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首页> 外文期刊>Biochemistry >Regulation of Photoactivation in Vertebrate Short Wavelength Visual Pigments Protonation of the Retinylidene Schiff Base and a Counterion Switch
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Regulation of Photoactivation in Vertebrate Short Wavelength Visual Pigments Protonation of the Retinylidene Schiff Base and a Counterion Switch

机译：视黄醛席夫碱和抗衡离子开关的脊椎动物短波长视觉色素的质子化中的光活化调控。

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Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambda_(max) = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambda_(max) = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.
机译：非洲爪蟾紫锥视蛋白（VCOP）及其抗衡离子变体（VCOP-D108A）在哺乳动物COS1细胞中表达，并用11-顺式-视网膜再生。通过低温电子光谱，同源性建模，分子动力学和分子轨道理论研究了VCOP-D108A中的光转导过程。 VCOP-D108A变体是一种类似UV的颜料，其光活化效率低于小鼠短波长敏感视觉颜料（MUV），并且光漂白特性显着不同。理论计算可追溯到附近的谷氨酸残基E176的质子化状态的差异，这是视紫红质中E181的同源性。我们发现E176在MUV中带负电，但在VCOP-D108A中为中性（质子化）。在黑暗状态下，VCOP-D108A具有未质子化的席夫碱（SB）生色团（λ_（max）= 357 nm）。 VCOP-D108A在70 K处发生光解，产生红移光平稳状态（λ_（max）= 380 nm）。我们确定了两个发光中间体，其中从深蓝过渡到发光中间体是依赖于温度和pH值的。蓝绿色中间物衰变成一个更红移的中间物，称为lumiI。SB在lumi I到lumi II的过渡过程中被质子化。亮度II的衰变形成meta I，然后形成meta II。我们得出的结论是，即使在VCOP-D108A中不存在主要抗衡离子的情况下，SB在光活化级联过程中也会质子化。我们检查这种观察与视觉色素活化的平衡离子转换机制的相关性。

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