Challenges in the interpretation of protein h/d exchange data: a molecular dynamics simulation perspective.

Many protein structural investigations involve the use of H/D exchange (HDX) techniques. It is commonly thought that amide backbone protection arises from intramolecular H-bonding and/or burial of NH sites. Recently, fundamental HDX-related tenets have been called into question. The current work focuses on ubiquitin for exploring the defining features that distinguish amides in "open" (exchange-competent) and "closed" (exchange-incompetent) environments. Instead of relying on static X-ray structures, we employ all-atom molecular dynamics (MD) simulations for obtaining a dynamic view of the protein ground state and its surrounding solvent. The HDX properties for 57 out of 72 NH sites can be readily explained on the basis of backbone and side chain H-bonding, as well as solvent accessibility considerations. Unexpectedly, the same criteria fail for predicting the HDX characteristics of the remaining 15 amides. Significant protection is seen for numerous exposed NH sites that are not engaged in intramolecular H-bonds, whereas other amides that seemingly share the same features are unprotected. We scrutinize the proposal that H-bonding to crystallographically defined water can cause the protection of surface amides. For ubiquitin, the positioning of crystal water is not compatible with this idea. To further explore possible solvation effects, we tested for the presence of partially immobilized water networks. Our MD data reveal no difference in the solvation properties of protected vs unprotected surface amides, making it unlikely that restricted water dynamics can cause anomalous amide protection. The findings reported here suggest that efforts to deduce protein structural features on the basis of HDX protection factors may yield misleading results. This conclusion is relevant for initiatives that rely on sparse structural data as constraints for elucidating protein conformations. It may be necessary to pursue detailed quantum mechanical studies of the protein, the solvent, and the hydroxide catalyst for obtaining a comprehensive understanding of the factors that govern HDX rates. The considerable size of the systems involved makes such endeavors a daunting task.