Stopped-flow analysis on anion binding to blue-form halorhodopsin from Natronobacterium pharaonis: comparison with the anion-uptake process during the photocycle.
Stopped-flow analysis on anion binding to blue-form halorhodopsin from Natronobacterium pharaonis: comparison with the anion-uptake process during the photocycle.
复制标题
阴离子与法老嗜碱杆菌蓝色型盐视紫红质结合的停流分析：与光循环过程中阴离子摄取过程的比较。
DOI：
10.1021/bi011788g
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
K. Nitta
中科院分区：
文献类型：
--
作者：
Maki Sato;T. Kanamori;N. Kamo;M. Demura;K. Nitta
法老钠杆菌c端组氨酸标签的光驱动氯离子泵Pharaonis halorhodopsin （phR）在大肠杆菌中表达。蛋白用0.1% n-十二烷基β -d -麦芽吡喃苷溶解，用镍柱纯化。从培养基中去除Cl-产生蓝色phR（phR（蓝色）），在发色团附近失去了Cl-。Cl-的加入将phR（蓝色）转化为红移的Cl键形式（phR(Cl)）。phR（蓝色）和phR（Cl）的圆二色光谱在视觉区域显示出一个双叶体，表明phR单体的特异性低聚。诱导phR（蓝色）向phR(X)转变的阴离子浓度顺序为Br- < Cl- < NO3- < N3-，与从褐藻膜中纯化的phR相同。采用停流快速混合的时间分辨吸收变化法测定氯化物结合动力学。氯离子结合速率由快慢两部分组成，快慢部分的变化幅度约占总变化幅度的90%。快组分在25℃下100 mM NaCl下的反应速率常数为260 s(-1)，表观活化能为35 kJ/mol。这些值与先前报道的光循环中Cl-吸收过程（O—> hR’反应）非常吻合[Váró et al. (1995) Biochemistry 34, 14500-14507]。此外，两种速率对Cl-浓度的依赖关系相似。这些观察结果表明，o -中间体与phR（蓝色）相似，并且光循环过程中的Cl-吸收可能是一个被动过程。
Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.