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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Effect of channel mutations on the uptake and release of the retinal ligand in opsin
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Effect of channel mutations on the uptake and release of the retinal ligand in opsin

机译：通道突变对视蛋白中视网膜配体摄取和释放的影响

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摘要

In the retinal binding pocket of rhodopsin, a Schiff base links the retinal ligand covalently to the Lys296 side chain. Light transforms the inverse agonist 11-c/s-retinal into the agonist all-trans-retinal, leading to the active Meta II state. Crystal structures of Meta II and the active conformation of the opsin apoprotein revealed two openings of the 7-transmembrane (TM) bundle towards the hydrophobic core of the membrane, one between TM1/TM7 and one between TM5/TM6, respectively. Computational analysis revealed a putative ligand channel connecting the openings and traversing the binding pocket. Identified constrictions within the channel motivated this study of 35 rhodopsin mutants in which single amino acids lining the channel were replaced. 11-c/s-retinal uptake and all-frans-retinal release were measured using UV/visible and fluorescence spectroscopy. Most mutations slow or accelerate both uptake and release, often with opposite effects. Mutations closer to the Lys296 active site show larger effects. The nucleophile hydroxylamine accelerates retinal release 80 times but the action profile of the mutants remains very similar. The data show that the mutations do not probe local channel permeability but rather affect global protein dynamics, with the focal point in the ligand pocket. We propose a model for retinal/receptor interaction in which the active receptor conformation sets the open state of the channel for 11-c/s-retinal and all-trans-retinal, with positioning of the ligand at the active site as the kinetic bottleneck. Although other G protein-coupled receptors lack the covalent link to the protein, the access of ligands to their binding pocket may follow similar schemes.
机译：在视紫红质的视网膜结合袋中，席夫碱将视网膜配体共价连接至Lys296侧链。光将反向激动剂11-c / s-视网膜转变为全跨视网膜激动剂，从而导致活跃的Meta II状态。 Meta II的晶体结构和视蛋白载脂蛋白的主动构象表明，7个跨膜（TM）束朝着膜的疏水核有两个开口，一个在TM1 / TM7之间，一个在TM5 / TM6之间。计算分析表明推定的配体通道连接开口并横穿结合袋。通道内的狭窄表明了对35个视紫红质突变体的研究，其中突变体位于通道内。使用UV /可见光和荧光光谱法测量11-c / s-视网膜摄取和全乳-视网膜释放。大多数突变通常会产生相反的效果，从而减慢或加速摄取和释放。靠近Lys296活性位点的突变显示出更大的作用。亲核羟胺可加速视网膜释放80次，但突变体的作用曲线仍然非常相似。数据显示，突变不会探测局部通道的通透性，而是会影响整体蛋白质动力学，而焦点会位于配体袋中。我们提出了一种视网膜/受体相互作用的模型，其中活性受体构象设定了11-c / s-视网膜和全跨视网膜通道的开放状态，配体在活性位点的定位是动力学瓶颈。尽管其他G蛋白偶联受体缺乏与蛋白质的共价连接，但配体接近其结合袋的途径可能遵循相似的方案。

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  • 作者

    Ronny Piechnick; Eglof Ritter; Peter W. Hildebrand; Oliver P. Ernst; Patrick Scheerer; Klaus Peter Hofmann; Martin Heck;

  • 作者单位

    Institut fuer Medizinische Physik und Biophysik, Charite-Universitaetsmedizin Berlin, 10117 Berlin, Germany;

    Institut fuer Medizinische Physik und Biophysik, Charite-Universitaetsmedizin Berlin, 10117 Berlin, Germany;

    Institut fuer Medizinische Physik und Biophysik, Charite-Universitaetsmedizin Berlin, 10117 Berlin, Germany;

    Institut fuer Medizinische Physik und Biophysik, Charite-Universitaetsmedizin Berlin, 10117 Berlin, Germany,Departments of Biochemistry and Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON, Canada M5S 1A8;

    Institut fuer Medizinische Physik und Biophysik, Charite-Universitaetsmedizin Berlin, 10117 Berlin, Germany;

    Institut fuer Medizinische Physik und Biophysik, Charite-Universitaetsmedizin Berlin, 10117 Berlin, Germany,Zentrum fuer Biophysik und Bioinformatik, Humboldt-Universitaet zu Berlin, 10115 Berlin, Germany;

    Institut fuer Medizinische Physik und Biophysik, Charite-Universitaetsmedizin Berlin, 10117 Berlin, Germany;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    G protein-coupled-receptor; regeneration; signal transduction;

    机译：G蛋白偶联受体;再生;信号转导;

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