PROTONATION STATES OF MEMBRANE-EMBEDDED CARBOXYLIC-ACID GROUPS IN RHODOPSIN AND METARHODOPSIN-II - A FOURIER-TRANSFORM INFRARED-SPECTROSCOPY STUDY OF SITE-DIRECTED MUTANTS
PROTONATION STATES OF MEMBRANE-EMBEDDED CARBOXYLIC-ACID GROUPS IN RHODOPSIN AND METARHODOPSIN-II - A FOURIER-TRANSFORM INFRARED-SPECTROSCOPY STUDY OF SITE-DIRECTED MUTANTS
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DOI：
10.1073/pnas.90.21.10206
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发表时间：
1993-11-01
影响因子：
11.1
通讯作者：
SIEBERT, F
中科院分区：
文献类型：
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作者：
FAHMY, K;JAGER, F;SIEBERT, F
开发了一种测量 COS 细胞中表达的去污剂溶解视紫红质的傅里叶变换红外 (FTIR) 差异光谱的方法。对天然牛视紫红质、在 COS 细胞中表达的视紫红质以及膜嵌入的羧酸基团进行氨基酸替换的三种表达的视紫红质突变体进行了实验：Asp-83 --> Asn (D83N)、Glu-122 --> Gln (E122Q) 和双突变体 D83N/E122Q。每个突变视蛋白均与 11-顺式视黄醛结合，产生可见光吸收色素。在光照下，每种突变色素形成一种类变视紫红质 II 的物质，在 380 nm 处具有最大吸收，能够通过转导蛋白激活鸟嘌呤核苷酸交换。记录每个样品的视紫红质与变视紫质 II 类光产物 FTIR 差异光谱。 COS 细胞视紫红质和突变体差异光谱与椎间盘膜的视紫红质的光谱密切对应。突变体 D83N 和 E122Q 的光谱中分别缺少 1767/1750 cm-1 和 1734/1745 cm-1 处的差异带（视紫红质/变视紫红质 II）。双突变体 D83N/E122Q 的光谱中不存在这两条带。这些结果表明 Asp-83 和 Glu-122 在视紫红质和变视紫红质 II 中均被质子化，这与 (H2)-H-2 中测量的光谱中观察到的同位素效应一致。 1712 cm-1 处的光产物带不受位置 83 和 122 处的单替换或双替换的影响。我们推断 1712 cm-1 带来自变视紫红质 II 中 Glu-113 的质子化。
A method was developed to measure Fourier-transform infrared (FTIR) difference spectra of detergent-solubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83 --> Asn (D83N), Glu-122 --> Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin II-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by transducin. Rhodopsin versus metarhodopsin II-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-cell rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes. Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm-1 and at 1734/1745 cm-1 were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin II, in agreement with the isotope effects observed in spectra measured in (H2)-H-2. A photo-product band at 1712 cm-1 was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-1 band arises from the protonation of Glu-113 in metarhodopsin II.