Ana A. Arteni

Structural organisation of phycobilisomes from Synechocystis sp strain PCC6803 and their interaction with the membrane

In cyanobacteria, the harvesting of light energy for photosynthesis is mainly carried out by the phycobilisome - a giant, multi-subunit pigment-protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides such that light absorption and energy transfer to photosystem II are optimised. In this work we have studied, using single particle electron microscopy, the phycobilisome structure in mutants lacking either two or all three of the phycocyanin hexamers. The images presented give much greater detail than those previously published, and in the best two-dimensional projection maps a resolution of 13 A was achieved. As well as giving a better overall picture of the assembly of phycobilisomes, these results reveal new details of the association of allophycocyanin trimers within the core. Insights are gained into the attachment of this core to the membrane surface, essential for efficient energy transfer to photosystem II. Comparison of projection maps of phycobilisomes with and without reconstituted ferredoxin:NADP oxidoreductase suggests a location for this enzyme within the complex at the rod-core interface.

Structure and functional role of supercomplexes of IsiA and Photosystem I in cyanobacterial photosynthesis

Cyanobacteria express large quantities of the iron stress‐inducible protein IsiA under iron deficiency. IsiA can assemble into numerous types of single or double rings surrounding Photosystem I. These supercomplexes are functional in light‐harvesting, empty IsiA rings are effective energy dissipaters. Electron microscopy studies of these supercomplexes show that Photosystem I trimers bind 18 IsiA copies in a single ring, whereas monomers may bind up to 35 copies in two rings. Work on mutants indicates that the PsaF/J and PsaL subunits facilitate the formation of closed rings around Photosystem I monomers but are not obligatory components in the formation of Photosystem I–IsiA supercomplexes.

The peripheral light-harvesting complexes from purple sulfur bacteria have different 'ring' sizes

The integral membrane light‐harvesting (LH) proteins from purple photosynthetic bacteria form circular oligomers of an elementary unit that is composed of two very hydrophobic polypeptides, termed α and β. These apoprotein dimers are known to associate into closed circular arrays of 8, 9 and 16 α/β‐mers. We report the existence of peripheral LH proteins purified from Allochromatium vinosum with two intermediate ring sizes and postulate that one is a 13 α/β‐mer. This shows that LH proteins are able to form membrane rings of continuously increasing diameter from 68 to 115 Å. The presence of these new ring sizes warrants further study, as it will help to further validate the structure–function models of LH proteins currently found in the literature.

Structural studies show energy transfer within stabilized phycobilisomes independent of the mode of rod-core assembly

The major light harvesting complex in cyanobacteria and red algae is the phycobilisome (PBS), comprised of hundreds of seemingly similar chromophores, which are protein bound and assembled in a fashion that enables highly efficient uni-directional energy transfer to reaction centers. The PBS is comprised of a core containing 2-5 cylinders surrounded by 6-8 rods, and a number of models have been proposed describing the PBS structure. One of the most critical steps in the functionality of the PBS is energy transfer from the rod substructures to the core substructure. In this study we compare the structural and functional characteristics of high-phosphate stabilized PBS (the standard fashion of stabilization of isolated complexes) with cross-linked PBS in low ionic strength buffer from two cyanobacterial species, Thermosynechococcus vulcanus and Acaryochloris marina. We show that chemical cross-linking preserves efficient energy transfer from the phycocyanin containing rods to the allophycocyanin containing cores with fluorescent emission from the terminal emitters. However, this energy transfer is shown to exist in PBS complexes of different structures as characterized by determination of a 2.4Å structure by X-ray crystallography, single crystal confocal microscopy, mass spectrometry and transmission electron microscopy of negatively stained and cryogenically preserved complexes. We conclude that the PBS has intrinsic structural properties that enable efficient energy transfer from rod substructures to the core substructures without requiring a single unique structure. We discuss the significance of our observations on the functionality of the PBS in vivo.

Mapping of the epitopes of poliovirus type 2 in complex with antibodies.

The inactivated polio vaccine (IPV) contains poliovirus (PV) samples that belong to serotypes 1, 2 and 3. All three serotypes contain the D-antigen, which induces protective antibodies. The antigenic structure of PVs consists of at least four different antigenic sites and the D-antigen content represents the combined activity of multiple epitopes (Ferguson et al., 1993; Minor, 1990; Minor et al., 1986). The potency of IPV vaccines is determined by measuring the D-antigen content. Several ELISA methods have been developed using polyclonal or monoclonal antibodies (Mabs) in order to quantify the D-antigen content. Characterization of the epitopes recognized by the different Mabs is crucial to map the entire virus surface and ensure the presence of epitopes able to induce neutralizing antibodies. Using a new approach that we developed to study the interaction between monoclonal antibodies and poliovirus type 2, which combines cryo-electron microscopy, image analysis and X-ray crystallography along with identification of exposed amino acids, we have mapped in 3D the epitope sites recognized by three specific Fabs at the surface of poliovirus type 2 (PV2) and characterized precisely the antigenic sites for these Fabs.