Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
European XFEL, 22869 Schenefeld, Germany.
J Phys Chem Lett. 2024 May 23;15(20):5510-5516. doi: 10.1021/acs.jpclett.4c00693. Epub 2024 May 15.
Viral rhodopsins are light-gated cation channels representing a novel class of microbial rhodopsins. For viral rhodopsin 1 subfamily members VirChR1 and OLPVR1, channel activity is abolished above a certain calcium concentration. Here we present a calcium-dependent spectroscopic analysis of VirChR1 on the femtosecond to second time scale. Unlike channelrhodopsin-2, VirChR1 possesses two intermediate states P and P on the ultrafast time scale, similar to J and K in ion-pumping rhodopsins. Subsequently, we observe multifaceted photocycle kinetics with up to seven intermediate states. Calcium predominantly affects the last photocycle steps, including the appearance of additional intermediates P and P representing the blocked channel. Furthermore, the photocycle of the counterion variant D80N is drastically altered, yielding intermediates with different spectra and kinetics compared to those of the wt. These findings demonstrate the central role of the counterion within the defined reaction sequence of microbial rhodopsins that ultimately defines the protein function.
病毒视紫红质是光门控阳离子通道，代表了一类新型的微生物视紫红质。对于病毒视紫红质 1 亚家族成员 VirChR1 和 OLPVR1，通道活性在超过一定的钙离子浓度时被废除。在这里，我们在飞秒到秒的时间尺度上呈现了 VirChR1 的钙离子依赖性光谱分析。与通道视紫红质-2 不同，VirChR1 在超快时间尺度上具有两个中间状态 P 和 P，类似于离子泵浦视紫红质中的 J 和 K。随后，我们观察到多达七个中间状态的多方面光循环动力学。钙离子主要影响最后一个光循环步骤，包括出现代表阻塞通道的额外中间状态 P 和 P。此外，抗衡离子变体 D80N 的光循环被严重改变，与 wt 相比，产生了具有不同光谱和动力学的中间状态。这些发现证明了在微生物视紫红质的定义反应序列中，抗衡离子处于核心地位，最终决定了蛋白质的功能。
