期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 113, 期 21, 页码 5910-5915出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1605363113
关键词
noncanonical amino acids; extended disulfide bonds; beta-lactamase; thermostability; evolutionary advantage
资金
- Department of Energy [DE-SC0011787]
- U.S. Department of Energy (DOE) [DE-SC0011787] Funding Source: U.S. Department of Energy (DOE)
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 beta-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by similar to 9 degrees 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.
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