Sudo Yuki, Nishihori Tatsuya, Iwamoto Masayuki, Shimono Kazumi, Kojima Chojiro, Kamo Naoki
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Biophys J. 2008 Jul;95(2):753-60. doi: 10.1529/biophysj.107.125294. Epub 2008 Mar 28.
Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II) is a seven transmembrane helical retinal protein. ppR forms a signaling complex with pharaonis Halobacterial transducer II (pHtrII) in the membrane that transmits a light signal to the sensory system in the cytoplasm. The M-state during the photocycle of ppR (lambda(max) = 386 nm) is one of the active (signaling) intermediates. However, progress in characterizing the M-state at physiological temperature has been slow because its lifetime is very short (decay half-time is approximately 1 s). In this study, we identify a highly stable photoproduct that can be trapped at room temperature in buffer solution containing n-octyl-beta-d-glucoside, with a decay half-time and an absorption maximum of approximately 2 h and 386 nm, respectively. HPLC analysis revealed that this stable photoproduct contains 13-cis-retinal as a chromophore. Previously, we reported that water-soluble hydroxylamine reacts selectively with the M-state, and we found that this stable photoproduct also reacts selectively with that reagent. These results suggest that the physical properties of the stable photoproduct (named the M-like state) are very similar with the M-state during the photocycle. By utilizing the high stability of the M-like state, we analyzed interactions of the M-like state and directly estimated the pK(a) value of the Schiff base in the M-like state. These results suggest that the dissociation constant of the ppR(M-like)/pHtrII complex greatly increases (to 5 muM) as the pK(a) value greatly decreases (from 12 to 1.5). The proton transfer reaction of ppR from the cytoplasmic to the extracellular side is proposed to be caused by this change in pK(a).
法老嗜盐菌视紫红质（ppR，也称为法老嗜盐菌传感视紫红质II）是一种七跨膜螺旋视网膜蛋白。ppR在膜中与法老嗜盐菌转导蛋白II（pHtrII）形成信号复合物，将光信号传递到细胞质中的传感系统。ppR光循环过程中的M态（λmax = 386 nm）是活性（信号）中间体之一。然而，在生理温度下对M态进行表征的进展一直很缓慢，因为其寿命非常短（衰减半衰期约为1秒）。在本研究中，我们鉴定出一种高度稳定的光产物，它可以在含有正辛基-β-D-葡萄糖苷的缓冲溶液中于室温下捕获，衰减半衰期和最大吸收波长分别约为2小时和386 nm。高效液相色谱分析表明，这种稳定的光产物含有13-顺式视黄醛作为发色团。此前，我们报道水溶性羟胺与M态选择性反应，并且我们发现这种稳定的光产物也与该试剂选择性反应。这些结果表明，稳定光产物（命名为M样态）的物理性质与光循环过程中的M态非常相似。通过利用M样态的高稳定性，我们分析了M样态的相互作用，并直接估计了M样态中席夫碱的pKa值。这些结果表明，随着pKa值大幅降低（从12降至1.5），ppR（M样态）/pHtrII复合物的解离常数大幅增加（至5 μM）。ppR从细胞质侧向细胞外侧的质子转移反应被认为是由这种pKa变化引起的。
