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 (C ₂) 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 C₂S ₂M₂ 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 C₂S ₂M₂ 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 C₂S₂M ₂ and C₂S₂ supercomplexes, approaching respectively 30 and 28 Å 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.