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

Journal Article (Journal Article)

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 β2-adrenergic receptor, including experiments that characterize the functional conformational changes associated with activation of distinct signaling pathways induced by different β-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.

Full Text

Duke Authors

Cited Authors

  • Kahsai, AW; Rajagopal, S; Sun, J; Xiao, K

Published Date

  • 2014

Published In

Volume / Issue

  • 9 / 6

Start / End Page

  • 1301 - 1319

PubMed ID

  • 24810039

Pubmed Central ID

  • PMC4367447

Electronic International Standard Serial Number (EISSN)

  • 1750-2799

Digital Object Identifier (DOI)

  • 10.1038/nprot.2014.075


  • eng

Conference Location

  • England