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首页> 外文期刊>Biochemistry >Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors
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Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors

机译：人绿色OPSIN的氢气/氘交换质谱显示在单稳态视觉G蛋白偶联受体的细胞外环2中的保守的Pro-Pro主题

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摘要

Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N-32 and N-34 are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.
机译：Opsins在眼视网膜中包含光敏G蛋白偶联受体（GPCR）的蛋白质组分，其负责将光转换为生化信号。这里，我们使用氢/氘（H / D）交换与质谱相结合，以在光活化时映射绿色锥形Opsin的构象变化。然后我们将这些发现与罗地普林报告的那些结果进行了比较。绿色锥形Opsin的H / D交换的程度大于黑暗和漂白状态的洛越蛋白，表明绿色锥形Opsin的结构性异质性。进一步的分析显示，绿色锥形Opsin作为暗（无活性）和漂白（活性）状态的二聚体存在，并且N-32和N-34的预测糖基化位点确实是糖基化的。氘摄取在绿色锥形Opsin的无活性和活性状态之间的比较还公开了细胞外N-末端区域的溶剂可渗透性和发色团结合位点的增加。在跨膜螺旋四（TM4）的细胞外侧增加H / D兑换的H / D置侧与序列比对的分析揭示了单稳态视觉GPCR的细胞外环2（EL2）中的保守Pro-Pro主题。这些数据在TM4和TM5的细胞外侧发出新的洞察力，并为未来的功能评估提供了基础。

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