Farahbakhsh Z T, Ridge K D, Khorana H G, Hubbell W L
Jules Stein Eye Institute, Los Angeles, California, USA.
Biochemistry. 1995 Jul 11;34(27):8812-9. doi: 10.1021/bi00027a033.
All 20 single cysteine substitution mutants in the sequence Y136-M155 of bovine rhodopsin have been prepared and modified with a sulfhydryl-specific nitroxide reagent. This sequence contains the C-D interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra were analyzed, both in the dark and after photoexcitation. Accessibility data show that the rhodopsin polypeptide crosses an aqueous/hydrophobic boundary near V138 and H152. The nitroxide mobilities inferred from the spectra are consistent with a model where the C helix extends to at least residue C140, with much of the helix surface in contact with protein rather than lipid near the cytoplasmic surface of the membrane. Upon photoexcitation, electron paramagnetic resonance spectral changes are observed at sites on the putative C helix surface that are in contact with the protein and at specific sites in the C-D interhelical loop. A simple interpretation of these results is that photoexcitation involves a rigid body movement of the C helix relative to the others in the helix bundle.
已制备了牛视紫红质Y136 - M155序列中的所有20个单半胱氨酸取代突变体，并用巯基特异性氮氧化物试剂进行了修饰。该序列包含C - D螺旋间环，一个转导素相互作用位点。测定了连接的氮氧化物在溶液中与顺磁探针碰撞的可及性，并在黑暗中和光激发后分析了电子顺磁共振光谱。可及性数据表明，视紫红质多肽在V138和H152附近穿过水/疏水边界。从光谱推断的氮氧化物迁移率与一个模型一致，即C螺旋至少延伸到C140残基，在膜的细胞质表面附近，螺旋的大部分表面与蛋白质而非脂质接触。光激发后，在假定的C螺旋表面与蛋白质接触的位点以及C - D螺旋间环的特定位点观察到电子顺磁共振光谱变化。对这些结果的一个简单解释是，光激发涉及C螺旋相对于螺旋束中其他螺旋的刚体运动。
