Combination of HDX-MS and in silico modeling to study enzymatic reactivity and stereo-selectivity at different solvent conditions

Publication date: Available online 1 February 2020Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Alexey A. Makarov, Roxana E. Iacob, Gregory F. Pirrone, Agustina Rodriguez-Granillo, Leo Joyce, Ian Mangion, Jeffrey C. Moore, Edward C. Sherer, John R. EngenAbstractThe higher-order structure of a protein defines its function, and protein structural dynamics are often essential for protein binding and enzyme catalysis. Methods for protein characterization in solution are continuously being developed to understand and explore protein conformational changes with regards to function and activity. The goal of this study was to survey the use of combining HDX-MS global conformational screening with in silico modeling and continuous labeling peptide-level HDX-MS as an approach to highlight regions of interest within an enzyme required for biocatalytic processes. We surveyed in silico modeling correlated with peptide level HDX-MS experiments to characterize and localize transaminase enzyme structural dynamics at different conditions. This approach was orthogonally correlated with a global Size-Exclusion-HDX (SEC-HDX) screen for global conformational comparison and global alpha-helical content measurements by circular dichroism. Enzymatic activity and stereo-selectivity of transaminases were compared at different reaction-solution conditions that forced protein conformational changes by increasing acetonitrile concentration. The experimental peptide-level HDX-MS resu...
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research