title: 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 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.
authors.affiliation: 1] Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA. [2] Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Alem W
authors.initials: AW
authors.lastname: Kahsai
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Sudarshan
authors.initials: S
authors.lastname: Rajagopal
authors.affiliation: Department of Biochemistry and Molecular Biology, Shandong University, School of Medicine, Jinan, China.
authors.forename: Jinpeng
authors.identifier: 0000000335721580
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Kunhong
authors.initials: K
authors.lastname: Xiao
mesh_terms: D005033:Ethylmaleimide; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D011487:Protein Conformation; D058849:Protein Refolding; D011506:Proteins / Q000737:chemistry*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry; D013387:Succinic Anhydrides
chemical_list: D011506:Proteins; D018343:Receptors, Adrenergic, beta-2; D013387:Succinic Anhydrides; C031801:succinic anhydride; D005033:Ethylmaleimide