Lawson M A, Zacks D N, Derguini F, Nakanishi K, Spudich J L
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Biophys J. 1991 Dec;60(6):1490-8. doi: 10.1016/S0006-3495(91)82184-1.
The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).
单细胞真核藻类莱茵衣藻的CC - 2359菌株最初被描述为低色素突变体，现已发现其在广泛的光照强度范围内对光刺激缺乏避光停止反应。通过外源添加全反式视黄醛可恢复该突变体的避光反应。我们将基于计算机的细胞追踪和运动分析与CC - 2359的视黄醛异构体和视黄醛类似物重构相结合，以研究避光反应受体的特性。与13 - 顺式、11 - 顺式和9 - 顺式视黄醛相比，全反式视黄醛能实现最快且最完全的重构。一种通过碳桥锁定在6 - s - 反式构象的类似物能够重构，而相应的6 - s - 顺式锁定类似物则不能。多烯链中带有五元环从而阻止围绕13 - 14双键异构化的视黄醛类似物（锁定在13 - 反式或13 - 顺式构型）不能恢复反应，但它们会进入生色团结合口袋，这可通过它们对反应中全反式视黄醛再生的抑制作用得到证明。全反式视黄醛与每种13 - 锁定类似物之间的竞争实验结果符合一个模型，即每个生色团与光受体脱辅基蛋白表现出可逆结合。一种竞争性抑制方案与数据高度吻合，并允许计算出全反式、13 - 反式锁定和13 - 顺式锁定类似物在体内重构过程的表观解离常数，分别为2.5×10⁻¹¹ M、5.2×10⁻¹⁰ M和5.4×10⁻⁹ M。生色团对反式构型和6 - s - 反式构象的要求，以及13位锁定类似物缺乏信号功能，是古细菌视紫红质的特征，而非先前研究的真核视紫红质（即视觉色素）。
