We have analyzed the structure, stability and folding kinetics of circularly permuted forms of alpha-spectrin SH3 domain. All the possible permutations involving the disruption of the covalent linkage between two beta-strands forming a beta-hairpin have been done. The different proteins constructed here fold to a native conformation similar to that of wild-type protein, as demonstrated by nuclear magnetic resonance and circular dichroism. Although all the mutants have similar stabilities (they are 1 to 2 kcal mol-1 less stable than the wild-type) their rate constants for folding and unfolding are quite different. Protein engineering, in combination with kinetics indicates that the folding pathway has been changed in the circularly permuted proteins. We conclude that neither the order of secondary structure elements, nor the preservation of any of the beta-hairpins present in this domain, is crucial for the ability of the polypeptide to fold, but they influence the folding and unfolding kinetics and could determine its folding pathway.

• PMID: 7723022
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Viguera AR, Blanco FJ, Serrano L

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