Abstract：
The creation of unidirectional ion transporters across membranes represents one of the greatest challenges in chemistry. Proton-pumping rhodopsins are composed of seven transmembrane helices with a retinal chromophore bound to a lysine side chain via a Schiff base linkage and provide valuable insights for designing such transporters. What makes these transporters particularly intriguing is the discovery of both outward and inward proton-pumping rhodopsins. Surprisingly, despite sharing identical overall structures and membrane topologies, these proteins facilitate proton transport in opposite directions, implying an underlying rational mechanism that can transport protons in different directions within similar protein structures. In this study, we unraveled this mechanism by examining the chromophore structures of deprotonated intermediates in schizorhodopsins, a recently discovered subfamily of inward proton-pumping rhodopsins, using time-resolved resonance Raman spectroscopy. The photocycle of schizorhodopsins revealed the cis-trans thermal isomerization that precedes reprotonation at the Schiff base of the retinal chromophore. Notably, this order has not been observed in other proton-pumping rhodopsins, but here, it was observed in all seven schizorhodopsins studied across the archaeal domain, strongly suggesting that cis-trans thermal isomerization preceding reprotonation is a universal feature of the schizorhodopsin family. Based on these findings, we propose a structural basis for the remarkable order of events crucial for facilitating inward proton transport. The mechanism underlying inward proton transport by schizorhodopsins is straightforward and rational. The insights obtained from this study hold great promise for the design of transmembrane unidirectional ion transporters.
摘要：
跨膜的单向离子转运蛋白的产生代表了化学中最大的挑战之一。质子泵送视紫质由七个跨膜螺旋组成，视网膜发色团通过席夫碱键与赖氨酸侧链结合，为设计此类转运蛋白提供了有价值的见解。使这些转运蛋白特别吸引人的是发现了向外和向内的质子泵吸视紫红质。令人惊讶的是,尽管共享相同的整体结构和膜拓扑，这些蛋白质促进相反方向的质子运输，暗示着一种潜在的合理机制，可以在相似的蛋白质结构内向不同方向运输质子。在这项研究中,我们通过检查裂孔视紫红质中去质子化中间体的发色团结构来揭示这种机制，最近发现的一个向内质子泵吸视紫红质亚科，使用时间分辨共振拉曼光谱。裂孔视紫红质的光循环揭示了顺式-反式热异构化，该异构化先于视网膜发色团的席夫碱进行再质子化。值得注意的是,在其他质子泵浦视紫红质中没有观察到这种顺序，但在这里,在整个古细菌领域研究的所有七个分裂视紫红质中都观察到了这一点，强烈表明顺式-反式热异构化在再质子化之前是分裂视紫红质家族的普遍特征。基于这些发现,我们为促进质子向内传输至关重要的事件的显着顺序提出了结构基础。裂孔视紫红质向内质子传输的机制是简单而合理的。从这项研究中获得的见解为跨膜单向离子转运蛋白的设计带来了巨大的希望。
