Research Article
Protein dynamics studied by neutron scattering
- Frank Gabel, Dominique Bicout, Ursula Lehnert, Moeava Tehei, Martin Weik, Giuseppe Zaccai
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- Published online by Cambridge University Press:
- 17 February 2003, pp. 327-367
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1. Introduction 328
2. Basic concepts of neutron scattering 329
2.1 Introduction 329
2.2 Neutron-scattering functions 331
2.3 Coherent and incoherent neutron scattering. The particular role of hydrogen in incoherent scattering 332
2.4 Total elastic scattering, EISF and mean square displacement (MSD) 333
2.5 Quasielastic scattering and relaxation function 334
2.6 Inelastic scattering and density of states 335
3. Experimental aspects and instruments 335
3.1 Energy and space resolution 335
3.2 General sample aspects 335
3.3 Potential effects of D2O on dynamics 336
3.4 Experimental 2H (deuterium) labelling 336
4. Physics of protein dynamics 336
4.1 Models 336
4.2 The dynamical transition 338
4.3 Effective force constants 339
5. Dynamics of hydrated protein powders 339
5.1 First experiments on myoglobin 340
5.2 Dynamical transitions in other proteins 340
5.3 The role of hydration water 341
5.4 Influence of the solvent 344
5.5 Diffusional motions within proteins by QENS 346
5.6 Inelastic neutron scattering and vibrational spectra 347
5.7 Conclusions 351
6. Membranes 352
6.1 Lipid bilayers 353
6.2 BR and the purple membrane (PM) 353
6.2.1 The dynamical transition in the PM 353
6.2.2 QENS from oriented PM 354
6.2.3 Hydration dependence of PM motions 355
6.2.4 Local dynamics in PM studied by isotope labelling 356
6.2.5 Dynamics of different BR conformations 357
7. Protein solutions 358
7.1 From powders to solutions 358
7.2 Water dynamics and solvent dependence of the dynamical transition in proteins 359
8. Comparing neutron scattering with other techniques 359
9. Biological relevance 360
9.1 Dynamics–activity relations 360
9.2 Dynamics–stability relations (adaptation to extreme environments) 360
9.3 Protein folding 361
10. Acknowledgements 364
11. References 364
This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.
What vibrations tell about proteins
- Andreas Barth, Christian Zscherp
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- Published online by Cambridge University Press:
- 17 February 2003, pp. 369-430
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1. Introduction 370
2. Infrared (IR) spectroscopy – general principles 372
2.1 Vibrations 372
2.2 Information that can be derived from the vibrational spectrum 372
2.3 Absorption of IR light 375
3. Protein IR absorption 376
3.1 Amino-acid side-chain absorption 376
3.2 Normal modes of the amide group 381
4. Interactions that shape the amide I band 382
4.1 Overview 382
4.2 Through-bond coupling 383
4.3 Hydrogen bonding 383
4.4 Transition dipole coupling (TDC) 383
5. The polarization and IR activity of amide I modes 387
5.1 The coupled oscillator system 387
5.2 Optically allowed transitions 388
5.3 The infinite parallel β-sheet 388
5.4 The infinite antiparallel β-sheet 389
5.5 The infinite α-helix 390
6. Calculation of the amide I band 391
6.1 Overview 391
6.2 Perturbation treatment by Miyazawa 393
6.3 The parallel β-sheet 394
6.4 The antiparallel β-sheet 395
6.5 The α-helix 396
6.6 Other secondary structures 398
7. Experimental analysis of protein secondary structure 398
7.1 Band fitting 398
7.2 Methods using calibration sets 401
7.3 Prediction quality 403
8. Protein stability 404
8.1 Thermal stability 404
8.2 1H/2H exchange 406
9. Molecular reaction mechanisms of proteins 408
9.1 Reaction-induced IR difference spectroscopy 408
9.2 The origin of difference bands 409
9.3 The difference spectrum seen as a fingerprint of conformational change 410
9.4 Molecular interpretation: strategies of band assignment 416
10. Outlook 419
11. Acknowledgements 420
12. References 420
This review deals with current concepts of vibrational spectroscopy for the investigation of protein structure and function. While the focus is on infrared (IR) spectroscopy, some of the general aspects also apply to Raman spectroscopy. Special emphasis is on the amide I vibration of the polypeptide backbone that is used for secondary-structure analysis. Theoretical as well as experimental aspects are covered including transition dipole coupling. Further topics are discussed, namely the absorption of amino-acid side-chains, 1H/2H exchange to study the conformational flexibility and reaction-induced difference spectroscopy for the investigation of reaction mechanisms with a focus on interpretation tools.
Helicase mechanisms and the coupling of helicases within macromolecular machines Part I: Structures and properties of isolated helicases
- Emmanuelle Delagoutte, Peter H. von Hippel
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- Published online by Cambridge University Press:
- 17 February 2003, pp. 431-478
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1. Mechanisms of nucleic acid (NA) unwinding by helicases 432
2. Helicases may take advantage of ‘breathing’ fluctuations in dsNAs 434
2.1 Stability and dynamics of dsNAs 434
2.2 dsNAs ‘breathe’ in isolation 435
2.3 Thermodynamics of terminal base pairs of dsNA 438
2.4 Thermal fluctuations may be responsible for sequential base-pair opening at replication forks 439
2.5 Helicases may capture single base-pair opening events sequentially 440
3. Biochemical properties of helicases 443
3.1 Binding of NAs 443
3.2 Binding and hydrolysis of NTP 445
3.3 Coordination between NA binding and NTP binding and hydrolysis activities 446
4. Helicase structures and mechanistic consequences 447
4.1 Amino-acid sequence analysis reveals conserved motifs that constitute the NTP-binding pocket and a portion of the NA-binding site 447
4.2 Organization of hepatitis virus C NS3 RNA helicase 449
4.2.1 Biochemical properties of HCV NS3 449
4.2.2 Crystal structures of HCV NS3 helicase 450
4.2.2.1 The apoprotein 450
4.2.2.2 The protein–dU8 complex 450
4.2.3 A possible unwinding mechanism 452
4.2.4 What is the functional oligomeric state of HCV NS3? 452
4.3 Organization of the PcrA helicase 453
4.3.1 The apoenzyme and ADP–PcrA complex 454
4.3.2 The protein–DNA–sulfate complex 456
4.3.3 The PcrA–DNA–ADPNP complex 456
4.3.4 A closer look at the NTP-binding site in the crystal structure of PcrA–ADPNP–DNA 457
4.3.5 Communication between domains A and B 457
4.3.6 How might ssDNA stimulate the ATPase activity of PcrA? 457
4.3.7 A possible helicase translocation mechanism 458
4.3.8 A possible unwinding mechanism 458
4.4 Organization of the Rep helicase 459
4.4.1 Biochemical properties 459
4.4.2 Crystal structure of Rep bound to ssDNA 462
4.5 Organization of the RecG helicase 462
4.6 Hexameric helicases 466
4.6.1 Insights from crystal structures of hexameric helicases 467
4.6.2 Possible translocation and unwinding mechanisms 468
5. Conclusions 469
6. Acknowledgments 472
7. References 472
Helicases are proteins that harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of double-stranded nucleic acids. These enzymes have been much studied in isolation, and here we review what is known about the mechanisms of the unwinding process. We begin by considering the thermally driven ‘breathing’ of double-stranded nucleic acids by themselves, in order to ask whether helicases might take advantage of some of these breathing modes. We next provide a brief summary of helicase mechanisms that have been elucidated by biochemical, thermodynamic, and kinetic studies, and then review in detail recent structural studies of helicases in isolation, in order to correlate structural findings with biophysical and biochemical results. We conclude that there are certainly common mechanistic themes for helicase function, but that different helicases have devised solutions to the nucleic acid unwinding problem that differ in structural detail. In Part II of this review (to be published in the next issue of this journal) we consider how these mechanisms are further modified to reflect the functional coupling of these proteins into macromolecular machines, and discuss the role of helicases in several central biological processes to illustrate how this coupling actually works in the various processes of gene expression.