Resek J F, Farahbakhsh Z T, Hubbell W L, Khorana H G
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1993 Nov 16;32(45):12025-32. doi: 10.1021/bi00096a012.
Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.
已经产生了视紫红质的五种突变体，每个突变体在细胞质结构域的第62、65、140、240或316位含有一个独特的半胱氨酸残基。用巯基特异性氮氧化物自旋标记对单个反应性半胱氨酸进行衍生化，并在黑暗中和光漂白后，在月桂基麦芽糖苷和洋地黄皂苷中分析电子顺磁共振（EPR）光谱。附着的氮氧化物与极性和非极性顺磁性试剂的碰撞速率表明它们都暴露于水环境中。在洋地黄皂苷中对突变体进行光漂白，使蛋白质停滞在间态I中间体，附着的氮氧化物的迁移率几乎没有变化。另一方面，在月桂基麦芽糖苷中进行光漂白产生了间态II中间体，并且附着在第140和316位的氮氧化物的EPR光谱发生了显著变化。这些数据直接揭示了在形成间态II时伴随发生的细胞质环中的光诱导构象变化。
