Resler Tom, Schultz Bernd-Joachim, Lórenz-Fonfría Víctor A, Schlesinger Ramona, Heberle Joachim
Experimental Molecular Biophysics, Freie Universität Berlin, Berlin, Germany.
Genetic Biophysics at Department of Physics, Freie Universität Berlin, Berlin, Germany.
Biophys J. 2015 Jul 21;109(2):287-97. doi: 10.1016/j.bpj.2015.06.023.
Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm(-1) to 1738 cm(-1) in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm(-1) in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm(-1) and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.
视紫红质通道蛋白（ChRs）是光门控阳离子通道。蓝光激发后，该蛋白质经历一个包含不同中间体的光循环。在此，我们以纳秒时间分辨率记录了莱茵衣藻中ChR2在可见光和红外区域的瞬态吸光度变化，后者是使用可调谐量子级联激光器实现的。由于质子转移反应在通道门控中起关键作用，我们通过监测各种关键天冬氨酸和谷氨酸残基羧基在H₂O和D₂O中的C=O伸缩振动，确定了它们的振动以及动力学同位素效应（VIEs和KIEs）。D156在其去质子化和再质子化过程中表现出显著的KIE（>2），这证实了它作为视网膜席夫碱内部质子供体的作用。通过研究D156E变体，仔细研究了D156在D₂O中异常的VIE，其从1736 cm⁻¹上移至1738 cm⁻¹。E156的C=O伸缩在D₂O中下移了8 cm⁻¹，为羧基的可及性提供了证据。在晚期脱敏状态的寿命期间，E90的C=O伸缩带在去质子化和再质子化反应中表现出9 cm⁻¹的VIE和约2的KIE。在20 ns至5 ms的时间范围内测定的1的KIE与E90的早期去质子化不相符。
