Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Department of Physics, Nagoya University, Nagoya 464-8602, Japan.
Biochemistry. 2022 Sep 20;61(18):1936-1944. doi: 10.1021/acs.biochem.2c00328. Epub 2022 Aug 25.
Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified as blue-absorbing PRs (BPR; λ ∼ 490 nm) and green-absorbing PRs (GPR; λ ∼ 525 nm). We previously converted BPR into GPR using an anomalous pH effect, which was achieved by an irreversible process at around pH 2. Recent size-exclusion chromatography (SEC) and atomic force microscopy (AFM) analyses of BPR from (VcBPR) revealed the anomalous pH effect owing to the irreversible transition from pentamer to monomer. Different p values of the Schiff base counterion between pentamer and monomer lead to different colors at the same pH. Here, we incorporate systematic mutation into VcBPR and examine the anomalous pH effect. The anomalous pH effect was observed for the mutants of key residues near the retinal chromophore such as D76N, D206N, and Q84L, indicating that the Schiff base counterions and the L/Q switch do not affect the irreversible transition from pentamer to monomer at pH ∼ 2. We then focus on the two specific interactions at the intermonomer interface in a pentamer, E29/R30/D31 and W13/H54. Single mutants such as E29Q, R30A, W13A, and H54A and the wild type (WT) exhibited an anomalous pH effect. In contrast, the anomalous pH effect was lost for E29Q/H54A, R30A/H54A, and W13A/E29Q. Size-exclusion chromatography (SEC) and atomic force microscopy (AFM) measurements showed monomer forms in the original states of the double mutants, being a clear contrast to the pentamer forms of all single mutants in the original states. It was concluded that the pentamer structure of VcBPR was stabilized by an electrostatic interaction in the E29/R30/D31 region and a hydrogen-bonding interaction in the W13/H54 region, which was disrupted at pH 2 and converted into monomers.
光驱动质子泵 Proteorhodopsin（PR）存在于海洋细菌中，数以千计的 PR 被分类为蓝光吸收 PR（BPR；λ∼490nm）和绿光吸收 PR（GPR；λ∼525nm）。我们之前利用异常 pH 效应将 BPR 转化为 GPR，该效应是通过约 pH 2 时的不可逆过程实现的。最近对来自 （VcBPR）的 BPR 的分子筛层析（SEC）和原子力显微镜（AFM）分析表明，异常 pH 效应是由于五聚体不可逆地转化为单体。五聚体和单体之间的席夫碱抗衡离子的不同 p 值在相同 pH 下导致不同的颜色。在这里，我们将系统突变整合到 VcBPR 中，并检查异常 pH 效应。在靠近视黄醛发色团的关键残基如 D76N、D206N 和 Q84L 的突变体中观察到异常 pH 效应，表明席夫碱抗衡离子和 L/Q 开关不会影响 pH∼2 时五聚体到单体的不可逆转变。然后，我们将重点放在五聚体中单体间界面的两个特定相互作用上，即 E29/R30/D31 和 W13/H54。单个突变体，如 E29Q、R30A、W13A 和 H54A 以及野生型（WT）都表现出异常 pH 效应。相比之下，E29Q/H54A、R30A/H54A 和 W13A/E29Q 的异常 pH 效应消失。分子筛层析（SEC）和原子力显微镜（AFM）测量显示，在原始状态下，双突变体形成单体形式，与所有原始状态下单突变体的五聚体形式形成鲜明对比。结论是，VcBPR 的五聚体结构通过 E29/R30/D31 区域的静电相互作用和 W13/H54 区域的氢键相互作用得到稳定，在 pH 2 时被破坏并转化为单体。
