Kinetic stabilization of Bacillus licheniformis  a-amylase through introduction of hydrophobic residues at the surface

Machius, M., Declerck, N., Huber, Robert and Wiegand, G. 2003. Kinetic stabilization of Bacillus licheniformis  a-amylase through introduction of hydrophobic residues at the surface. Journal of Biological Chemistry 278 (13) , pp. 11546-11553. 10.1074/jbc.M212618200

Full text not available from this repository.

Abstract

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis α-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 °C higher than the wild-type. Crystal structure analysis at 1.7 Å resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a β-sheet by introducing a residue with high β-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute “weak points” where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	American Society for Biochemistry and Molecular Biology
ISSN:	0021-9258
Last Modified:	24 Jun 2017 11:00
URI:	https://orca.cardiff.ac.uk/id/eprint/70194

Citation Data

Cited 102 times in Scopus. View in Scopus. Powered By Scopus® Data

Actions (repository staff only)

Edit Item