Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry.

ABSTRACT: An understanding of the mechanism accompanying functional conformational changes associated with protein activation has important implications for drug design. Here we describe a powerful method, conformational changes and dynamics using stable-isotope labeling and mass spectrometry (CDSiL-MS), which involves chemical labeling by isotope-coded forms of N-ethylmaleimide or succinic anhydride to site-specifically label the side chains of cysteines or lysines, respectively, in native proteins. Subsequent MS analysis allows the quantitative monitoring of reactivity of residues as a function of time, providing a measurement of the labeling kinetics and thereby enabling elucidation of conformational changes of proteins. We demonstrate the utility of this method using a model G protein-coupled receptor, the ?2-adrenergic receptor, including experiments that characterize the functional conformational changes associated with activation of distinct signaling pathways induced by different ?-adrenoceptor ligands. The procedure requires 5 d, and it can easily be adapted to systems in which soluble and detergent-solubilized membrane protein targets, which undergo function-dependent conformational changes, can be interrogated structurally to allow drug screening.

SUBMITTER: Kahsai AW 

PROVIDER: S-EPMC4367447 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature