Unusual Effects of an Engineered Disulfide on Global and Local Protein Stability

• Published in: Biochemistry (Easton) Vol. 35; no. 23; pp. 7422 - 7428
• Main Authors: Betz, Stephen F, Marmorino, Jennifer L, Saunders, Aleister J, Doyle, Donald F, Young, Gregory B, Pielak, Gary J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.06.1996
• Subjects: Amino Acid Sequence; Calorimetry; Circular Dichroism; Cytochrome c Group - biosynthesis; Cytochrome c Group - chemistry; Cytochromes c; Disulfides; Drug Stability; Genetic Variation; Magnetic Resonance Spectroscopy; Protein Conformation; Protein Engineering; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Saccharomyces cerevisiae Proteins; Thermodynamics; Threonine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9528558

The global and local stabilities of a eukaryotic ferricytochrome c variant with an engineered disulfide are examined. The disulfide connects position 20, which is usually a valine, to position 102, which is usually a threonine. The cross-linked variant is ∼1.2 kcal mol-1 less stable than the wild-type protein at 298 K, pH 4.6, in H2O and D2O. Circular dichroism studies show that the decreased stability results from structure-induced stabilization of the denatured state [Betz, S. F., & Pielak, G. J. (1992) Biochemistry 31, 12337−12344]. Here, we use proton chemical shift, paramagnetic shift, and amide proton exchange data to obtain atomic level structural and energetic information. Chemical and paramagnetic shift data indicate only minor native state structural changes. Local stability is obtained from amide proton−deuterium exchange data, using model peptide intrinsic exchange rates. As expected, the exchange data indicate that cross-link incorporation decreases the majority of local stabilities. Near the cross-link, however, local stability seems to increase despite the overall global stability decrease. Furthermore, local stability changes for hydrophobic core residues seem to be greater than the global stability change. We interpret these observations as cross-link-induced changes in exchange competent states and relate them to changes in the denatured state.