Time-resolved spectroscopy of the early photolysis intermediates of rhodopsin Schiff base counterion mutants.
Time-resolved spectroscopy of the early photolysis intermediates of rhodopsin Schiff base counterion mutants.
复制标题
视紫红质席夫碱抗衡离子突变体早期光解中间体的时间分辨光谱。
DOI：
10.1021/bi962320u
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Kliger,DS
中科院分区：
文献类型：
--
作者：
Jager,S;Lewis,JW;Zvyaga,TA;Szundi,I;Sakmar,TP;Kliger,DS
用波长为477 nm的7 ns脉冲激光光解COS细胞表达的视紫红质及其突变体（E113 D、E113 A/A117 E和G90 D），在20 ns ~ 510 ms范围内采集其时间分辨吸收差光谱。使用奇异值分解（SVD）后的全局指数拟合程序分析数据。在整个时间范围内，COS细胞和视杆外段视紫红质的动力学和中间体的λ max值的结果之间实现了极好的一致性。Schiff碱互补突变体E113 D显示出与视紫红质至lumi的强烈相似性，遵循既定的方案：batho lubsi → lumi。包括后期延迟时间（超过1 μs），突变体E113 D lumi衰减为变视紫红质II（MII），表明去污剂强烈偏好MII而不是变视紫红质I（MI）。然而，观察到从MII到Lumi的反反应，而对于视紫红质则没有观察到。突变体E113 A/A117 E和G90 D的动力学模式与视紫红质的动力学模式明显不同。在这两种突变体中，batho衰减到与bsi平衡的速度太快而无法分辨（<20 ns）。batho/bsi混合物的衰变过程如下：batho/bsi混合物的衰变过程为类Batho/bsi混合物的衰变过程。然而，在G90 D中未观察到从MI样到Lumi的反反应。在两种突变体中都出现了在460 nm附近吸收的MI样光谱中间体。它们已被证明是转导素活化物质（R*）。结合先前的NMR、FTIR和拉曼光谱数据，这些数据与一幅图相一致，在这幅图中，batho衰变的动力学取决于视网膜发色团C12− C13附近蛋白质诱导的扰动。突变体色素中bsi和lumi中间体的λ max值被解释为Schiff碱相对于其相对分子的运动。
Time-resolved absorption difference spectra of COS-cell expressed rhodopsin and rhodopsin mutants (E113D, E113A/A117E, and G90D), solubilized in detergent, were collected from 20 ns to 510 ms after laser photolysis with 7 ns pulses (λmax= 477 nm). The data were analyzed using a global exponential fitting procedure following singular value decomposition (SVD). Over the entire time range excellent agreement was achieved between results for COS-cell and rod outer segment rhodopsin both in kinetics and in the λmaxvalues of the intermediates. The Schiff base counterion mutant E113D showed strong similarities to rhodopsin up to lumi, following the established scheme: batho ⇌ bsi → lumi. Including late delay times (past 1 μs), the mutant E113D lumi decayed to metarhodopsin II (MII), showing that the detergent strongly favors MII over metarhodopsin I (MI). However, a back-reaction from MII to lumi was observed that was not seen for rhodopsin. The kinetic schemes for the mutants E113A/A117E and G90D were significantly different from that of rhodopsin. In both mutants batho decay into an equilibrium with bsi was too fast to resolve (<20 ns). The batho/bsi mixtures decayed with the following reaction scheme: batho/bsi ⇌ lumi ⇌ MI-like ⇌ MII-like. However, the back-reaction from MI-like to lumi was not seen in G90D. MI-like spectral intermediates absorbing around 460 nm appeared in both mutants. They have been shown to be the transducin-activating species (R*). These data, interpreted in the context of previous NMR, FTIR, and Raman data, are consistent with a picture in which the kinetics of batho decay is dependent on a protein-induced perturbation near C12−C13of the retinal chromophore. The λmaxvalues of the bsi and lumi intermediates in the mutant pigments are interpreted in terms of movement of the Schiff base relative to its counterion.