The effects of disulfide bonds on the denatured state of barnase

The effects of engineered disulfide bonds on protein stability are poorly u nderstood because they can influence the structure, dynamics, and energetic s of both the native and denatured states. To explore the effects of two en gineered disulfide bonds on the stability of barnase, we have conducted a c ombined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. Ho wever, specific extended beta -sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predi cted. Our study suggests a possible cause of the very high stability confer red by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.