Efremov R, Gordeliy V I, Heberle J, Büldt G
Forschungszentrum Jülich, IBI-2: Structural Biology, 52425 Jülich, Germany.
Biophys J. 2006 Aug 15;91(4):1441-51. doi: 10.1529/biophysj.106.083345. Epub 2006 May 26.
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.
细菌视紫红质光循环中间态结构的确定使人们对膜蛋白催化过程的理解达到了前所未有的水平。然而，只有当工作循环不受晶格影响时，中间态的晶体结构才具有相关性。因此，我们应用了具有微秒级时间分辨率的可见光谱和傅里叶变换红外光谱（FTIR）显微光谱，以比较单个细菌视紫红质晶体与天然紫膜中细菌视紫红质的光反应。对解析出的中间态的FTIR差光谱分析表明，晶体和紫膜中发生的结构变化具有很大的相似性。然而，与紫膜相比，三维晶体中的光循环动力学发生了显著改变。令人惊讶的是，晶体中L态的衰减加速，而M态的衰减延迟。讨论了这种偏差的物理起源以及对中间态捕获的影响。作为一项方法学进展，首次展示了对单个蛋白质晶体进行时间分辨步进扫描FTIR光谱，未来可用于以振动光谱的分子分辨率评估其他结晶蛋白的功能。
