title: Protein folding transition states: elicitation of Hammond effects by 2,2,2-trifluoroethanol.
abstract: Adaptation of the techniques of classical physical-organic chemistry to the study of protein folding has led to our current detailed understanding of the transition states. Here, we have applied a series of structure--activity relationships to analyse the effects on protein folding transition states of 2,2,2-trifluoroethanol (TFE), a reagent that is usually assumed to act by stabilising secondary structure. The folding and unfolding of the highly alpha-helical tetramerisation domain of p53 provides a useful paradigm for analysing its effects on kinetics: The first step of its folding consists of an association reaction with little, if any, formation of secondary structure in the transition state; and the final step of the folding reaction involves just the formation of bonds at subunit interfaces, with the alpha-helical structure being completely formed. We have systematically measured the effects of TFE on two sets of structure--activity relationships. The first is for Phi values, which measure the degree of non-covalent bond formation at nearly every position in the transition state. The second is for relative effects of the denaturant, guanidinium chloride, on kinetics and equilibria, which measure the gross position of the transition state on the reaction co-ordinate. We find that TFE modulated the kinetics by a variety of effects other than that on secondary structure. In particular, there were Hammond effects, movement of the position of the transition state along the reaction co-ordinate, which either significantly speeded up or slowed down protein unfolding, depending on the particular mutant examined. The gross effects of TFE on protein folding kinetics are thus not a reliable guide to the structures of transition states.
authors.affiliation: Cambridge University Chemical Laboratory and Centre for Protein Engineering, MRC Centre Hills Road, Cambridge CB12QH UK.
authors.forename: C P
authors.initials: CP
authors.lastname: Yiu
authors.forename: M G
authors.initials: MG
authors.lastname: Mateu
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D002942:Circular Dichroism; D019791:Guanidine; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012997:Solvents / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014270:Trifluoroethanol / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000378:metabolism
chemical_list: D012997:Solvents; D016159:Tumor Suppressor Protein p53; D014867:Water; D014270:Trifluoroethanol; D019791:Guanidine