Protein engineering on thermostability of subtilisin J

Abstract

Subtilsin J is an extracellular serine protease from Bacillus stearothermophilus. Subtilisin is one of the most intensively studied and widely used enzymes. In application to industry, stability of subtilisin is important. Many factors influence on stab ility of protein. Among them, this study focused on stability of helix and interhelical interaction. The segments of $Ser^{105}-Asn^{117}$ and $Ser^{132}-Gly^{146}$ of subtilisin J form α-helices. These two α-helices closely interact each other just as coiled-coil structure in surface of the molecule. First of all, to determine the role of N-terminus of α-helix in protein stability $Ser^{105}$ and $Ser^{132}$, N-termini of two neighboring α-heilces, were changed into Ala, Asp and Lys by site-directed mutagen sis. To determine the role of interaction between two N-termini of α-helices, four double mutant(S105A/S132A, S105D/S132D, S105D/S132K, S105K/S132D) were constructed. The half-life at 60℃ of S105A mutant protein was improved without changing the catalytic efficiency of the enzyme. Alanine replacement may result in more stable packing against hydrophobic core and decreased helical flexibility. S105D and S132D mutant protein showed improvement of thermostability through strengthened dipole moment. However thermostability of S105D/S132D double mutant protein was radically decreased. While S105K and S132K mutant proteins showed destabilization, the thermostability of S105K/S132D double mutant was nearly equal to that of wild-type protein. These results suggest that transition to alanine or aspartic acid in N-terminus of α-helix may improve protein stability through decrease of helical flexibility or increase of dipole moment and interaction of 105-residue and 132-residue has important influence on protein stability.