Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein.

@article{citeulike:439376, abstract = {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.}, address = {Department of Chemistry, Stanford University, Stanford, CA 94305, USA.}, author = {Peleg, G. and Ghanouni, P. and Kobilka, B. K. and Zare, R. N. }, citeulike-article-id = {439376}, doi = {http://dx.doi.org/10.1073/pnas.151239698}, issn = {0027-8424}, journal = {Proc Natl Acad Sci U S A}, keywords = {b2ar, fcs, membrane-protein}, month = {July}, number = {15}, pages = {8469--8474}, posted-at = {2007-07-05 20:17:19}, priority = {2}, title = {Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein.}, url = {http://dx.doi.org/10.1073/pnas.151239698}, volume = {98}, year = {2001} }

TY - JOUR ID - citeulike:439376 L3 - citeulike-article-id:439376 TI - Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein. JF - Proc Natl Acad Sci U S A VL - 98 IS - 15 SP - 8469 EP - 8474 AD - Department of Chemistry, Stanford University, Stanford, CA 94305, USA. SN - 0027-8424 N2 - 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. KW - b2ar KW - fcs KW - membrane-protein AU - Peleg, G AU - Ghanouni, P AU - Kobilka, BK AU - Zare, RN PY - 2001/07/17/ UR - http://dx.doi.org/10.1073/pnas.151239698 ER -