期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 98, 期 15, 页码 8469-8474出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.151239698
关键词
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资金
- NIDA NIH HHS [DA09873] Funding Source: Medline
- NIGMS NIH HHS [F32 GM019835, 5T32GM07365, F32 GM19835-02, T32 GM007365] Funding Source: Medline
- NINDS NIH HHS [5R01 NS28471, R01 NS028471] Funding Source: Medline
Single-molecule studies of the conformations of the intact beta (2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta (2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.
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