Elucidation of the Cl-transport mechanism of halorthodopsin by time-resolved electric and spectroscopic measurements

首先，我们改进了光电压测量系统，使其能够检测从1微秒到500毫秒的光电压产生。然后，使用时间分辨光电压测量系统，我们研究了盐生盐杆菌盐视蛋白的光电压动力学。作为氯化物浓度的函数。光电压由两个主要分量组成;一个具有亚ms范围时间常数，另一个具有不同幅度的ms范围时间常数。，这些组分表现出不同的Cl^-浓度依赖性（0.1 M至9 M）。我们发现，快成分的时间常数与Cl^-浓度相对无关，而慢成分的时间常数在Cl^-浓度较高时呈S形增加。快和慢过程分别归因于蛋白质内的电荷（Cl^-）运动和Cl^-排出。SHR-膜悬浮液的激光光解研究表明， ...更多信息 它们对应于N中间体的形成和衰变。慢分量的光电压幅值与Cl^-浓度呈钟形关系，其时间常数与Cl^-浓度的关系表明在胞质侧存在一个弱的、高度合作的Cl^-结合位点（表观K_D约为5 M，Hill系数约为5）。光电压幅度和慢过程时间常数的Cl^-浓度依赖性表明自发弛豫和离子移位之间存在竞争。弛豫时间常数大于100 ms。同时，还研究了鲨鱼卤视紫红质（hR）的生电活性。在将过量产生hR的膜部分吸收到薄聚合物膜上之后，我们测量了在不同Cl^-浓度下照射时的光电流。光电流的振幅呈现出钟形的Cl^-浓度依赖性。发现R108 Q突变体在高Cl^-浓度下也表现出光电流。结果表明，R108对于从培养基中摄取Cl^-是重要的，但是一旦Cl^-位于Schiff碱附近，即使没有R108，hR也可以表现出产电性。少

First of all, we improved the photovoltage measurements system so that it enabled us to examine the photovoltage generation from 1 μsec to 500 msec.Then, using the time-resolved photovoltage measurement system, we examined photovoltage kinetics of halorthodopsin from Halobacterium salinarum. As a function of chloride concentrations. The photovoltage consisted of two major components ; one with a sub-ms range time constant and the other with ms range time constant with different amplitudes., These components exhibited different Cl^- concentration dependencies (0.1 M to 9 M). We found that the time constant for the fast component was relatively independent of the Cl^- concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl^- concentrations. The fast and the slow processes were attributed to charge (Cl^-) movements within the protein and related to Cl^- ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that … More they correspond to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl^- concentration dependence and the Cl^- concentration dependence of its time constant suggested a weak and highly cooperative Cl^- binding site(s) at cytoplasmic side (apparent K_D of about 5 M and Hill coefficient ≧ 5). The Cl^- concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be > 100 ms.Electrogenic activity of the halorhodopsin (hR) from shark strain was also examined. After absorbing hR-overproduced membrane fraction onto a thin polymer film, we measured photoelectric current upon illumination at various Cl^- concentrations. The amplitude of the photoelectric current exhibited a bell-shaped Cl^- concentration dependency. It was found that R108Q mutant also exhibited photoelectric current at a high Cl^- concentration. The result indicates that R108 is important for uptaking Cl^- from the medium, but once Cl^- is located near the Schiff base, hR can exhibit electrogenicity even without R108. Less