Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy.
Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy.
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DOI：
10.1021/bi010670x
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发表时间：
2001-07
期刊：
影响因子：
2.9
通讯作者：
D. Pan;R. Mathies
中科院分区：
文献类型：
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作者：
D. Pan;R. Mathies
在室温下，对视紫红质的光视紫质(Lumi)和后视紫红质I(Meta I)光中间体进行了时间分辨共振拉曼芯片流动实验，以阐明每种物质中发色团的结构以及蛋白质-发色团相互作用的变化。利用微探头系统聚焦微细加工流道中位移的泵浦和探测光束并探测散射，获得了延时分别为16微秒和1ms的Lumi和Meta I的瞬时拉曼光谱。这两个物种的指纹模式非常相似，具有全反式发色团的特征。LUMI在951/959 cm(-1)处有一个相对正常的氢离面(环状)双峰，而Meta I在957 cm(-1)处有一个单环带。这些结果表明，从底紫红质到Lumi和Meta I的转变包括发色团的松弛到更平坦的全反式构象，以及消除了使底紫红质中11H和12H摆动解偶联的结构扰动。令人惊讶的是，LUMI的质子化Schiff碱C=N伸缩模式(1638 cm(-1))比视紫红质和底紫红质的伸缩模式低得多，在D2O缓冲液中C=ND伸缩模式仅下移7 cm(-1)。这表明在底紫红质到Lumi的转变过程中，Schiff碱氢键被显著削弱。然而，在1654 cm(-1)处发现了Meta I的C=N伸缩模式，该伸缩模式与全反式质子化Schiff碱的伸缩模式相同，表现出正常的氢化引起的下移24 cm(-1)。因此，在底紫红质到Lumi转变过程中，发色团-蛋白质复合体的结构松弛似乎将Schiff碱基赶出了Glu113附近的氢键环境，氢键恢复到正常的溶剂化PSB值，但随着Meta I的形成，可能是一个不同的氢键受体。
Time-resolved resonance Raman microchip flow experiments have been performed on the lumirhodopsin (Lumi) and metarhodopsin I (Meta I) photointermediates of rhodopsin at room temperature to elucidate the structure of the chromophore in each species as well as changes in protein-chromophore interactions. Transient Raman spectra of Lumi and Meta I with delay times of 16 micros and 1 ms, respectively, are obtained by using a microprobe system to focus displaced pump and probe laser beams in a microfabricated flow channel and to detect the scattering. The fingerprint modes of both species are very similar and characteristic of an all-trans chromophore. Lumi exhibits a relatively normal hydrogen-out-of-plane (HOOP) doublet at 951/959 cm(-1), while Meta I has a single HOOP band at 957 cm(-1). These results suggest that the transitions from bathorhodopsin to Lumi and Meta I involve a relaxation of the chromophore to a more planar all-trans conformation and the elimination of the structural perturbation that uncouples the 11H and 12H wags in bathorhodopsin. Surprisingly, the protonated Schiff base C=N stretching mode in Lumi (1638 cm(-1)) is unusually low compared to those in rhodopsin and bathorhodopsin, and the C=ND stretching mode shifts down by only 7 cm(-1) in D2O buffer. This indicates that the Schiff base hydrogen bonding is dramatically weakened in the bathorhodopsin to Lumi transition. However, the C=N stretching mode in Meta I is found at 1654 cm(-1) and exhibits a normal deuteration-induced downshift of 24 cm(-1), identical to that of the all-trans protonated Schiff base. The structural relaxation of the chromophore-protein complex in the bathorhodopsin to Lumi transition thus appears to drive the Schiff base group out of its hydrogen-bonded environment near Glu113, and the hydrogen bonding recovers to a normal solvated PSB value but presumably a different hydrogen bond acceptor with the formation of Meta I.