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.