title: Role of trimer-trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy.
abstract: Bacteriorhodopsin (bR) trimers form a two-dimensional hexagonal lattice in the purple membrane of Halobacterium salinarum. However, the physiological significance of forming the lattice has long been elusive. Here, we study this issue by comparing properties of assembled and non-assembled bR trimers using directed mutagenesis, high-speed atomic force microscopy (HS-AFM), optical spectroscopy, and a proton pumping assay. First, we show that the bonds formed between W12 and F135 amino acid residues are responsible for trimer-trimer association that leads to lattice assembly; the lattice is completely disrupted in both W12I and F135I mutants. HS-AFM imaging reveals that both crystallized D96N and non-crystallized D96N/W12I mutants undergo a large conformational change (i.e., outward E-F loop displacement) upon light-activation. However, lattice disruption significantly reduces the rate of conformational change under continuous light illumination. Nevertheless, the quantum yield of M-state formation, measured by low-temperature UV-visible spectroscopy, and proton pumping efficiency are unaffected by lattice disruption. From these results, we conclude that trimer-trimer association plays essential roles in providing bound retinal with an appropriate environment to maintain its full photo-reactivity and in maintaining the natural photo-reaction pathway.
authors: Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
authors: Hayato
authors: H
authors: Yamashita
authors: Keiichi
authors: K
authors: Inoue
authors: Mikihiro
authors: M
authors: Shibata
authors: Takayuki
authors: T
authors: Uchihashi
authors: Jun
authors: J
authors: Sasaki
authors: Hideki
authors: H
authors: Kandori
authors: Toshio
authors: T
authors: Ando
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D003460:Crystallization / Q000379:methods; D019617:Halobacterium salinarum / Q000737:chemistry / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods; D009154:Mutation; D061848:Optical Imaging / Q000379:methods; D017494:Proton Pumps / Q000737:chemistry; D018717:Purple Membrane / Q000737:chemistry / Q000378:metabolism
chemical_list: D017494:Proton Pumps; D001436:Bacteriorhodopsins
keywords: AFM; Bacteriorhodopsin; HPLC; HS-AFM; High-speed atomic force microscopy; Low-temperature UV–visible spectroscopy; PM; Point mutation; Proton pumping efficiency; Trimer; WT; atomic force microscopy; bR; bacteriorhodopsin; high-performance liquid chromatography; high-speed atomic force microscopy; purple membrane; wild-type