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image/svg+xml Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao Closed Access logo, derived from PLoS Open Access logo. This version with transparent background. http://commons.wikimedia.org/wiki/File:Closed_Access_logo_transparent.svg Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao Photosynthesis Resea...arrow_drop_down
image/svg+xml Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao Closed Access logo, derived from PLoS Open Access logo. This version with transparent background. http://commons.wikimedia.org/wiki/File:Closed_Access_logo_transparent.svg Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao
Photosynthesis Research
Article . 2005 . Peer-reviewed
License: Springer TDM
Data sources: Crossref
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Characterization of a Highly Purified, Fully Active, Crystallizable RC–LH1–PufX Core Complex from Rhodobacter sphaeroides

Authors: E. C. Abresch; Herbert L. Axelrod; Mark L. Paddock; J. T. Beatty; Rachel Nechushtai; J.A. Johnson;

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 .

Keywords

Bacterial Proteins, Spectrum Analysis, Light-Harvesting Protein Complexes, Rhodobacter sphaeroides, Crystallization, Protein Binding

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