The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis

Abstract

We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a T_m of 60 °C and a ΔG_u of 3.08 kcal/mol. Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild-type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment. Three of the eight mutants constructed in this way displayed increases in T_m ranging from 8 to 15 °C with concomitant increases in ΔG_u of up to 1.4 kcal/mol. The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a T_m greater than 90 °C and a ΔG_u more than double that of the wild-type domain. The folding rate of this hyperstable mutant was 10-fold faster than wild-type, while its unfolding rate was fivefold slower. All of the stabilized mutants were still able to bind a target peptide with wild-type affinity. We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies. In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.