The effects of engineered disulfide bonds on protein
stability are poorly understood because they can influence
the structure, dynamics, and energetics of both the native
and denatured states. To explore the effects of two engineered
disulfide bonds on the stability of barnase, we have conducted
a combined molecular dynamics and NMR study of the denatured
state of the two mutants. As expected, the disulfide bonds
constrain the denatured state. However, specific extended
β-sheet structure can also be detected in one of the
mutant proteins. This mutant is also more stable than would
be predicted. Our study suggests a possible cause of the
very high stability conferred 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.