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