Rothschild K J, Marrero H
Proc Natl Acad Sci U S A. 1982 Jul;79(13):4045-9. doi: 10.1073/pnas.79.13.4045.
It is possible, by using Fourier-transform infrared (FTIR) difference spectroscopy, to detect the conformational changes occurring in both the protein and the chromophore of bacteriorhodopsin during the photocycle. In contrast to Raman spectroscopy, a laser is unnecessary and hence the problem of a perturbing probe beam is eliminated. Furthermore, the relatively high signal-to-noise ratio obtainable with FTIR enables measurements to be made in minutes over a large spectral range. In the study reported in this paper, we used this method to examine the state of protonation of the retinylidene Schiff base in light-adapted bR570 and in K, the first intermediate in the photocycle. Resonance Raman spectroscopy provides evidence that bR570 is protonated, but these results have been questioned on the basis of theoretical and experimental grounds. FTIR difference spectral changes in the bR570-to-K transition clearly indicate that bR570 contains a protonated Schiff base. In contrast, the K intermediate displays a Schiff base that is altered but still is associated to some degree with a proton. Because the low-temperature FTIR difference spectrum of bR570 and K is similar to the recently reported low-temperature resonance Raman spectra of bR570 and K [Braiman, M. & Mathies, R. (1982) Proc. Natl. Acad. Sci. USA 79, 403-407], we can assign most, but not all, vibrational changes in the bR570-to-K transition to the chromophore. These results are consistent with a simple model of the first step in the photocycle which involves a movement of the Schiff base proton away from a counterion.
通过使用傅里叶变换红外（FTIR）差示光谱法，可以检测在光循环过程中细菌视紫红质的蛋白质和发色团中发生的构象变化。与拉曼光谱不同，不需要激光，因此消除了干扰探测光束的问题。此外，FTIR可获得相对较高的信噪比，从而能够在几分钟内在较大的光谱范围内进行测量。在本文报道的研究中，我们使用这种方法来研究光适应的bR570以及光循环中的第一个中间体K中视黄叉席夫碱的质子化状态。共振拉曼光谱提供了bR570被质子化的证据，但这些结果在理论和实验基础上受到了质疑。bR570到K转变过程中的FTIR差示光谱变化清楚地表明bR570含有质子化的席夫碱。相比之下，K中间体显示出一个改变了的席夫碱，但仍然在一定程度上与一个质子相关。因为bR570和K的低温FTIR差示光谱与最近报道的bR570和K的低温共振拉曼光谱相似[Braiman, M. & Mathies, R. (1982) Proc. Natl. Acad. Sci. USA 79, 403 - 407]，所以我们可以将bR570到K转变过程中大部分（但不是全部）的振动变化归因于发色团。这些结果与光循环第一步的简单模型一致，该模型涉及席夫碱质子从抗衡离子移开。
