Experimental Biophysics, Institute for Biology, Humboldt-Universität zu Berlin, Invalidenstr. 42, 10115 Berlin, Germany.
Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Institute for Medical Physics and Biophysics, Group Protein X-ray Crystallography & Signal Transduction, Charitéplatz 1, D-10117 Berlin, Germany.
Sci Signal. 2019 Mar 19;12(573):eaav4203. doi: 10.1126/scisignal.aav4203.
The light-driven proton pump rhodopsin (CsR) provides-because of its high expression in heterologous host cells-an opportunity to study active proton transport under controlled electrochemical conditions. In this study, solving crystal structure of CsR at 2.0-Å resolution enabled us to identify distinct features of the membrane protein that determine ion transport directivity and voltage sensitivity. A specific hydrogen bond between the highly conserved Arg and the nearby nonconserved tyrosine (Tyr) guided our structure-based transformation of CsR into an operational light-gated proton channel (CySeR) that could potentially be used in optogenetic assays. Time-resolved electrophysiological and spectroscopic measurements distinguished pump currents from channel currents in a single protein and emphasized the necessity of Arg mobility in CsR as a dynamic extracellular barrier to prevent passive conductance. Our findings reveal that molecular constraints that distinguish pump from channel currents are structurally more confined than was generally expected. This knowledge might enable the structure-based design of novel optogenetic tools, which derive from microbial pumps and are therefore ion specific.
光驱动质子泵视紫红质（CsR）在异源宿主细胞中高表达，为在可控电化学条件下研究主动质子转运提供了机会。在这项研究中，我们以 2.0-Å 的分辨率解析了 CsR 的晶体结构，从而能够确定决定离子传输方向性和电压敏感性的膜蛋白的独特特征。高度保守的精氨酸（Arg）和附近非保守的酪氨酸（Tyr）之间的特定氢键指导了我们基于结构的 CsR 转化为可操作的光门控质子通道（CySeR），该通道可潜在用于光遗传学测定。时间分辨的电生理和光谱测量在单个蛋白中将泵电流与通道电流区分开来，并强调了 CsR 中 Arg 移动性作为动态细胞外屏障以防止被动电导的必要性。我们的研究结果表明，区分泵电流和通道电流的分子限制在结构上比通常预期的更受限制。这一知识可能使基于结构的新型光遗传学工具的设计成为可能，这些工具源自微生物泵，因此具有离子特异性。
