title: Enhancing protein stability with extended disulfide bonds.
abstract: Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Xiaozhou
authors.initials: X
authors.lastname: Luo
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Jack
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Sean A
authors.initials: SA
authors.lastname: Reed
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Han
authors.initials: H
authors.lastname: Xiao
authors.affiliation: California Institute for Biomedical Research , La Jolla, CA 92037.
authors.forename: Travis S
authors.initials: TS
authors.lastname: Young
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; California Institute for Biomedical Research , La Jolla, CA 92037 schultz@scripps.edu.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D055550:Protein Stability; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D001618:beta-Lactamases
keywords: evolutionary advantage; extended disulfide bonds; noncanonical amino acids; thermostability; β-lactamase