Mechanism of folding/unfolding of Rd-apocyt $b_{562}$: a molecular dynamics simulation study

The four-helix bundle protein Rd-apocyt $b_{562}$, a redesigned stable variant of apocytochrome$ b_{562}$, has been shown to exhibit two-state folding kinetics. Its transition-state ensemble has been characterized by \textit{$\Phi $}-value analysis and native-state hydrogen exchange. To elucidate the molecular basis of the transition-state ensemble, we have carried out high-temperature molecular dynamics simulations of the unfolding process. In agreement with experiment, helix one was found to melt first, followed by helix four, and then by helices three and two. This ordering among the four helices could be rationalized by differences in helix propensities and by the presence of tertiary contacts.