Isolation, Structural and Biochemical Characterization of Plant PSII-LHCII Supercomplexes

In the first part of this thesis work, they have been investigated the solubilizing properties of -DM and -DM on the isolation of photosynthetic complexes from pea thylakoids membranes maintaining their native architecture of stacked grana and stroma lamellae. Exposure of these stacked thylakoids to a single step treatment with increasing concentrations (5–100 mM) of -DM or -DM resulted in a quick partial or complete solubilization of the membranes. Regardless of the isomeric form used: 1) at the lowest DM concentrations only a partial solubilization of thylakoids was achieved, giving rise to the release of mainly small protein complexes mixed with membrane fragments enriched in PSI from stroma lamellae; 2) at concentrations above 30 mM a complete solubilization occurred with the further release of high molecular weight protein complexes identified as dimeric PSII, PSI-LHCI and PSII–LHCII supercomplexes. It has been identified in 50 mM of both detrgents the minimal concentration that fully solubilized PSII-LHCII supercomplexes. In the second part of this work they have been characterized, in terms of polypeptide composition, purity and functionality by using biochemical techniques, two forms of PSII-LHCII supercomplexes isolated via sucrose gradient centrifugation from solubilized Pisum sativum thylakoid with 50 mM- and -n-dodecil maltoside. In according to the previous results, to isolate the supercomplexes they have been solubilized directly thylakoids instead of PSII membranes (BBYs), in order to minimize the possibility of detergent-induced artefacts and they have been used stacked membranes in order to avoid destabilization of the interaction of LHCII with the PSII core. The isolated supercomplexes haven‟t shown any ATP-ase and PSI contaminations. Both supercomplexes have shown an identical set of LMM subunits. The supercomplex isolated with -DM was larger and more intact, in terms of OEC and Lhcb subunits, than the supercomplex isolated with -DM. The supercomplex isolated with -DM has shown an higher activity than of those isolated with -DM. In the last part of the work they have been structurally characterized both isolated supercomplexes by using EM single particle analysis. The EM data have shown that the supercomplexes isolated with -DM was a C2S2M2 while those isolated with -DM was a C2S2 supercomplex. They have been obtained the 2D projection maps and the 3D reconstructions maps and angular reconstitution at 30 Å (for the C2S2M2) and at 28 Å (for the C2S2).