The folding transition-state ensemble of a four-helix bundle protein: helix propensity as a determinant and macromolecular crowding as a probe.

The four-helix bundle protein Rd-apocyt b(562), a redesigned stable variant of apocytochrome b(562), exhibits two-state folding kinetics. Its transition-state ensemble has been characterized by Phi-value analysis. To elucidate the molecular basis of the transition-state ensemble, we have carried out high-temperature molecular dynamics simulations of the unfolding process. In six parallel simulations, unfolding started with the melting of helix I and the C-terminal half of helix IV, and followed by helix III, the N-terminal half of helix IV and helix II. This ordered melting of the helices is consistent with the conclusion from native-state hydrogen exchange, and can be rationalized by differences in intrinsic helix propensity. Guided by experimental Phi-values, a putative transition-state ensemble was extracted from the simulations. The residue helical probabilities of this transition-state ensemble show good correlation with the Phi-values. To further validate the putative transition-state ensemble, the effect of macromolecular crowding on the relative stability between the unfolded ensemble and the transition-state ensemble was calculated. The resulting effect of crowding on the folding kinetics agrees well with experimental observations. This study shows that molecular dynamics simulations combined with calculation of crowding effects provide an avenue for characterize the transition-state ensemble in atomic details. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.