title: Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by (13)C-NMR.
abstract: We have examined the (13)C-NMR spectra of [3-(13)C] 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 alpha helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal alpha 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 alpha 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 alpha 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.
authors: Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo, Japan.
authors: S
authors: S
authors: Yamaguchi
authors: K
authors: K
authors: Yonebayashi
authors: H
authors: H
authors: Konishi
authors: S
authors: S
authors: Tuzi
authors: A
authors: A
authors: Naito
authors: J K
authors: JK
authors: Lanyi
authors: R
authors: R
authors: Needleman
authors: H
authors: H
authors: Saitô
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D009682:Magnetic Resonance Spectroscopy; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D012492:Salts; D013696:Temperature
chemical_list: D007477:Ions; D012492:Salts; D001436:Bacteriorhodopsins