Off-pathway intermediate(s) in the refolding of alkaline ferrocytochrome c: A pulse-labeling HX NMR study

Abstract

Proteins meet with the stipulations of Levinthal. The classical mechanism involving a pre-transition search topology step can explain adequately how the biologically relevant time scale is achieved. Association of the classical mechanism of chemical kinetics naturally allows depiction of folding pathways with dead-end intermediates. By using the approach of hydrogen exchange (HX) pulse labeling in conjunction with NMR spectroscopy, it is shown here that carbonmonoxycytochrome c when allowed to refold from a strongly alkaline medium to neutral pH folds via a highly misfolded state. The misfolded state placed at the dead end of the folding pathway must then melt to the initial unfolded state to have a chance to refold to the correct native state.