Ion channels, like many other proteins, have moving parts that perform useful functions. The channel proteins contain an aqueous, ion-selective pore that crosses the plasma membrane, and they use a number of distinct ‘gating’ mechanisms to open and close this pore in response to biological stimuli such as the binding of a ligand or a change in the transmembrane voltage.

This review is written at a watershed in our understanding of ion channels.

1. INTRODUCTION 240

1.1 Basic structure of voltage-activated channels 241

1.2 What are the physical motions of the channel protein during gating? 243

1.3 Gating involves several distinct mechanisms of activation and inactivation 246

2. ACTIVATION GATING 246

2.1 Early evidence for an activation gate at the intracellular mouth 247

2.1.1 Open channel blockade 247

2.1.2 The ‘ foot-in-the-door’ effect 249

2.1.3 Trapping of blockers behind closed activation gates 249

2.2 Site-directed mutagenesis and the difficulty of inferring structural roles from functional effects 250

2.3 State-dependent cysteine modification as a reporter of position and motion 251

2.4 Localization of activation gating 254

2.4.1 The trapping cavity 254

2.4.2 The activation gate 255

2.4.3 Is there more than one site of activation gating? 258

3. INACTIVATION GATING 259

3.1 Ball-and-chain (N-type) inactivation 261

3.1.1 Nature of the ‘ball’ – a tethered blocking particle 262

3.1.2 The ball receptor 263

3.1.3 The chain 264

3.1.4 Variations on the N-type inactivation theme: multiple balls, foreign balls, anti-balls 265

3.2 C-type inactivation 266

3.2.1 C-type inactivation and the outer mouth of the K+channel 266

3.2.2 The selectivity filter participates in C-type inactivation 267

3.2.3 A consistent structural picture of C-type inactivation 269

3.3 By what mechanism do other voltage-gated channels inactivate? 272

4. THE VOLTAGE SENSOR 273

4.1 Quantitative principles of voltage-dependent gating 276

4.2 S4 (and its neighbours) as the principal voltage sensor 277

4.2.1 Mutational effects on voltage-dependence and charge movement 277

4.2.2 Evidence for the translocation of S4 279

4.2.3 Real-time monitoring of S4motion by fluorescence 282

4.3 Coupling between the voltage sensor and gating 283

5. CONCLUSION 284

6. ACKNOWLEDGEMENTS 287

7. REFERENCES 287

Alcohol based cosolvents, such as trifluoroethanol (TFE) have been used for many decades to denature proteins and to stabilize structures in peptides. Nuclear magnetic resonance spectroscopy and site directed mutagenesis have recently made it possible to characterize the effects of TFE and of other alcohols on polypeptide structure and dynamics at high resolution. This review examines such studies, particularly of hen lysozyme and β-lactoglobulin. It presents an overview of what has been learnt about conformational preferences of the polypeptide chain, the interactions that stabilize structures and the nature of the denatured states. The effect of TFE on transition states and on the pathways of protein folding and unfolding are also reviewed. Despite considerable progress there is as yet no single mechanism that accounts for all of the effects TFE and related cosolvents have on polypeptide conformation. However, a number of critical questions are beginning to be answered. Studies with alcohols such as TFE, and ‘cosolvent engineering’ in general, have become valuable tools for probing biomolecular structure, function and dynamics.

1. COSOLVENTS: OLD HAT? 298

2. HOW DOES TFE WORK? 299

2.1 Effect on hydrogen bonding 300

2.2 Effect on non-polar sidechains 301

2.3 Effect on solvent structure 302

3. EFFECTS OF TFE ON (UN-)FOLDING TRANSITIONS 303

3.1 Pretransition 303

3.2 Transition 304

3.3 Posttransition 305

3.4 Far UV CD spectroscopic detection of structure 306

3.5 Effect with temperature 306

3.6 Effect with additional denaturants 306

4. THERMODYNAMIC PARAMETERS FROM STRUCTURAL TRANSITIONS OF PEPTIDES AND PROTEINS IN TFE 307

5. ADVANCES IN NMR SPECTROSCOPY 310

5.1 Chemical shifts 310

5.2 3[Jscr ]HNHαcoupling constants 311

5.3 Amide hydrogen exchange 312

5.4 Nuclear Overhauser Effects (NOEs) 312

6. α-HELIX – EVERYWHERE? 313

6.1 Intrinsic helix propensity equals helix content? 313

6.2 A helix propensity scale for the amino acids in TFE 314

6.3 Capping motifs and stop signals 315

6.4 Limits and population of helices as seen by CD and NMR 316

7. TURNS 317

8. β-HAIRPINS AND SHEETS 317

9. ‘CLUSTERS’ OF SIDECHAINS 320

10. THE TFE DENATURED STATE OF β-LACTOGLOBULIN 321

11. THE TFE DENATURED STATE OF HEN LYSOZYME 324

12. TERTIARY STRUCTURE, DISULPHIDES, DYNAMICS AND COMPACTNESS 327

13. PROSPECTS FOR STRUCTURE CALCULATION 328

14. EFFECT OF TFE ON QUATERNARY STRUCTURE 329

15. EFFECT ON TFE ON UN- AND REFOLDING KINETICS 330

16. OTHER USES 336

16.1 Mimicking membranes and protein receptors 336

16.2 Solubilizing peptides and proteins 336

16.3 Cosolvents as helpers for protein folding? 338

16.4 Modifying protein dynamics and catalysis 338

16.5 Effects on nucleic acids 339

16.6 Effects on lipid bilayers and micelles 339

16.7 Future applications 339

17. CONCLUSIONS: TFE – WHAT IS IT GOOD FOR? 340

18. ACKNOWLEDGMENTS 340

19. REFERENCES 340