Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan.
Biochemistry. 2012 Jun 12;51(23):4677-84. doi: 10.1021/bi300485r. Epub 2012 May 25.
Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR. Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.
细菌视紫红质（BR）和盐细菌视紫红质（HR）分别是光驱动的质子外排泵和氯离子内流泵。它们具有相似的蛋白质结构，由七个跨膜螺旋组成，结合全反式视黄醛。BR 可以通过在 85 位的单个氨基酸替换转化为氯离子泵，这表明 BR 和 HR 共享共同的运输机制，离子特异性由该位置的氨基酸决定。然而，相应的反向突变不能使 HR 转化为质子泵。在这里，我们将 HR 的 6 位和 10 位氨基酸突变为 BR 样，而这些 HR 多重突变体从未泵出过质子。光诱导傅里叶变换红外光谱显示，BR 的视黄醛席夫碱和水的氢键均较强，而 HR 的氢键均较弱。HR 的多重突变体表现出较强的席夫碱氢键，但水的氢键仍然较弱。我们得出结论，HR 非功能性转化的原因是缺乏强氢键水，这是质子泵的功能决定因素。
