Securing Native Disulfide Bonds in Disulfide-Coupled Protein Folding Reactions: The Role of Intrinsic and Extrinsic Elements vis-a-vis Protein Aggregation and Neurodegeneration.
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ABSTRACT:
SUBMITTER: Narayan M
PROVIDER: S-EPMC8637583 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 ?-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.
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Project description:The Anfinsen principle that the protein sequence uniquely determines its structure is based on experiments on oxidative refolding of a protein with disulfide bonds. The problem of how protein folding drives disulfide bond formation is poorly understood. Here, we have solved this long-standing problem by creating a general method for implementing the chemistry of disulfide bond formation and rupture in coarse-grained molecular simulations. As a case study, we investigate the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI). After confirming the experimental findings that the multiple routes to the folded state contain a network of states dominated by native disulfides, we show that the entropically unfavorable native single disulfide [14-38] between Cys14 and Cys38 forms only after polypeptide chain collapse and complete structuring of the central core of the protein containing an antiparallel β-sheet. Subsequent assembly, resulting in native two-disulfide bonds and the folded state, involves substantial unfolding of the protein and transient population of nonnative structures. The rate of [14-38] formation increases as the β-sheet stability increases. The flux to the native state, through a network of kinetically connected native-like intermediates, changes dramatically by altering the redox conditions. Disulfide bond formation between Cys residues not present in the native state are relevant only on the time scale of collapse of BPTI. The finding that formation of specific collapsed native-like structures guides efficient folding is applicable to a broad class of single-domain proteins, including enzyme-catalyzed disulfide proteins.
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2011-12-01 | E-GEOD-27062 | biostudies-arrayexpress
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