Identifying the site of initial tertiary structure disruption during apomyoglobin unfolding.

Structural characterization of protein unfolding intermediates [Kiefhaber et al. (1995) Nature 375, 513; Hoeltzli et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318], which until recently were thought to be nonexistent, is beginning to give information on the mechanism of unfolding. To test for apomyoglobin unfolding intermediates, we monitored kinetics of urea-induced denaturation by stop-flow tryptophan fluorescence and quench-flow amide hydrogen exchange. Both measurements yield a single, measurable kinetic phase of identical rate, indicating that the reaction is highly cooperative. A burst phase in fluorescence, however, suggests that an intermediate is rapidly formed. To structurally characterize it, we carried out stop-flow thiol-disulfide exchange studies of 10 single cysteine-containing mutants. Cysteine probes buried at major sites of helix-helix pairing revealed that side chains throughout the protein unpack and become accessible to the labeling reagent [5, 5'-dithiobis (2-nitrobenzoic acid)] with one of two rates. Probes located at all helical-packing interfaces-except for one-become exposed at the rate of global unfolding as determined by fluorescence and hydrogen exchange measurements. In contrast, probes located at the A-E helical interface undergo complete thiol-disulfide exchange within the mixing dead time of 6 ms. These results point to the existence of a burst-phase unfolding intermediate that contains globally intact hydrogen bonds but locally disrupted side-chain packing interactions. Dissolution of secondary and tertiary structure are therefore not tightly coupled processes. We suggest that disruption of tertiary structure may be a stepwise process that begins at the weakest point of the native fold, as determined by native-state hydrogen-exchange parameters.