title: Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides.
abstract: Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1-PufX complex (RC-LH1-PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQA --> D+QA -) in the RC as a function of light intensity. The RC-LH1-PufX complex had a quantum efficiency of 0.95 +/- 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 +/- 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 +/- 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 A. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 A, b = 137.2 A, c = 137.5 A; alpha = 60.0 degrees , beta = 89.95 degrees , gamma =90.02 degrees .
authors.affiliation: Department of Physics, University California San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA.
authors.forename: E C
authors.initials: EC
authors.lastname: Abresch
authors.forename: H L A
authors.initials: HL
authors.lastname: Axelrod
authors.forename: J T
authors.initials: JT
authors.lastname: Beatty
authors.forename: J A
authors.initials: JA
authors.lastname: Johnson
authors.forename: R
authors.initials: R
authors.lastname: Nechushtai
authors.forename: M L
authors.initials: ML
authors.lastname: Paddock
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003460:Crystallization; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012242:Rhodobacter sphaeroides / Q000737:chemistry*; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; C088749:PufX protein, Rhodobacter