UNUSUAL EFFECTS OF AN ENGINEERED DISULFIDE ON GLOBAL AND LOCAL PROTEIN STABILITY

The global and local stabilities of a eukaryotic ferricytochrome c var iant with an engineered disulfide are examined. The disulfide connects position 20, which is usually a valine, to position 102, which is usu ally a threonine. The cross-linked variant is similar to 1.2 kcal mol( -1) less stable than the wild-type protein at 298 K, pH 4.6, in H2O an d D2O. Circular dichroism studies show that the decreased stability re sults from structure-induced stabilization of the denatured state [Bet z, S. F., & Pielak, G. J. (1992) Biochemistry 31, 12337-12344]. Here, we use proton chemical shift, paramagnetic shift, and amide proton exc hange data to obtain atomic level structural and energetic information . Chemical and paramagnetic shift data indicate only minor native stat e structural changes. Local stability is obtained from amide proton-de uterium exchange data, using model peptide intrinsic exchange rates. A s expected, the exchange data indicate that cross-link incorporation d ecreases the majority of local stabilities. Near the cross-link, howev er, local stability seems to increase despite the overall global stabi lity decrease. Furthermore, local stability changes for hydrophobic co re residues seem to be greater than the global stability change. We in terpret these observations as cross-link-induced changes in exchange c ompetent states and relate them to changes in the denatured state.