Use of Protein Crosslinking and Tandem Mass Spectrometry to Study the PsbO, PsbP and PsbQ Extrinsic Proteins of Higher Plant Photosystem II

• Main Author: Mummadisetti, Manjula Priyadarshini
• Other Authors: Larkin, John C.
• Format: Others
• Language: en
• Published: LSU 2016
• Subjects: Biochemistry (Biological Sciences)
• Online Access: http://etd.lsu.edu/docs/available/etd-07112016-131235/

Photosystem II (PSII) is a light-driven, water plastoquinone oxidoreductase present in all oxygenic photosynthetic organisms. The oxygen evolution process is catalyzed by the Mn4CaO5 cluster and an ensemble of intrinsic and extrinsic proteins which are associated with the photosystem. This metal cluster is stabilized and protected from exogenous reductants by the extrinsic proteins, PsbO, PsbP and PsbQ in higher plants, which are present on the lumenal face of PSII. No crystal structure for the higher plant PSII is currently available; consequently, the binding locations of these extrinsic proteins in PSII remain elusive. We have used chemical-crosslinkers Bis (sulfosuccinimidyl) suberate (BS3) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to crosslink the extrinsic proteins in their bound state to PSII followed by identification of the crosslinked products by tandem mass spectrometry. BS3 crosslinking identified the interacting domain of PsbP with PsbQ involving the PsbP residues 93Y, 96K and 97T (located in the 17-residue loop 3A, 89G-105S) which are in close proximity (<11.4Å) to the N-terminal 1E residue of PsbQ. We also found that this PsbP assumes a compact structure from the nine independent crosslinked residues between the N- and C-terminus of PsbP. This suggests that the N-terminus of PsbP, 1A-11K (which is not resolved in the current crystal structures), is closely associated with the C-terminal domain 170K-186A. Additionally, interacting domains of two PsbQ copies from different PSII monomers were identified. The residue pairs 98K-133Y and 101K-133Y of PsbQ were crosslinked. These residues are >30 Å apart when mapped onto the PsbQ crystal structure. Since BS3 can only crosslink residues which are within 11.4 Å, these residues are hypothesized as inter-molecular crosslinks of PsbQ. Furthermore, EDC crosslinking provided structural information pertaining to the organization of the N-terminus, absent in the cyanobacterial-PsbO. In this study, twenty-four crosslinked residues located in the N-terminal, loop and the β-barrel region of PsbO were identified. The models incorporating crosslinking data suggests several differences in cyanobacterial- and higher plant-PsbO. The results on extrinsic proteins provide significant new information concerning the association of the extrinsic proteins with PSII and are valuable while proposing overall models of higher plant PSII.