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.

We have examined the 13 C-NMR spectra of [3-13 C] Alalabeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal a helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal a helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mm NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) 13 C signals from the C-terminal a helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal a helix is deleted or sitedirected mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.

We examined how the local conformation and dynamics of [3-13C]Ala-labeled bacteriorhodopsin (bR) are altered as viewed from 13C NMR spectra when the natural membrane lipids are partly or completely replaced with detergents. It turned out that the major conformational features of bR, the alphaII-helices, are generally unchanged in the delipidated or solubilized preparations. Upon partial delipidation or detergent solubilization, however, a significant conformational change occurs, ascribed to local conversion of alphaII-->alphaI-helix (one Ala residue involved), evident from the upfield displacement of the transmembrane helical peak from 16.4 ppm to 14.5 ppm, conformational change (one or two Ala residues) within alphaII-helices from 16.4 to 16.0 ppm, and acquired flexibility in the loop region (especially at the F-G loop) as manifested from suppressed peak-intensities in cross-polarization magic angle spinning (CP-MAS) NMR spectra. On the other hand, formation of monomers as solubilized by Triton X-100, Triton N-101 and n-dodecylmaltoside is characterized by the presence of a peak at 15.5 ppm and a shifted absorption maximum (550 nm). The size of micelles under the first two conditions was small enough to yield 13C NMR signals observable by a solution NMR spectrometer, although 13C CP-MAS NMR signals were also visible from a fraction of large-sized micelles. We found that the 16.9 ppm peak (three Ala residues involved), visible by CP-MAS NMR, was displaced upfield when Schiff base was removed by solubilization with sodium dodecyl sulfate, consistent with our previous finding of bleaching to yield bacterioopsin.