Architectural study of photosynthetic complexes

Photosynthesis is a process by which the absorbed fight goves the sufficient energy to a reacton centre to split a water molecule, release oxygen, assimilate carbon dioxide and produce chemically fixed energy The Earth fundamental reaction that we know is mainly shaped due this fundamental reaction performed by cyanobacteria. On the larger scale, there are photosystems where the change separation takes place. On the small scale, there are large numbers of pigment molecules bound to these photosystems and their antenna complexes, which are involved in light absorption. There are two types of photosystems, Photosystem I (PSI) and Photosystem II (PSII) which are embedded in the thrylakpid membrane and are working in series. The goal of this thesis is to provide a structural and PSII complexes and their associations with light harvesting complexes, Lhca and LHC6 proteins respectively. The study of the architecture of supercomplexes is helpful for a better understanding of protein-protein interactions within the complex

Functional architecture of higher plant photosystem II supercomplexes

Photosystem II (PSII) is a large multiprotein complex, which catalyses water splitting and plastoquinone reduction necessary to transform sunlight into chemical energy. Detailed functional and structural studies of the complex from higher plants have been hampered by the impossibility to purify it to homogeneity. In this work, homogeneous preparations ranging from a newly identified particle composed by a monomeric core and antenna proteins to the largest C2S2M2 supercomplex were isolated. Characterization by biochemical methods and single particle electron microscopy allowed to relate for the first time the supramolecular organization to the protein content. A projection map of C2S2M2 at 12 Å resolution was obtained, which allowed determining the location and the orientation of the antenna proteins. Comparison of the supercomplexes obtained from WT and Lhcb-deficient plants reveals the importance of the individual subunits for the supramolecular organization. The functional implications of these findings are discussed and allow redefining previous suggestions on PSII energy transfer, assembly, photoinhibition, state transition and non-photochemical quenching

The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts

The PsbS protein is a critical component in the regulation of non-photochemical quenching (NPQ) in higher plant photosynthesis. Electron microscopy and image analysis of grana membrane fragments from wild type and mutant Arabidopsis plants showed that the semi-crystalline domains of photosystem II supercomplexes were identical in the presence and absence of PsbS. However, the frequency of the domains containing crystalline arrays was increased in the absence of PsbS. Conversely, there was a complete absence of such arrays in the membranes of plants containing elevated amounts of this protein. It is proposed that PsbS controls the macro-organisation of the grana membrane, providing an explanation of its role in NPQ.

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

AbstractThe 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 115Å. 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

The cryo-EM structure of the S-layer deinoxanthin-binding complex of Deinococcus radiodurans informs properties of its environmental interactions

The radiation-resistant bacterium Deinococcus radiodurans is known as the world's toughest bacterium. The S-layer of D. radiodurans, consisting of several proteins on the surface of the cellular envelope and intimately associated with the outer membrane, has therefore been useful as a model for structural and functional studies. Its main proteinaceous unit, the S-layer deinoxanthin-binding complex (SDBC), is a hetero-oligomeric assembly known to contribute to the resistance against environmental stress and have porin functional features; however, its precise structure is unknown. Here, we resolved the structure of the SDBC at ∼2.5 Å resolution by cryo-EM and assigned the sequence of its main subunit, the protein DR_2577. This structure is characterized by a pore region, a massive β-barrel organization, a stalk region consisting of a trimeric coiled coil, and a collar region at the base of the stalk. We show that each monomer binds three Cu ions and one Fe ion and retains one deinoxanthin molecule and two phosphoglycolipids, all exclusive to D. radiodurans. Finally, electrophysiological characterization of the SDBC shows that it exhibits transport properties with several amino acids. Taken together, these results highlight the SDBC as a robust structure displaying both protection and sieving functions that facilitates exchanges with the environment

Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis.

Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages

Stimuli-Responsive Triblock Terpolymer Conversion into Multi-Stimuli-Responsive Micelles with Dynamic Covalent Bonds for Drug Delivery through a Quick and Controllable Post-Polymerization Reaction

Stimuli-responsive copolymers are of great interest for targeted drug delivery. This study reports on a controllable post-polymerization quaternization with 2-bromomethyl-4-fluorophenylboronic acid of the poly(4-vinyl pyridine) (P4VP) block of a common poly(styrene)-b-poly(4-vinyl pyridine)-b-poly(ethylene oxide) (SVE) triblock terpolymer in order to achieve a selective responsivity to various diols. For this purpose, a reproducible method was established for P4VP block quaternization at a defined ratio, confirming the reaction yield by 11B, 1H NMR. Then, a reproducible self-assembly protocol is designed for preparing stable micelles from functionalized stimuli-responsive triblock terpolymers, which are characterized by light scattering and by cryogenic transmission electron microscopy. In addition, UV-Vis spectroscopy is used to monitor the boron-ester bonding and hydrolysis with alizarin as a model drug and to study encapsulation and release of this drug, induced by sensing with three geminal diols: fructose, galactose and ascorbic acid. The obtained results show that only the latter, with the vicinal diol group on sp2-hybridized carbons, was efficient for alizarin release. Therefore, the post-polymerization method for triblock terpolymer functionalization presented in this study allows for preparation of specific stimuli-responsive systems with a high potential for targeted drug delivery, especially for cancer treatment

Discontinuous transcription of ribosomal DNA in human cells.

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events