Structural Basis of the Green-Blue Color Switching in Proteorhodopsin as Determined by NMR Spectroscopy
Structural Basis of the Green-Blue Color Switching in Proteorhodopsin as Determined by NMR Spectroscopy
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DOI：
10.1021/ja5097946
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发表时间：
2014-12-17
影响因子：
15
通讯作者：
Glaubitz, Clemens
中科院分区：
文献类型：
--
作者：
Mao, Jiafei;Nhu-Nguyen Do;Glaubitz, Clemens
在海洋微生物中发现的视紫红质（PR）是这个星球上最丰富的视网膜光感受器。PR变体显示出高水平的环境适应性，因为它们的颜色被调整到可用光的最佳波长。两个主要的绿色和蓝色亚家族可以通过位置105处的L/Q点突变相互转化。在这里，我们揭示了这种有趣的颜色调节效应背后的结构基础。高场固态NMR光谱用于可视化结构的变化，直接在脂质双层内的绿色PR后，引入的绿色-蓝色L105 Q突变。所观察到的效应位于结合口袋内，靠近视网膜碳C14和C15。随后，魔角旋转（MAS）核磁共振光谱与灵敏度增强的动态核极化（DNP）被应用于精确地确定视网膜结构周围的C14-C15。突变后，观察到C14-C15键的显著拉伸、C15的去屏蔽和视网膜链平面外扭曲的轻微改变。因此，L105 Q蓝色开关局部作用于视网膜本身，并诱导异构化区域和亚胺键之间的共轭缺陷。因此，S-0-S-1能隙增加，导致观察到的蓝移。发色团结构的扭曲也提供了一个解释，为延长的初级反应检测泵探测光谱，而蛋白质内的化学位移扰动可以链接到后期的光循环中间体研究的闪光光解延长。除了解决一个长期存在的问题，这项研究还表明，从高场和DNP增强的MAS NMR光谱与时间分辨光谱获得的数据相结合，使膜蛋白的深入功能研究具有强大的协同作用。
Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two major green and blue subfamilies can be interconverted through a L/Q point mutation at position 105. Here we reveal the structural basis behind this intriguing color-tuning effect. High-field solid-state NMR spectroscopy was used to visualize structural changes within green PR directly within the lipid bilayer upon introduction of the green-blue L105Q mutation. The observed effects are localized within the binding pocket and close to retinal carbons C14 and C15. Subsequently, magic-angle spinning (MAS) NMR spectroscopy with sensitivity enhancement by dynamic nuclear polarization (DNP) was applied to determine precisely the retinal structure around C14-C15. Upon mutation, a significantly stretched C14-C15 bond, deshielding of C15, and a slight alteration of the retinal chains out-of-plane twist was observed. The L105Q blue switch therefore acts locally on the retinal itself and induces a conjugation defect between the isomerization region and the imine linkage. Consequently, the S-0-S-1 energy gap increases, resulting in the observed blue shift. The distortion of the chromophore structure also offers an explanation for the elongated primary reaction detected by pump-probe spectroscopy, while chemical shift perturbations within the protein can be linked to the elongation of late-photocycle intermediates studied by flash photolysis. Besides resolving a long-standing problem, this study also demonstrates that the combination of data obtained from high-field and DNP-enhanced MAS NMR spectroscopy together with time-resolved optical spectroscopy enables powerful synergies for in-depth functional studies of membrane proteins.