Biochemistry 2017 Nov 14. Epub 2017 Nov 14.
Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
A review of the previous stabilization of α/β-hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme. For this reason, we compared five strategies to identify stabilizing mutations in a model α/β-hydrolase fold enzyme, salicylic acid binding protein 2, to reversible denaturation by urea and to irreversible denaturation by heat. The five strategies included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus). Read More