Time-resolved microspectroscopy on a single crystal of bacteriorhodopsin reveals lattice-induced differences in the photocycle kinetics
Time-resolved microspectroscopy on a single crystal of bacteriorhodopsin reveals lattice-induced differences in the photocycle kinetics
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DOI：
10.1529/biophysj.106.083345
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发表时间：
2006-08-15
影响因子：
3.4
通讯作者：
Bueldt, G.
中科院分区：
文献类型：
--
作者：
Efremov, R.;Gordeliy, V. I.;Bueldt, G.
细菌视紫红质光循环的中间态结构的确定使人们对膜蛋白所发挥的催化过程有了前所未有的了解。然而，只有当工作循环不受晶格损害时，中间态的晶体结构才有意义。因此，我们应用具有微秒时间分辨率的可见光和傅里叶变换红外光谱（FTIR）显微光谱来比较单个细菌视紫红质晶体的光反应与驻留在天然紫色膜中的细菌视紫红质的光反应。对解析的中间态的 FTIR 差异光谱的分析表明，晶体和 PM 中发生的结构变化非常相似。然而，与 PM 相比，三维晶体中的光循环动力学发生了显着改变。引人注目的是，晶体中 L 态衰变加速，而 M 态衰变延迟。讨论了这种偏差的物理起源以及中间态捕获的影响。作为方法论的进步，首次证明了单个蛋白质晶体上的时间分辨步进扫描 FTIR 光谱，将来可用于通过振动光谱的分子分辨率来测量其他结晶蛋白质的功能。
The determination of the intermediate state structures of the bacteriorhodopsin photocycle has lead to an unprecedented level of understanding of the catalytic process exerted by a membrane protein. However, the crystallographic structures of the intermediate states are only relevant if the working cycle is not impaired by the crystal lattice. Therefore, we applied visible and Fourier transform infrared spectroscopy (FTIR) microspectroscopy with microsecond time resolution to compare the photoreaction of a single bacteriorhodopsin crystal to that of bacteriorhodopsin residing in the native purple membrane. The analysis of the FTIR difference spectra of the resolved intermediate states reveals great similarity in structural changes taking place in the crystal and in PM. However, the kinetics of the photocycle are significantly altered in the three-dimensional crystal as compared to PM. Strikingly, the L state decay is accelerated in the crystal, whereas the M decay is delayed. The physical origin of this deviation and the implications for trapping of intermediate states are discussed. As a methodological advance, time-resolved step-scan FTIR spectroscopy on a single protein crystal is demonstrated for the first time which may be used in the future to gauge the functionality of other crystallized proteins with the molecular resolution of vibrational spectroscopy.