Ramos Lavoisier S, Chen Min-Hsuan, Knox Barry E, Birge Robert R
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.
Biochemistry. 2007 May 8;46(18):5330-40. doi: 10.1021/bi700138g. Epub 2007 Apr 18.
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 (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 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.
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-顺式视黄醛再生。通过低温电子光谱、同源建模、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变体是一种类似紫外线的色素，其光激活效率低于小鼠短波长敏感视觉色素（MUV），且光漂白特性明显不同。理论计算将这种差异追溯到附近谷氨酸残基E176的质子化状态，E176是视紫红质中E181的同源等效物。我们发现E176在MUV中带负电荷，但在VCOP-D108A中呈中性（质子化）。在黑暗状态下，VCOP-D108A具有未质子化的席夫碱（SB）发色团（λmax = 357 nm）。VCOP-D108A在70 K下的光解产生一个红移的光稳态（λmax = 380 nm）。我们鉴定出两种发光中间体，其中从嗜碱性中间体到发光中间体的转变取决于温度和pH值。嗜碱性中间体衰变为一个红移更大的中间体，称为发光I。在从发光I到发光II的转变过程中，SB质子化。发光II的衰变形成间体I，随后形成间体II。我们得出结论，即使在VCOP-D108A中没有初级抗衡离子，SB在光激活级联过程中也会质子化。我们研究了这一观察结果与视觉色素激活的抗衡离子开关机制的相关性。
