Misra Ramprasad, Das Ishita, Dér András, Steinbach Gábor, Shim Jin-Gon, Busse Wayne, Jung Kwang-Hwan, Zimányi László, Sheves Mordechai
Department of Molecular Chemistry and Materials Science, Weizmann Institute of Science Rehovot 76100 Israel
Institute of Biophysics, Biological Research Centre, Eötvös Loránd Research Network Szeged H-6726 Hungary
Chem Sci. 2023 Sep 6;14(36):9951-9958. doi: 10.1039/d3sc02961a. eCollection 2023 Sep 20.
The function of microbial as well as mammalian retinal proteins ( rhodopsins) is associated with a photocycle initiated by light excitation of the retinal chromophore of the protein, covalently bound through a protonated Schiff base linkage. Although electrostatics controls chemical reactions of many organic molecules, attempt to understand its role in controlling excited state reactivity of rhodopsins and, thereby, their photocycle is scarce. Here, we investigate the effect of highly conserved tryptophan residues, between which the all- retinal chromophore of the protein is sandwiched in microbial rhodopsins, on the charge distribution along the retinal excited state, quantum yield and nature of the light-induced photocycle and absorption properties of rhodopsin (GR). Replacement of these tryptophan residues by non-aromatic leucine (W222L and W122L) or phenylalanine (W222F) does not significantly affect the absorption maximum of the protein, while all the mutants showed higher sensitivity to photobleaching, compared to wild-type GR. Flash photolysis studies revealed lower quantum yield of - photoisomerization in W222L as well as W222F mutants relative to wild-type. The photocycle kinetics are also controlled by these tryptophan residues, resulting in altered accumulation and lifetime of the intermediates in the W222L and W222F mutants. We propose that protein-retinal interactions facilitated by conserved tryptophan residues are crucial for achieving high quantum yield of the light-induced retinal isomerization, and affect the thermal retinal re-isomerization to the resting state.
微生物以及哺乳动物视网膜蛋白（视紫红质）的功能与一个光循环相关，该光循环由蛋白质中视网膜发色团的光激发引发，发色团通过质子化席夫碱连接共价结合。尽管静电作用控制着许多有机分子的化学反应，但试图理解其在控制视紫红质激发态反应性以及由此控制其光循环方面作用的研究却很少。在这里，我们研究了高度保守的色氨酸残基的作用，在微生物视紫红质中，蛋白质的全反式视网膜发色团夹在这些色氨酸残基之间，它们对沿视网膜激发态的电荷分布、量子产率以及光诱导光循环的性质和视紫红质（GR）的吸收特性的影响。用非芳香族亮氨酸（W222L和W122L）或苯丙氨酸（W222F）取代这些色氨酸残基不会显著影响蛋白质的最大吸收峰，而与野生型GR相比，所有突变体对光漂白表现出更高的敏感性。闪光光解研究表明，相对于野生型，W222L和W222F突变体中光异构化的量子产率较低。这些色氨酸残基也控制着光循环动力学，导致W222L和W222F突变体中中间体的积累和寿命发生改变。我们提出，由保守色氨酸残基促进的蛋白质 - 视网膜相互作用对于实现光诱导视网膜异构化的高量子产率至关重要，并影响视网膜热再异构化为静止状态。
