Marrero H, Rothschild K J
Department of Physics, Boston University, Massachusetts 02215.
Biophys J. 1987 Oct;52(4):629-35. doi: 10.1016/S0006-3495(87)83254-X.
We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments. An increase in the intensity of peaks in the amide I and II regions agrees with recent time-resolved kinetic FTIR-difference measurements and indicates that a localized protein conformational change involving the peptide backbone of bR occurs which is not evident at the lower temperature.
我们报道了一种基于傅里叶变换红外（FTIR）差示光谱的新方法，用于研究细菌视紫红质光循环过程中发生的构象变化。以往的研究是通过测量透过薄水合紫膜的红外（IR）光束的吸光度来进行的。相比之下，本研究采用了衰减全反射（ATR）技术。紫膜固定在锗内反射晶体表面，并浸入pH值和离子组成可变化的缓冲液中。用与晶体表面平行和成45度角偏振的光测量酰胺I和II的吸光度，结果表明膜具有高度的取向性。光到暗（bR570到bR548）转变的ATR-FTIR差示光谱与透射FTIR差示光谱相似但不完全相同。两种方法之间的这种差异在ATR情况下被证明是由于主要垂直于膜平面取向的跃迁矩的吸收。将La3+取代的紫膜的pH值从6.8提高到8.0，足以减缓M衰变率，从而在室温下通过稳态光照获得bR570到M412的差示光谱。将该差示光谱与使用透射法在-23℃下获得的差示光谱进行比较，发现了一些不能归因于平面外跃迁矩的变化。酰胺I和II区域峰强度的增加与最近的时间分辨动力学FTIR差示测量结果一致，表明发生了涉及bR肽主链的局部蛋白质构象变化，而在较低温度下这种变化并不明显。
