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

• Published in: Protein science Vol. 9; no. 4; pp. 776 - 785
• Main Authors: BURTON, RANDALL E., HUNT, JENNIFER A., FIERKE, CAROL A., OAS, TERRENCE G.
• Format: Journal Article
• Language: English
• Published: Bristol Cambridge University Press 01-04-2000; Cold Spring Harbor Laboratory Press
• Subjects: Carbonic Anhydrases - chemistry; Circular Dichroism; Crystallography, X-Ray; Database Management Systems; disulfide bond; Disulfides - chemistry; Humans; Mutagenesis; Protein Engineering; protein stability; protein stability; disulfide bond; protein engineering
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.9.4.776

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.