Proteomic characterization and three-dimensional electron microscopy study of PSII-LHCII supercomplexes from higher plants.
In higher plants a variable number of peripheral LHCII trimers can strongly (S), moderately (M) or loosely (L) associate with the dimeric PSII core (C2) complex via monomeric Lhcb proteins to form PSII-LHCII supercomplexes with different structural organizations. By solubilizing isolated stacked pea thylakoid membranes either with the α or β isomeric forms of the detergent n-dodecyl-D-maltoside, followed by sucrose density ultracentrifugation, we previously showed that PSII-LHCII supercomplexes of types C2S2M2 and C2S2, respectively, can be isolated [S. Barera et al., Phil. Trans. R Soc. B 67 (2012) 3389-3399]. Here we analysed their protein composition by applying extensive bottom-up and top-down mass spectrometry on the two forms of the isolated supercomplexes. In this way, we revealed the presence of the antenna proteins Lhcb3 and Lhcb6 and of the extrinsic polypeptides PsbP, PsbQ and PsbR exclusively in the C2S2M2 supercomplex. Other proteins of the PSII core complex, common to the C2S2M2 and C2S2 supercomplexes, including the low molecular mass subunits, were also detected and characterized. To complement the proteomic study with structural information, we performed negative stain transmission electron microscopy and single particle analysis on the PSII-LHCII supercomplexes isolated from pea thylakoid membranes solubilized with n-dodecyl-α-D-maltoside. We observed the C2S2M2 supercomplex in its intact form as the largest PSII complex in our preparations. Its dataset was further analysed in silico, together with that of the second largest identified sub-population, corresponding to its C2S2 subcomplex. In this way, we calculated 3D electron density maps for the C2S2M2 and C2S2 supercomplexes, approaching respectively 30 and 28Å resolution, extended by molecular modelling towards the atomic level. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
Applied Science and Technology Department, BioSolar Lab, Politecnico di Torino, Viale T. Michel 5, 15121 Alessandria, Italy. Electronic address: cristina.pagliano@polito.it.
Cristina
C
Pagliano
School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, UK. Electronic address: j.nield@qmul.ac.uk.
Jon
J
Nield
Department of Science and Technological Innovation, University of Piemonte Orientale, Viale T. Michel 11, 15121 Alessandria, Italy. Electronic address: francesco.marsano@mfn.unipmn.it.
Francesco
F
Marsano
Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address: t.pape@imperial.ac.uk.
Tillmann
T
Pape
Department of Science and Technological Innovation, University of Piemonte Orientale, Viale T. Michel 11, 15121 Alessandria, Italy. Electronic address: simone.barera@mfn.unipmn.it.
Simone
S
Barera
Applied Science and Technology Department, BioSolar Lab, Politecnico di Torino, Viale T. Michel 5, 15121 Alessandria, Italy. Electronic address: guido.saracco@polito.it.
Guido
G
Saracco
Applied Science and Technology Department, BioSolar Lab, Politecnico di Torino, Viale T. Michel 5, 15121 Alessandria, Italy; Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address: j.barber@imperial.ac.uk.
James
J
Barber
D045342:Light-Harvesting Protein Complexes / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D045332:Photosystem II Protein Complex / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020524:Thylakoids / Q000737:chemistry
D045342:Light-Harvesting Protein Complexes; D045332:Photosystem II Protein Complex
PSII–LHCII supercomplex; Proteomics; Single particle analysis; Structure; Thylakoids; Transmission electron microscopy