Location of a cation-binding site in the loop between helices F and G of bacteriorhodopsin as studied by C-13 NMR

The high-affinity cation-binding sites of bacteriorhodopsin (bR) were exami ned by solid-state C-13 NMR of samples labeled with [3-C-13]Ala and [1-C-13 ]Val. We found that the C-13 NMR spectra of two kinds of blue membranes, de ionized (pH 4) and acid blue at pH 1.2, were very similar and different fro m 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, sub stantial change is induced in the secondary structure of the protein. Parti al replacement of the bound cations with Na+, Ca2+, or Mn2+ produced additi onal spectral changes in the C-13 NMR spectra. The following conclusions we re made. First, there are high-affinity cation-binding sites in both the ex tracellular and the cytoplasmic regions, presumably near the surface, and o ne of the preferred cation-binding sites is located at the loop between the helix F and G (F-G loop) near Ala(196), consistent with the 3D structure o f bR from x-ray diffraction and cryoelectron microscopy. Second, the bound cations undergo rather rapid exchange (with a lifetime shorter than 3 ms) a mong various types of cation-binding sites. As expected from the location o f one of the binding sites, cation binding induced conformational alteratio n of the F-G interhelical loop.