Abundance of protein components in photosystem II protein complex purified from mutant cyanobacterial cells lacking individual subunit protein
EMSL Project ID
3682
Abstract
Photosystem II (PSII) is the pigment-protein complex responsible for the process of converting water to molecular oxygen and subsequent carbon fixation during oxygenic photosynthesis. PSII consists of more than 30 protein components that all work in concert to drive the light-induced extraction of electrons from water before depositing them in the plastoquinone pool within the photosynthetic membrane. In both cyanobacteria and chloroplasts, there are soluble, membrane-extrinsic PSII subunits required for optimal activity of the oxygen evolution complex. Individual knockout mutations reveal that each protein is important to optimal PSII activity, but the interdependence of these proteins is not clear. Based on SDS PAGE analysis, it appears that the relative abundance of some PSII components is altered when individual subunits are removed. We propose to examine the role each extrinsic subunit plays in the assembly of the other components in cyanobacterial PSII by quantifying the peptide constituents of PSII in mutants lacking individual extrinsic proteins. The cyanobacterium, Synechocystis 6803, was chosen because it is easy to make genetic deletions of individual PSII components without relying on biochemical methods to remove subunits. Deletions in the extrinsic proteins, PsbP, PsbQ, and PsbV were introduced into a background where CP47, one of the major membranous components of PSII, was labeled with a six-histidine tag at the carboxyl terminus. PSII complex from each mutant has been purified using a one-step Ni-affinity chromatography procedure. Dr. Richard Smith, Dr. David Camp and colleagues at EMSL will analyze these purified complexes using high-resolution capillary liquid chromatography and Fourier transform-ion cyclotron resonance mass spectrometry to determine the abundance of each subunit. Ultimately, the data will be correlated with the activity data from the mutants to determine the relative contribution each extrinsic subunit makes to PSII oxygen evolution activity.
Project Details
Project type
Exploratory Research
Start Date
2003-08-26
End Date
2005-12-15
Status
Closed
Released Data Link
Team
Principal Investigator