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Tracking G-protein-coupled receptor activation using genetically encoded infrared probes

机译：使用遗传编码的红外探针跟踪G蛋白偶联受体的活化

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摘要

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-L-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplas-mic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.
机译：视紫红质是负责昏暗视觉的原型七螺旋家族A G蛋白偶联受体（GPCR）。光使视紫红质的视网膜发色团异构化，并触发跨膜螺旋的一致运动，包括螺旋6（H6）的向外倾斜和H5的较小运动，从而形成G蛋白结合和激活的位点。但是，这些螺旋重排的确切时间序列和机制尚不清楚。我们使用定点非天然氨基酸诱变来工程化视紫红质，并在选定位点掺入对叠氮基-L-苯丙氨酸残基，并使用视紫红质沿其激活途径进行红外光谱监测叠氮基振动特征。在这里，我们报告了叠氮探针在静电环境中的显着变化，甚至在无活性的光产物Meta I中，也早于活性受体状态形成之前。这些早期的变化表明H6发生了明显的旋转，并使H5的细胞质部分远离H3。随后，H6的大向外倾斜导致细胞质表面开放，形成活性受体光产物Meta II。因此，我们的结果揭示了较大的刚体螺旋运动之前的早期构象变化，并为解释最新的GPCR晶体结构和了解在其他GPCR激活期间观察到的构象亚状态提供了基础。

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  • 来源
    《Nature》 |2010年第7293期|p.1386-1389|共4页
  • 作者

    Shixin Ye; Ekaterina Zaitseva; Gianluigi Caltabiano; Gebhard F. X. Schertler; Thomas P. Sakmar; Xavier Deupi; Reiner Vogel;

  • 作者单位

    Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA;

    Biophysics Section, Institute of Molecular Medicine and Cell Research, University of Freiburg, Hermann Herder Str. 9, D-79104 Freiburg, Germany;

    Laboratori de Medicina Computacional, Unitat de Bioestadistica, Universitat Autonoma de Barcelona, 08193 Bellaterra, Catalunya, Spain;

    MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen, Switzerland;

    Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA;

    Laboratori de Medicina Computacional, Unitat de Bioestadistica, Universitat Autonoma de Barcelona, 08193 Bellaterra, Catalunya, Spain;

    Biophysics Section, Institute of Molecular Medicine and Cell Research, University of Freiburg, Hermann Herder Str. 9, D-79104 Freiburg, Germany;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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