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

通过时间分辨电学和光谱测量阐明盐紫质的 Cl 传输机制

基本信息

批准号：
11680653
负责人：
MUNEYUKI Eiro
金额：
$ 2.37万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680653/
关键词：
halorhodopsin ion pump photocych photovoltage spectroscopic measurements フォトサイクル電荷移動

项目摘要

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

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，我们检测了来自盐杆菌的盐视紫红质的光电压动力学。光电压由两个主要部分组成；一个具有亚毫秒范围的时间常数，另一个具有不同的毫秒范围时间常数。这些成分表现出不同的 Cl^- 浓度依赖性（0.1 M 到 9 M），我们发现快成分的时间常数相对独立于 Cl^- 浓度，而慢成分的时间常数呈 S 形增加。在较高的 Cl^- 浓度下，快速和缓慢的过程分别归因于蛋白质内的电荷 (Cl^-) 运动以及与 Cl^- 喷射相关的 shR 膜悬浮液的激光光解研究表明……更多地，它们对应于N中间体的形成和衰变，慢分量的光电压幅度显示出扭曲的钟形Cl^-浓度依赖性，并且其时间常数的Cl^-浓度依赖性表明弱且高度合作的Cl。 ^- 结合位点位于细胞质侧（表观 K_D 约为 5 M，希尔系数 ≧ 5）。光电压幅度和缓慢过程的时间常数的 Cl^- 浓度依赖性表明两者之间存在竞争。自发弛豫和离子易位估计弛豫时间常数 > 100 ms。在将 hR 过量产生的膜部分吸收到聚合物薄膜上后，我们还检测了来自鲨鱼品系的盐视紫红质 (hR) 的电活性。不同Cl^-浓度下光照下的光电流显示出钟形Cl^-浓度依赖性。发现R108Q突变体也显示出光电流。结果表明，R108 对于从培养基中吸收 Cl^- 很重要，但一旦 Cl^- 位于席夫碱附近，即使没有 R108，hR 也能表现出电性。