Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal α helix, modified by a variety of environmental factors as studied by 13C-NMR

Abstract

We have examined the 13C-NMR spectra of [3-13C] Ala-labeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal α helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal α 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) 13C signals from the C-terminal α 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 α helix is deleted or site-directed 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.