title: Overexpression of Rhodobacter sphaeroides PufX-bearing maltose-binding protein and its effect on the stability of reconstituted light-harvesting core antenna complex.
abstract: The PufX protein, encoded by the pufX gene of Rhodobacter sphaeroides, plays a key role in the organization and function of the core antenna (LH1)-reaction centre (RC) complex, which collects photons and triggers primary photochemical reactions. We synthesized a PufX/maltose-binding protein (MBP) fusion protein to study the effect of the PufX protein on the reconstitution of B820 subunit-type and LH1-type complexes. The fusion protein was synthesized using an Escherichia coli expression system and purified by affinity chromatography. Reconstitution experiments demonstrated that the MBP-PufX protein destabilizes the subunit-type complex (20°C), consistent with previous reports. Interestingly, however, the preformed LH1-type complex was stable in the presence of MBP-PufX. The MBP-PufX protein did not influence the preformed LH1-type complexes (4°C). The LH1-type complex containing MBP-PufX showed a unique temperature-dependent structural transformation that was irreversible. The predominant form of the complex at 4°C was the LH1-type. When shifted to 20°C, subunit-type complexes became predominant. Upon subsequent cooling back to 4°C, instead of re-forming the LH1-type complexes, the predominant form remained the subunit-type complexes. In contrast, reversible transformation of LH1 (4°C) and subunit-type complexes (20°C) occurs in the absence of PufX. These results are consistent with the suggestion that MBP-PufX interacts with the LH1α- polypeptide in the subunit (α/β)-type complex (at 20°C), preventing oligomerization of the subunit to form LH1-type complexes.
authors.affiliation: Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, 466-8555, Japan.
authors.forename: Shunnsuke
authors.initials: S
authors.lastname: Sakai
authors.forename: Akito
authors.initials: A
authors.lastname: Hiro
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kondo
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Mizuno
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tanaka
authors.forename: Takehisa
authors.initials: T
authors.lastname: Dewa
authors.forename: Mamoru
authors.initials: M
authors.lastname: Nango
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D045342:Light-Harvesting Protein Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D055550:Protein Stability; D011993:Recombinant Fusion Proteins; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism*; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D058619:Maltose-Binding Proteins; D010455:Peptides; C088749:PufX protein, Rhodobacter; D011993:Recombinant Fusion Proteins; D008320:Maltose