Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side-chain geometry database.

Journal Article

An analysis of the pairwise side-chain packing geometries of cysteine residues observed in high-resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double-cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross-link.

Full Text

Duke Authors

Cited Authors

  • Burton, RE; Hunt, JA; Fierke, CA; Oas, TG

Published Date

  • April 2000

Published In

Volume / Issue

  • 9 / 4

Start / End Page

  • 776 - 785

PubMed ID

  • 10794421

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1110/ps.9.4.776

Language

  • eng

Conference Location

  • United States