Abstract
Knowledge of the structure and dynamics of proteins and protein assemblies is critical both for understanding the molecular basis of physiological and patho-physiological processes and for guiding drug design. While X-ray crystallography and nuclear magnetic resonance spectroscopy are both excellent techniques for this purpose, both suffer from limitations, including the requirement for high quality crystals and large amounts of material. Recently, hydrogen/deuterium exchange measured using mass spectrometry (HXMS) has emerged as a powerful new tool for the study of protein structure, dynamics and interactions in solution. HXMS exploits the fact that backbone amide hydrogens can exchange with deuterium when a protein is incubated in D2O, and that the rate of the exchange process is highly dependent on the local structural environment. Several features of HXMS make it an especially attractive approach, including small sample requirements and the ability to study extremely large protein assemblies that are not amenable to other techniques. Here, we provide an overview of HXMS and describe several recent applications to problems of medical interest. After reviewing the molecular basis of the H/D exchange process, the different steps of the HXMS experiment - labeling, rapid proteolysis, fragment separation and mass measurement - are described, followed by a discussion of data analysis methods. Finally, we describe recent results on the application of HXMS to 1) mapping drug/inhibitor binding sites and detecting drug induced conformational changes, 2) studying viral capsid structure and assembly, and 3) characterizing the structure of pathological protein conformations, specifically amyloid fibrils.
Keywords: Hydrogen exchange, mass spectrometry, protein dynamics, amyloid, drug screening, viral capsid
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