Probing residue-specific interactions in the stabilization of proteins using high-resolution NMR: a study of...

It is well established that the oxidation state of cysteine residues in proteins is critical to overall physical stability. Disulfide bonds most often impart thermodynamic stability, but in some cases, diminish it. Predicting the circumstances that lead to each outcome is difficult because mechanistic information is lacking. Because the techniques typically used to study protein stability do not provide sufficient detail, high-resolution NMR was used in combination with low-resolution analysis to obtain mechanistic information regarding disulfide bond formation in a model protein. T(m) (CD) and T(onset) (SLS) for the reduced and oxidized wild type and C104S and C49S mutants were measured. The mutant proteins have altered T(m)s and T(onset)s compared to the reduced wild type, indicating that differences in local interactions of the Cys side chains are important for stability. The NMR spectra clearly show distinct differences in the chemical environment surrounding these Cys residues and the overall tertiary structure. The C49S protein, which is less stable and more aggregation prone than reduced wild type, lacks a hydrogen bond between Y53 and H103. Increased flexibility of the Y53-containing loop is correlated with increased dynamics and unraveling of alpha2, which likely leads to edge strand initiated aggregation of the central beta-sheet.