Abstract
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). All strategies identified stabilizing mutations, but the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus. A web-based automated program that predicts substitutions needed to mutate to consensus is available at http://kazlab.umn.edu.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 6521-6532 |
| Number of pages | 12 |
| Journal | Biochemistry |
| Volume | 56 |
| Issue number | 50 |
| DOIs | |
| State | Published - Dec 19 2017 |
Bibliographical note
Funding Information:*Phone: +1-612-624-5904. E-mail: rjk@umn.edu. ORCID Romas J. Kazlauskas: 0000-0002-3570-2411 Funding The authors thank the U.S. National Science Foundation (Grant CHE-1152804), the U.S. National Institutes of Health (Grants 1R01GM102205-01 and 5T32 GM08347), and the China National Natural Science Foundation (Grant 31470793; fellowship to J.H.) for funding. Notes The authors declare no competing financial interest.
Publisher Copyright:
© 2017 American Chemical Society.