SOLID-STATE C-13 NMR DETECTION OF A PERTURBED 6-S-TRANS CHROMOPHORE IN BACTERIORHODOPSIN
SOLID-STATE C-13 NMR DETECTION OF A PERTURBED 6-S-TRANS CHROMOPHORE IN BACTERIORHODOPSIN
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DOI：
10.1021/bi00345a031
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
GRIFFIN, RG
中科院分区：
文献类型：
--
作者：
HARBISON, GS;SMITH, SO;GRIFFIN, RG
本文用固体~（13）C魔角样品自旋核磁共振波谱研究了细菌视紫红质发色团的紫罗酮环部分。从用在位置C-5、C-6、C-7、C-8和C-18处标记的视黄醇13 C再生的完全水合样品和从用在C-9和C-13处标记的视黄醇再生的冻干样品获得光谱。研究了冻干和水合形式的C-15标记样品。三个独立的NMR参数（低场元素的C-5化学位移张量，C-8各向同性化学位移，和C-18纵向弛豫时间）表明，生色团具有6-S-反式构象的蛋白质，在相反的6-S-顺式构象，是积极有利于维甲酸在溶液中。我们还观察到一个额外的27 ppm的低场位移的C-5位移张量的中间元素，这为C-5附近的带负电荷的蛋白质残基的存在提供了支持。在C-7附近的正电荷的证据，可能是为负电荷的coupons，也进行了讨论。在这些结果的基础上，我们提出了一个新的模型的视网膜结合位点，这具有重要意义的“视蛋白移位”的机制中观察到的细菌视紫红质。
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 couterion 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.