Harbison G S, Smith S O, Pardoen J A, Courtin J M, Lugtenburg J, Herzfeld J, Mathies R A, Griffin R G
Biochemistry. 1985 Nov 19;24(24):6955-62. doi: 10.1021/bi00345a031.
Solid-state 13C magic angle sample spinning NMR spectroscopy has been used to study the ionone ring portion of the chromophore of bacteriorhodopsin. Spectra were obtained from fully hydrated samples regenerated with retinals 13C labeled at positions C-5, C-6, C-7, C-8, and C-18 and from lyophilized samples regenerated with retinals labeled at C-9 and C-13. C-15-labeled samples were studied in both lyophilized and hydrated forms. Three independent NMR parameters (the downfield element of the C-5 chemical shift tensor, the C-8 isotropic chemical shift, and the C-18 longitudinal relaxation time) indicate that the chromophore has a 6-s-trans conformation in the protein, in contrast to the 6-s-cis conformation that is energetically favored for retinoids in solution. We also observe an additional 27 ppm downfield shift in the middle element of the C-5 shift tensor, which provides support for the existence of a negatively charged protein residue near C-5. Evidence for a positive charge near C-7, possibly the counterion for the negative charge, is also discussed. On the basis of these results, we present a new model for the retinal binding site, which has important implications for the mechanism of the "opsin shift" observed in bacteriorhodopsin.
固态13C魔角样品旋转核磁共振光谱已被用于研究细菌视紫红质发色团的紫罗酮环部分。光谱是从用C-5、C-6、C-7、C-8和C-18位置标记13C的视黄醛再生的完全水合样品以及用C-9和C-13位置标记视黄醛再生的冻干样品中获得的。对C-15标记的样品进行了冻干和水合两种形式的研究。三个独立的核磁共振参数（C-5化学位移张量的低场分量、C-8各向同性化学位移和C-18纵向弛豫时间）表明，发色团在蛋白质中具有6-s-反式构象，这与溶液中类视黄醇在能量上更有利的6-s-顺式构象形成对比。我们还观察到C-5位移张量中间分量有额外的27 ppm低场位移，这为C-5附近存在带负电荷的蛋白质残基提供了支持。还讨论了C-7附近存在正电荷的证据，可能是负电荷的抗衡离子。基于这些结果，我们提出了一个新的视黄醛结合位点模型，这对视紫红质中观察到的“视蛋白位移”机制具有重要意义。
