To examine the relationship between protein structural dynamics and measurable hydrogen exchange (HX) data, the detailed exchange behavior of most of the backbone amide hydrogens of Staphylococcal nuclease was compared with that of their neighbors, with their structural environment, and with other information. Results show that H-bonded hydrogens are protected from exchange, with HX rate effectively zero, even when they are directly adjacent to solvent. The transition to exchange competence requires a dynamic structural excursion that removes H-bond protection and allows exposure to solvent HX catalyst. The detailed data often make clear the nature of the dynamic excursion required. These range from whole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as a single peptide group or side chain or water molecule. The particular motion that dominates the exchange of any hydrogen is the one that allows the fastest HX rate. The motion and the rate it produces are determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Many of these motions occur over time scales that are appropriate for biochemical function.

To investigate the determinants of protein hydrogen exchange (HX), HX rates of most of the backbone amide hydrogens of Staphylococcal nuclease were measured by NMR methods. A modified analysis was used to improve accuracy for the faster hydrogens. HX rates of both near surface and well buried hydrogens are spread over more than 7 orders of magnitude. These results were compared with previous hypotheses for HX rate determination. Contrary to a common assumption, proximity to the surface of the native protein does not usually produce fast exchange. The slow HX rates for unprotected surface hydrogens are not well explained by local electrostatic field. The ability of buried hydrogens to exchange is not explained by a solvent penetration mechanism. The exchange rates of structurally protected hydrogens are not well predicted by algorithms that depend only on local interactions or only on transient unfolding reactions. These observations identify some of the present difficulties of HX rate prediction and suggest the need for returning to a detailed hydrogen by hydrogen analysis to examine the bases of structure-rate relationships, as described in the companion paper (Skinner et al., Protein Sci 2012;21:996-1005).

Native-state hydrogen exchange experiments under EX1 conditions can distinguish partially unfolded intermediates by their formation rates and identify the amide hydrogens exposed and protected in each. Results obtained define a cytochrome c intermediate seen only poorly before and place it early on the major unfolding pathway. Four distinct unfolding steps are found to be kinetically ordered in the same pathway sequence inferred before.