Department of Physics and Astronomy , Vrije Universiteit , Amsterdam 1081 HV , The Netherlands.
Department of Biophysical Organic Chemistry, Leiden Institute of Chemistry, Gorlaeus Laboratories , Leiden University , Leiden 2300 RA , The Netherlands.
J Phys Chem B. 2019 May 16;123(19):4242-4250. doi: 10.1021/acs.jpcb.9b01136. Epub 2019 May 7.
Microbial rhodopsins constitute a key protein family in optobiotechnological applications such as optogenetics and voltage imaging. Spectral tuning of rhodopsins into the deep-red and near-infrared spectral regions is of great demand in such applications because more bathochromic light into the near-infrared range penetrates deeper in living tissue. Recently, retinal analogues have been successfully used in ion transporting and fluorescent rhodopsins to achieve red-shifted absorption, activity, and emission properties. Understanding their photochemical mechanism is essential for further design of appropriate retinal analogues but is yet only poorly understood for most retinal analogue pigments. Here, we report the photoreaction dynamics of red-shifted analogue pigments of the proton pump proteorhodopsin (PR) containing A2 (all- trans-3,4-dehydroretinal), MOA2 (all- trans-3-methoxy-3,4-dehydroretinal), or DMAR (all- trans-3-dimethylamino-16-nor-1,2,3,4-didehydroretinal), utilizing femto- to submillisecond transient absorption spectroscopy. We found that the A2 analogue photoisomerizes in 1.4, 3.0, and/or 13 ps upon 510 nm light illumination, which is comparable to the native retinal (A1) in PR. On the other hand, the deprotonation of the A2 pigment Schiff base was observed with a dominant time constant of 67 μs, which is significantly slower than the A1 pigment. In the MOA2 pigment, no isomerization or photoproduct formation was detected upon 520 nm excitation, implying that all the excited molecules returned to the initial ground state in 2.0 and 4.2 ps. The DMAR pigment showed very slow excited state dynamics similar to the previously studied MMAR pigment, but only very little photoproduct was formed. The low efficiency of the photoproduct formation likely is the reason why DMAR analogue pigments of PR showed very weak proton pumping activity.
微生物视紫红质构成了光生物技术应用（如光遗传学和电压成像）中的关键蛋白家族。在这些应用中，将视紫红质的光谱调谐到深红色和近红外光谱区域是非常需要的，因为更多的远红光是近红外光可以穿透更深的活组织。最近，视网膜类似物已成功用于离子转运和荧光视紫红质中，以实现红移吸收、活性和发射特性。了解它们的光化学反应机制对于进一步设计合适的视网膜类似物至关重要，但对于大多数视网膜类似物色素来说，这仍然理解得很差。在这里，我们报告了质子泵视蛋白（PR）中含有 A2（全反式-3,4-脱氢视黄醛）、MOA2（全反式-3-甲氧基-3,4-脱氢视黄醛）或 DMAR（全反式-3-二甲氨基-16-去甲-1,2,3,4-二脱氢视黄醛）的红移类似物的光反应动力学，利用飞秒至亚毫秒瞬态吸收光谱法。我们发现，A2 类似物在 510nm 光照射下，1.4、3.0 和/或 13ps 内光异构化，这与 PR 中的天然视黄醛（A1）相当。另一方面，A2 色素的质子化Schiff 碱的去质子化观察到的主要时间常数为 67μs，明显慢于 A1 色素。在 MOA2 色素中，在 520nm 激发下没有检测到异构化或光产物形成，这意味着所有激发的分子在 2.0 和 4.2ps 内都返回初始基态。DMAR 色素表现出与之前研究的 MMAR 色素非常相似的缓慢激发态动力学，但仅形成很少的光产物。光产物形成的低效率可能是 PR 的 DMAR 类似物色素表现出非常弱的质子泵活性的原因。
