Mechanism of folding and stability of Met80Gly mutant of cytochrome-c

Abstract

The present study deals with identifying conformational ensembles underlying the folding pathway of a model protein, the wild type (WT) horse cytochrome-c (cyt-c) and its mutant (M80G) in water and 8 M urea at different temperatures (300–450 K). Outcomes of this study revealed that the structural dynamics of both proteins remain restricted to the marginal changes in water and urea at 300 and 350 K. The dynamic equilibrium shifted towards unfolding at 400 K, and structures of both proteins are lost completely at 450 K, however, it occurred distinctly in water and urea. Free energy landscape analyses suggested that the unfolding of the WT and M80G proteins happens through different conformational states, the native (N), meta-stable intermediate (IN), intermediate (I), partially unfolded (IU) and completely unfolded (U) states. In urea at 400 K, IN state of M80G mutant is relatively long-lived as compared to the WT protein. However, the structure of M80G protein is lost more frequently in urea at 450 K. Our in vitro studies of the urea-induced denaturation of the WT cyt-c and its M80G mutant at pH 6.0 and 25 °C showed that the mutant is more stable than the WT protein. This conclusion is reached from the analysis of the denaturation curve (mean residue ellipticity at 222 nm versus urea concentration plot).