Schädel Sandra A, Heck Martin, Maretzki Dieter, Filipek Slawomir, Teller David C, Palczewski Krzysztof, Hofmann Klaus Peter
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany.
International Institute of Molecular and Cell Biology and the Department of Chemistry, University of Warsaw, 1 Pasteur St, PL-02109 Warsaw, Poland.
J Biol Chem. 2003 Jul 4;278(27):24896-24903. doi: 10.1074/jbc.M302115200. Epub 2003 Apr 21.
Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.
光激活视紫红质（变视紫红质II）的失活过程，在视紫红质激酶和抑制蛋白相互作用之后，涉及全反式视黄醛与脱辅基蛋白之间共价键的水解。尽管会短暂形成寿命较长的储存形式变视紫红质III，但全反式视黄醛最终会从活性位点释放。在此，我们探讨这样一个问题：这种释放是否会产生一种能在光感受器膜脂质相中自由扩散的视黄醛。释放反应伴随着内在蛋白荧光的增加（释放信号），这是由于活性位点中的视黄醛所施加的荧光猝灭得到缓解而产生的。当外源性类视黄醇与天然视蛋白膜非共价结合时，会引发类似的荧光降低（摄取信号）。11-顺式视黄醛的摄取比其与脱辅基蛋白形成视黄叉键的速度更快。在变视紫红质II衰变过程中从活性位点释放的内源性全反式视黄醛并未产生摄取信号。数据表明，除了视黄叉口袋（位点I）之外，视蛋白内还有另外两个类视黄醇结合位点。参与摄取信号的位点II是一个入口位点，而当视黄醛从位点I释放后仍保持结合状态时，出口位点（位点III）会被占据。对视黄醛通道机制的支持来自视紫红质晶体结构，该结构揭示了两个假定的疏水结合位点。这种机制使得光异构化发色团的释放以及新合成的11-顺式视黄醛的摄取成为一个单向过程用于视紫红质的再生。
