Identifying the Site of Initial Tertiary Structure Disruption during Apomyoglobin Unfolding

Abstract

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.

Structural Details, Pathways, and Energetics of Unfolding Apomyoglobin

“…Experimental characterization of such a barrier state is very difficult because of its extremely low population, but the presence of globally intact hydrogen bonds alongside locally disrupted packing interactions in the early unfolding of apomyoglobin 27 and DHFR 28 has been observed, and is consistent with the present simulation results, as well as with earlier MD simulations on BPTI 29 that used thermal unfolding. In their studies of partially unfolded conformations of barnase by molecular dynamics at various temperatures, Caflisch & Karplus 13 observed a delay in the entrance of water into the protein interior upon unfolding at 360 K and low pH, but no such delay at 600 K. In light of these results, our simulations at 300 K are of special interest.…”

Solvation and Desolvation Dynamics in Apomyoglobin Folding Monitored by Time-resolved Infrared Spectroscopy

“…67 The stop-flow thiol-disulfide exchange study demonstrated that the side-chain contacts at the C-terminal end of helix E are disrupted at the beginning of the unfolding. 68 It was suggested that the entry point of water in the unfolding pathway of apoMb was localized at the interface between helices A and E. 68 The current results demonstrating the presence of water until the last step of apoMb folding support the repeated suggestions assuming the involvement of water in the initial unfolding phase of apoMb.…”

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

“…4), In support, Δω I1;MG is large for both 15 N and 1 H (1.0 and 0.5 ppm, respectively). Recent studies of apoMb folding and unfolding kinetics (26,27) suggest that the initial event during unfolding and the final event during refolding involve solvation and desolvation, respectively. Indeed, it has been suggested that desolvation of backbone amides may contribute to the energy barrier in the final ratelimiting step of the folding pathway (27).…”