期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 287, 期 43, 页码 36305-36311出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.406371
关键词
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资金
- National Institutes of Health [NH028471]
- Natural Sciences and Engineering Research Council of Canada (NSERC) [261980]
The G protein-coupled beta(2)-adrenoreceptor (beta(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and beta-arrestin. As such, the energy landscape of beta(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of beta(2)AR with a trifluoromethyl probe, F-19 NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in beta(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of beta(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.
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