Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.
Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.
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细菌视紫红质中氨基酸 Asp-85、Asp-212 和 Arg-82 的取代会影响泵的质子释放相和席夫碱的 pK。
DOI：
10.1073/pnas.87.3.1018
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发表时间：
1990
影响因子：
11.1
通讯作者：
Heyn,MP
中科院分区：
文献类型：
--
作者：
Otto,H;Marti,T;Holz,M;Mogi,T;Stern,LJ;Engel,F;Khorana,HG;Heyn,MP
研究了光循环和闪光诱导的细菌视紫红质突变体的质子释放和吸收，其中Asp-85被Ala、Asn或Glu取代; Asp-212被Asn或Glu取代; Asp-115被Ala、Asn或Glu取代; Asp-96被Ala、Asn或Glu取代;在pH7.3的二肉豆蔻酰磷脂酰胆碱/3-[（3-胆酰胺丙基）二甲铵]-1-丙磺酸胶束中，Arg-82被Ala或Gln取代。在Asp-85-Ala和Asp-85-Asn突变体中，不存在带电荷的羧基分别导致在600和595 nm处的蓝色发色团，并将席夫碱去质子化的pK分别降低至8.2和7，这表明Asp-85作为席夫碱的抗衡物的作用。的早期部分的光周期的Asp-85-Ala和Asp-85-Asn突变体的强烈扰动，形成一个弱的M-样中间减慢约100倍，野生型。在这两种突变体中，质子释放也较慢，但明显先于M的上升。在Asp-85-Asn突变体中早期（小于0.2微秒）反向光电压分量的幅度非常大，净电荷位移接近于零，表明质子在膜的细胞质侧释放和摄取。这些数据表明，一个强制性的作用，天冬氨酸-85在有效的去质子化的席夫碱和在质子释放阶段，可能作为质子受体。在Asp-212-Asn突变体中，在410 nm处的吸光度变化的上升减慢至220微秒，其幅度小，并且质子的释放延迟至1.9 ms。在650 nm处的吸光度变化表明在早期时间范围内具有缓慢K中间体的扰动。因此，Asp-212也参与了席夫碱的电荷转移和去质子化的早期事件。在Arg-82-Gln突变体中，没有观察到净瞬时质子释放，而在Arg-82-Ala突变体中，摄取和释放被逆转。在Asp-85-Glu、Arg-82-Ala和Arg-82-Gln突变体中紫-蓝转变的pK位移以及Asp-85-Ala、Asp-85-Asn和Asp-212-Asn突变体的光循环和光电信号的相似性表明Asp-85、Arg-82、Asp-212、和席夫碱是质子释放的关键。
Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.