Structural characterization of short-lived protein unfolding intermediates by laser-induced oxidative labeling and mass spectrometry.

The structural characterization of short-lived intermediates provides insights into the mechanisms of protein folding and unfolding. Using holo-myoglobin as a model system, this work reports the application of oxidative pulse labeling for experiments of this kind. Protein unfolding is triggered by a pH jump from 6.5 to 3.2 in 150 mM NaCl. Subsequent (.-)OH exposure at various time points using laser photolysis of H2O2 leads to covalent modifications of solvent-exposed side chains within approximately 1 mus (Hambly, D. M.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2005, 16, 2057-2063). Most of these modifications appear as 16 Da adducts in the mass spectrum of the intact protein. The overall extent of labeling increases with time, reflecting the exposure of reactive side chains that had previously been buried. Unfolding and disruption of heme-protein interactions go to completion within approximately 10 s. Spatially resolved information is obtained by monitoring the signal intensities of unmodified tryptic peptides. After 50 ms, many regions have lost most of their protection, whereas structure is retained in the B, E, F, and G helices. The BEF core remains partially folded, even after 500 ms, at which point helix G is fully unprotected. The observation of an "early" (BEFG) and a "late" (BEF) intermediate is in accord with optical stopped-flow measurements. Formation of these transient species is attributed to the persistence of heme-protein interactions during the early stages of the reaction. Overall, this work demonstrates the feasibility of laser-induced oxidative labeling as a tool for characterizing the structure of short-lived protein conformers. The combination of this approach with ultrarapid mixing or photochemical triggering should allow folding experiments in the submillisecond range.