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 alpha-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 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A 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 beta-sheet by introducing a residue with high beta-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.
Highlights
Some general rules for increasing the stability of proteins have been derived from a large number of comparative structural and mutagenesis studies [1, 2]
We have identified hyperthermostable variants of Bacillus licheniformis ␣-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface
Surface Hydrophobicity and Protein Stability—It is generally assumed that amino acid substitutions on the surface do not affect the stability of proteins by large amounts
Summary
Unfolding and Inactivation Kinetics—BLA variants containing various combinations of the mutations, H133V, N190F, A209V, Q264S, and N265Y were constructed as described [18]. Pseudomelting curves were recorded by heating BLA with a rate of 1°/min from 20 to 95 °C and following the CD signal at 222 nm. After normalizing the raw data for sample concentration, this point was derived by calculating the intersection between the CD baseline at low temperatures (between 20 and 60 °C) and a line drawn through the linear part of the transition region. In order to minimize model bias, ten residues around the individual mutation sites were removed from the starting structure, which was subjected to simulated annealing at a starting temperature of 3000 K, followed by cycles of energy-restrained positional refinement of the coordinates, calculation of individual B factors, as well as visual inspection and manual correction of the model using the program O [27].
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