Hatanaka M, Kandori H, Maeda A
Department of Biophysics, Graduate School of Science, Kyoto University, Japan.
Biophys J. 1997 Aug;73(2):1001-6. doi: 10.1016/S0006-3495(97)78133-5.
Linear dichroic difference Fourier transform infrared spectra upon formation of the M photointermediate were recorded with oriented purple membranes. The purpose was to determine the angle of the directions of the dipole moments of 1) the water molecule whose O-H stretching vibration appears at 3643 cm-1 for the unphotolyzed state and 3671 cm-1 for the M intermediate, and 2) the C=O bond of protonated Asp85 in the M intermediate. The angle of 36 degrees we find for the C=O of the protonated Asp85 in the M intermediate is not markedly different from 26 degrees for unprotonated Asp85 in the model based on cryoelectron diffraction, indicating the absence of gross orientation changes in Asp85 upon its protonation. The O-H band at 3671 cm-1 of a water molecule in the M intermediate, although its position has not determined, is fixed almost parallel to the membrane plane. For the unphotolyzed state the angle of the water O-H to the membrane normal was determined to be 60 degrees. On the basis of these data and the structural model, we place the water molecule in the unphotolyzed state at a position where it forms hydrogen bonds with the Schiff base, Asp85, Asp212, and Trp86.
用取向紫膜记录了形成M光中间体时的线性二色性差示傅里叶变换红外光谱。目的是确定以下两种情况偶极矩方向的夹角：1）未光解状态下O-H伸缩振动出现在3643cm-1处、M中间体中出现在3671cm-1处的水分子；2）M中间体中质子化的Asp85的C=O键。我们发现M中间体中质子化的Asp85的C=O键夹角为36度，这与基于低温电子衍射的模型中未质子化的Asp85的26度夹角没有明显差异，表明Asp85质子化时没有明显的取向变化。M中间体中一个水分子在3671cm-1处的O-H带，尽管其位置尚未确定，但几乎与膜平面平行。对于未光解状态，水分子O-H与膜法线的夹角确定为60度。基于这些数据和结构模型，我们将未光解状态下的水分子置于与席夫碱、Asp85、Asp212和Trp86形成氢键的位置。
