Seminars Archive

Hydrogen Deuterium eXchange Mass Spectrometry (HDX MS) for protein higher order structure analysis

Abstract
In solution, proteins exist in many conformations dominated by the lowest free energy folded form. Because so many functional aspects of proteins are tied to higher order structural changes, it is just as important to investigate and characterize these structural changes as it is to understand general features of the protein structure. Hydrogen deuterium exchange (HDX), which probes for local dynamics of polypeptide chains, is a labeling approach to study mechanisms of protein folding, allosteric regulation, impact of post-translational modifications and other ligand binding to conformational dynamics, define truncation points for enhancing crystallization success and mapping interactions between proteins. Reproducibility of the obtained results highly depend on tight control of the experimental parameters because incorporated deuterium is partially washed off during subsequent LC-MS analysis. Hydroxyl radical footprinting is a labeling approach complementary to HDX were solvent exposed amino acids in the protein structure are chemically labeled allowing detailed LC-MS analysis without compromising reproducibility. However, it is known that chemical modification can alter protein structure and if hydroxyl radical for labeling is produced relatively slowly one can never be sure about the result: is it a consequence of labeling modification procedure or actual protein conformation? For successful footprinting experiment and reliable results it is crucial to use high flux radiation of the synchrotron beamline to produce abundant hydroxyl radical in a short period of time allowing rapid labeling before chemical labeling induced change in protein structure will occur. With the advent of monitoring reaction progress with mass spectrometry both methods became generally more applicable reaching possibility to analyze larger proteins at lower concentrations. In this presentation I will explain theoretical basis and practical steps carried out in typical continuous HDX experiment. In the later part I will illustrate how HDX was utilized to characterize active site conformation dynamics of E. coli purine nucleoside phosphorylase and point out benefits we hope to gain by proposing synchrotron footpriniting (SF) experiment with this sample.