Stabilisation of α-helices by site-directed mutagenesis reveals the importance of secondary structure in the transition state for acylphosphatase folding

• Published in: Journal of molecular biology 2000-07, Vol.300 (3), p.633-647
• Main Authors: Taddei, Niccolò, Chiti, Fabrizio, Fiaschi, Tania, Bucciantini, Monica, Capanni, Cristina, Stefani, Massimo, Serrano, Luis, Dobson, Christopher M, Ramponi, Giampietro
• Format: Article
• Language: English
• Subjects: Acid Anhydride Hydrolases - chemistry; Acid Anhydride Hydrolases - genetics; Acid Anhydride Hydrolases - metabolism; Acylphosphatase; Circular Dichroism; Dose-Response Relationship, Drug; Enzyme Stability; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Mutagenesis, Site-Directed - genetics; Mutation - genetics; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Denaturation - drug effects; Protein Folding; Protein Structure, Secondary - drug effects; Thermodynamics; transition state for folding; trifluoroethanol; Trifluoroethanol - pharmacology; Urea - pharmacology; α-helix stabilisation
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.3870

The effects of stabilising mutations on the folding process of common-type acylphosphatase have been investigated. The mutations were designed to increase the helical propensity of the regions of the polypeptide chain corresponding to the two α-helices of the native protein. Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism. The most substantial increase in helical content is found for the peptide carrying five mutations in the second α-helix. Acylphosphatase variants containing the corresponding mutations display, to different extents, enhanced conformational stabilities as indicated by equilibrium urea denaturation experiments monitored by changes of intrinsic fluorescence. All the protein variants studied here refold with apparent two-state kinetics. Mutations in the first α-helix are responsible for a small increase in the refolding rate, accompanied by a marked decrease in the unfolding rate. On the other hand, multiple mutations in the second helix result in a considerable increase in the refolding rate without any significant effect on the unfolding rate. Addition of trifluoroethanol was found to accelerate the folding of the acylphosphatase variants, the extent of the acceleration being inversely proportional to the intrinsic rate of folding of the corresponding mutant. The trifluoroethanol-induced acceleration is far less marked for those variants whose α-helical structure is efficiently stabilised by amino acid replacements. This observation suggests that trifluoroethanol acts in a similar manner to the stabilising mutations in promoting native-like secondary structure. Analysis of the kinetic data indicates that the second helix is fully consolidated in the transition state for folding of acylphosphatase, whereas the first helix is only partially formed. These data suggest that the second helix is an important element in the folding process of the protein.