Fahmy K, Siebert F, Sakmar T P
Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
Biochemistry. 1994 Nov 22;33(46):13700-5. doi: 10.1021/bi00250a021.
In the rhodopsin mutant E113A/A117E the position of the protonated Schiff base counterion, Glu113, is moved by one helix turn from position 113 to 117. The photoreaction of this mutant pigment was studied by Fourier-transform infrared (FTIR) difference spectroscopy. At acidic pH, formation of a 474-nm absorbing photoproduct previously characterized biochemically as a species that activates transducin caused infrared absorption changes typical of metarhodopsin II (MII) formation in native rhodopsin. Specific spectral alterations revealed a localized perturbation near the protonated Schiff base in the dark state. In addition, an infrared band assigned to the C = O stretching vibration of Glu113 in MII of rhodopsin was abolished in the mutant. Absorption changes caused by Asp83 and Glu122 C = O stretching vibrations characteristic of rhodopsin MII formation were not affected. At alkaline pH, mutant E113A/A117E formed predominantly a 382-nm absorbing photoproduct. It displayed infrared-difference absorption bands significantly different from those of native MII over a large spectral range. These results support the conclusion that the 474-nm photoproduct of mutant E113A/A117E, despite a protonated Schiff base linkage, displays a predominantly MII-like conformation capable of catalyzing guanine-nucleotide exchange by transducin.
在视紫红质突变体E113A/A117E中，质子化席夫碱抗衡离子Glu113的位置从第113位移动了一个螺旋圈至第117位。通过傅里叶变换红外（FTIR）差光谱研究了该突变色素的光反应。在酸性pH下，形成了一种474纳米吸收光产物，该产物先前在生物化学上被表征为一种激活转导蛋白的物质，导致了天然视紫红质中视紫红质II（MII）形成的典型红外吸收变化。特定的光谱变化揭示了在黑暗状态下质子化席夫碱附近的局部扰动。此外，在突变体中，视紫红质MII中Glu113的C = O伸缩振动所对应的红外波段消失了。视紫红质MII形成所特有的由Asp83和Glu122的C = O伸缩振动引起的吸收变化不受影响。在碱性pH下，突变体E113A/A117E主要形成一种382纳米吸收光产物。在很大的光谱范围内，它显示出与天然MII明显不同的红外差吸收带。这些结果支持了这样的结论：突变体E113A/A117E的474纳米光产物尽管有质子化席夫碱连接，但仍表现出主要为MII样的构象，能够催化转导蛋白进行鸟嘌呤核苷酸交换。
