Jäger F, Fahmy K, Sakmar T P, Siebert F
Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs-Universität, Freiburg, Federal Republic of Germany.
Biochemistry. 1994 Sep 13;33(36):10878-82. doi: 10.1021/bi00202a005.
In order to investigate the molecular mechanism of rhodopsin photoactivation, site-directed mutants of bovine rhodopsin were studied by Fourier-transform infrared (FTIR) difference spectroscopy. Rhodopsin mutants E113D and E113A were prepared in which the retinylidene Schiff base counterion, Glu113, was replaced by Asp and Ala, respectively. FTIR difference spectra were recorded and compared with spectra of recombinant native rhodopsin. Both mutant pigments formed photoproducts at 0 degrees C with vibrational absorption bands typical of the metarhodopsin II (MII) state of rhodopsin. The FTIR difference spectrum of E113D was nearly identical to that of rhodopsin. A positive band at 1712 cm-1 caused by the protonation of an internal carboxylic acid in rhodopsin was shifted slightly to 1709 cm-1 in mutant E113D. E113A was studied at acidic pH in the presence of chloride as an inorganic counterion to the protonated Schiff base. The 1712-cm-1 (1709-cm-1) band was absent in the FTIR difference spectrum of mutant E113A. Therefore, we have assigned the 1712-cm-1 absorbance band to the C = O stretching vibration of protonated Glu113 in MII of rhodopsin. These results show that the Schiff base counterion of rhodopsin, the carboxylate side chain of Glu113, becomes protonated during MII formation.
为了研究视紫红质光激活的分子机制，利用傅里叶变换红外（FTIR）差光谱对牛视紫红质的定点突变体进行了研究。制备了视紫红质突变体E113D和E113A，其中视黄醛席夫碱抗衡离子Glu113分别被Asp和Ala取代。记录FTIR差光谱并与重组天然视紫红质的光谱进行比较。两种突变色素在0℃时均形成光产物，具有视紫红质的变视紫红质II（MII）状态典型的振动吸收带。E113D的FTIR差光谱与视紫红质的几乎相同。视紫红质中内部羧酸质子化引起的1712 cm-1处的正带在突变体E113D中略微移至1709 cm-1。在酸性pH下，在存在作为质子化席夫碱无机抗衡离子的氯离子的情况下研究了E113A。突变体E113A的FTIR差光谱中不存在1712-cm-1（1709-cm-1）带。因此，我们将1712-cm-1吸收带归属于视紫红质MII中质子化的Glu113的C = O伸缩振动。这些结果表明，视紫红质的席夫碱抗衡离子，即Glu113的羧酸盐侧链，在MII形成过程中发生质子化。
