title: A heterogeneous tag-attachment to the homodimeric type 1 photosynthetic reaction center core protein in the green sulfur bacterium Chlorobaculum tepidum.
abstract: The 6xHis-tag-pscA gene, which was genetically engineered to express N-terminally histidine (His)-tagged PscA, was inserted into a coding region of the recA gene in the green sulfur bacterium Chlorobaculum tepidum (C. tepidum). Although the inactivation of the recA gene strongly suppressed a homologous recombination in C. tepidum genomic DNA, the mutant grew well under normal photosynthetic conditions. The His-tagged reaction center (RC) complex could be obtained simply by Ni(2+)-affinity chromatography after detergent solubilization of chlorosome-containing membranes. The complex consisted of three subunits, PscA, PscB, and PscC, in addition to the Fenna-Matthews-Olson protein, but there was no PscD. Low-temperature EPR spectroscopic studies in combination with transient absorption measurements indicated that the complex contained all intrinsic electron transfer cofactors as detected in the wild-type strain. Furthermore, the LC/MS/MS analysis revealed that the core protein consisted of a mixture of a His-/His-tagged PscA homodimer and a non-/His-tagged PscA heterodimer. The development of the pscA gene duplication method presented here, thus, enables not only a quick and large-scale preparation of the RC complex from C. tepidum but also site-directed mutagenesis experiments on the artificially incorporated 6xHis-tag-pscA gene itself, since the expression of the authentic PscA/PscA homodimeric RC complex could complement any defect in mutated His-tagged PscA. This method would provide an invaluable tool for structural and functional analyses of the homodimeric type 1 RC complex.
authors.affiliation: Department of Biological Sciences, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Azai
authors.forename: Kwang
authors.initials: K
authors.lastname: Kim
authors.forename: Toru
authors.initials: T
authors.lastname: Kondo
authors.forename: Jiro
authors.initials: J
authors.lastname: Harada
authors.forename: Shigeru
authors.initials: S
authors.lastname: Itoh
authors.forename: Hirozo
authors.initials: H
authors.lastname: Oh-oka
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019414:Chlorobi / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005818:Genetic Engineering; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010641:Phenotype; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D020033:Protein Isoforms; D021122:Protein Subunits; C096017:PscA protein, green sulfur bacteria; D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases