamics of slow events: translational diffusion......Page 1075
J3.4 Solid-state NMR......Page 1077
J3.4.1 Solution and solid-state NMR: comparative analysis......Page 1078
Orientation-dependent approaches......Page 1079
Dipole interactions, CSA and quadrupole interactions......Page 1080
Distance-dependent approaches......Page 1081
J3.4.3 Structure of membrane proteins by NMR......Page 1082
J3.5 NMR and X-ray crystallography......Page 1083
J3.5.1 Structure of macromolecules in crystal and in solution: comparative studies......Page 1084
J3.5.2 NMR and structural genomics......Page 1086
J3.6 NMR imaging......Page 1088
J3.7 Checklist of key ideas......Page 1091
Three-dimensional structure of biological macromolecules......Page 1092
NMR imaging......Page 1093
References......Page 1094
Index of eminent scientists......Page 1121
Subject index......Page 1126