Amide protection in an early folding intermediate of cytochrome c

Abstract

For many proteins, compact states appear long before the rate-limiting step in the formation of the native structure. A key issue is whether the initial collapse of the chain is driven by random or more specific hydrophobic interactions. Hydrogen-exchange labeling coupled with NMR was used to monitor the formation of stable hydrogen-bonded and solvent-excluded structure in horse cytochrome c (cyt c). Protection was measured using a hydrogen exchange/folding competition protocol at variable pH and short competition time (2 ms). Protection factors of threefold to eightfold were observed in all three alpha helices of cyt c, whereas other regions showed no significant protection. This suggests that the compact states that are present contain segments of marginally stable hydrogen-bonded structure. When the intermediate(s) are destabilized, only amide protons from Cys14, Ala15 and His18 show significant protection, indicating a region of persistent residual structure near the covalently bound heme group in the unfolded protein. Fluorescence-detected stopped-flow studies showed that the maximum protection factor in the early intermediate is consistent with its unfolding equilibrium constant. Together with previous fluorescence and CD results, the observed pattern of amide protection is consistent with the early formation of an alpha-helical core domain in an ensemble of compact states, indicating that efficient folding is facilitated by stepwise acquisition of native structural elements. These specific early interactions are established on the sub-millisecond time scale, prior to the rate-limiting step for folding.