Protein folding: Molecular dynamics simulations and in vitro studies for probing mechanism of urea- and guanidinium chloride-induced unfolding of horse cytochrome-c

Abstract Urea- and guanidinium chloride (GdmCl)-induced denatured states of horse cytochrome- c (cyt- c ) are structurally identical. It is then expected that estimates of ∆ G 0 N→U (Gibbs free energy change in the absence of denaturants) from GdmCl- and urea-induced denaturation curves should be identical, if denaturation induced by them follows a two-state mechanism. That denaturation of cyt- c by urea or GdmCl follows a two-state mechanism is reported by some in vitro studies while other in vitro studies reported contradictory observations. Molecular dynamic (MD) simulation is a technique that could reveal the mechanism of unfolding/folding of proteins in the absence and presence of chemical denaturants at the amino acid residue level. We therefore performed multiple unconstrained MD simulations of cyt- c (PDB ID: 1HRC) in water and aqueous mixtures of GdmCl and urea for the period of 0-500 ns at 300, 400 and 450 K, which showed that denaturation of cyt- c by urea and GdmCl is a two-state and three-state process, respectively. To corroborate these findings, we measured urea- and GdmCl-induced denaturation curves of different optical properties (circular dichroism at 222, 405 and 416 nm and absorbance at 405 nm) and analyzed them for ∆ G 0 N→U . These studies supported conclusions reached from MD simulation studies.