Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins

Abstract

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