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Title: Enhancing protein stability with extended disulfide bonds

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. In this paper, 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. Finally, 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.
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [3]
  1. The Scripps Research Institute, La Jolla, CA (United States)
  2. California Institute for Biomedical Research, La Jolla, CA (United States)
  3. The Scripps Research Institute, La Jolla, CA (United States); California Institute for Biomedical Research, La Jolla, CA (United States)
Publication Date:
Grant/Contract Number:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 113; Journal Issue: 21; Journal ID: ISSN 0027-8424
National Academy of Sciences, Washington, DC (United States)
Research Org:
Scripps Research Inst., La Jolla, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; noncanonical amino acids; extended disulfide bonds; β-lactamase; thermostability; evolutionary advantage
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1347591