Conformer-specific characterization of nonnative protein states using hydrogen exchange and top-down mass spectrometry.

Characterization of structure and dynamics of nonnative protein states is important for understanding molecular mechanisms of processes as diverse as folding, binding, aggregation, and enzyme catalysis to name just a few; however, selectively probing local minima within rugged energy landscapes remains a problem. Mass spectrometry (MS) coupled with hydrogen/deuterium exchange (HDX) offers a unique advantage of being able to make a distinction among multiple protein conformers that coexist in solution; however, detailed structural interrogation of such states previously remained out of reach of HDX MS. In this work, we exploited the aforementioned unique feature of HDX MS in combination with the ability of MS to isolate narrow populations of protein ions to characterize individual protein conformers coexisting in solution in equilibrium. Subsequent fragmentation of the protein ions using electron-capture dissociation allowed us to allocate the deuterium distribution along the protein backbone, yielding a backbone-amide protection map for the selected conformer unaffected by contributions from other protein states present in solution. The method was tested with the small regulatory protein ubiquitin (Ub), which is known to form nonnative intermediate states under a variety of mildly denaturing conditions. Protection maps of these intermediate states obtained at residue-level resolution provide clear evidence that they are very similar to the so-called A-state of Ub that is formed in solutions with low pH and high alcohol. Method validation was carried out by comparing the backbone-amide protection map of native Ub with those deduced from high-resolution NMR measurements.