Tuzi S, Yamaguchi S, Tanio M, Konishi H, Inoue S, Naito A, Needleman R, Lanyi J K, Saitô H
Department of Life Science, Himeji Institute of Technology, Harima Science Garden City, Kamigori, Hyogo, Japan 678-1297, USA.
Biophys J. 1999 Mar;76(3):1523-31. doi: 10.1016/S0006-3495(99)77311-X.
The high-affinity cation-binding sites of bacteriorhodopsin (bR) were examined by solid-state 13C NMR of samples labeled with [3-13C]Ala and [1-13C]Val. We found that the 13C NMR spectra of two kinds of blue membranes, deionized (pH 4) and acid blue at pH 1.2, were very similar and different from that of the native purple membrane. This suggested that when the surface pH is lowered, either by removal of cations or by lowering the bulk pH, substantial change is induced in the secondary structure of the protein. Partial replacement of the bound cations with Na+, Ca2+, or Mn2+ produced additional spectral changes in the 13C NMR spectra. The following conclusions were made. First, there are high-affinity cation-binding sites in both the extracellular and the cytoplasmic regions, presumably near the surface, and one of the preferred cation-binding sites is located at the loop between the helix F and G (F-G loop) near Ala196, consistent with the 3D structure of bR from x-ray diffraction and cryoelectron microscopy. Second, the bound cations undergo rather rapid exchange (with a lifetime shorter than 3 ms) among various types of cation-binding sites. As expected from the location of one of the binding sites, cation binding induced conformational alteration of the F-G interhelical loop.
通过对用[3-13C]丙氨酸和[1-13C]缬氨酸标记的样品进行固态13C核磁共振，研究了细菌视紫红质（bR）的高亲和力阳离子结合位点。我们发现，去离子化（pH 4）的蓝色膜和pH 1.2的酸性蓝色膜这两种蓝色膜的13C核磁共振谱非常相似，且与天然紫色膜的谱不同。这表明，当通过去除阳离子或降低整体pH来降低表面pH时，蛋白质的二级结构会发生显著变化。用Na+、Ca2+或Mn2+部分取代结合的阳离子会在13C核磁共振谱中产生额外的光谱变化。得出了以下结论。首先，在细胞外区域和细胞质区域都存在高亲和力阳离子结合位点，大概靠近表面，其中一个优先的阳离子结合位点位于靠近Ala196的螺旋F和G之间的环（F-G环）处，这与X射线衍射和冷冻电子显微镜得出的bR的三维结构一致。其次，结合的阳离子在各种类型的阳离子结合位点之间进行相当快速的交换（寿命短于3毫秒）。正如从其中一个结合位点的位置所预期的那样，阳离子结合诱导了F-G螺旋间环的构象改变。
