Thirteen versions of a
-sheet protein have been constructed, each with a single, surface-exposed disulfide bridge. A comparison of folding kinetics, in oxidizing and reducing conditions, is usedto elucidate the order in which
-strands become associated during the folding process and, hence, therelationship between topology and folding dynamics. In common with the wild-type molecule, all theproteins fold through a two-step (three state) mechanism with a rapidly formed intermediate which slowlyconverts to the native state. In a majority of cases, the bridge is seen to stabilize the folded state, and forfive of the modified proteins, the additional stability is greater than 3 kcal/mol. Surprisingly, cross-linkswhich connect
-strands which are distant in sequence predominantly stabilize the rapidly formedintermediate state, suggesting that these strand-strand interactions occur in the initial stages of folding.Cross-links which stabilize local hairpins have their major influence on the second, rate-determining stepleading to significant enhancements in the folding rate. We find that enhancement of the folding rate inthe second, rate-limiting step is correlated with a reduction in contact order in the same way as in naturallyoccurring proteins of different folds. The large increases in native-state stability resulting from the insertionof disulfide bridges on the surface of
-sheet structures have implications for enhancing the robustness ofproteins by molecular engineering.
