Solar-driven hydrogen production from the abundant and cheap electron source water is a promising way to produce renewable energy. Plants and cyanobacteria have developed a water splitting enzyme which is able to oxidize water into molecular oxygen, protons and electrons using visible light energy within the membrane protein photosystem II. The heart of the enzyme is a Mn4CaO5 cluster at which water oxidation takes place following four sequential light-induced steps. Reactions at the Mn4CaO5 cluster consist of concerted electron and proton transfer, and form intermediate states that minimize the activation energy necessary for the water oxidation process. Photosystem II is thus a paradigm for engineering bio-inspired solar energy converting applications. A recent high-resolution three-dimensional structure of photosystem II gave a precise arrangement of the Mn-Ca cluster necessary for water oxidation. In addition, the combination of theoretical catalytic models with experimental data from numerous state-of-the-art spectroscopic techniques have given a possible view of Mn oxidation states during water oxidation, of water fixation steps, have revealed the importance of a set of amino acids in the catalytic mechanism, and given hints on proton transfer reactions involving extended hydrogen bonding networks. Despite these remarkable progresses in recent years, key questions remain opened. They concern the position of reactive molecules, the formation mechanisms of the oxygen molecule itself, and relaxation processes at the Mn4CaO5 cluster involving spin-state transitions and concerted electron and proton transfer. The PS2FIR project will contribute to answer these questions. We will gather the complementary expertise of three research teams: Team 1, R. Hienerwadel & C. Berthomieu, UMR 7265; Team 2, A. Boussac UMR 9198; and Team 3, J.B. Brubach & P. Roy, Synchrotron SOLEIL, to probe light- and near infared (NIR)- induced transitions and spin conversions at the Mn4CaO5 cluster, using state-of-the-art far-infrared FTIR difference spectroscopy. Vibrational modes in the far-infrared down to 10 cm-1 will allow probing the valence state of the Mn ions, cluster conformation, and Mn-O/Ca-O interactions. Of particular interest will be the identification of libration and connectivity modes of water molecules associated to the cluster below 300 cm-1 during the reaction cycle. To overcome the challenge of exploiting small-bands in the Far-infrared domain, setups will be optimized to probe different samples in parallel and to optimize NIR-induced spin-state transitions by controlled temperature jumps. We will also benefit from the brilliance of the synchrotron AILES beamline at SOLEIL. Highly resistant photosystem II from Thermosynechoccocus elongatus prepared to precisely select within heterogeneous oxidation or spin states of photosystem II will allow to decipher the molecular origin of different Mn4CaO5 cluster conformations, and ultimately to contribute to our understanding of water oxidation and O-O bond formation.

Disulfide-rich peptides (DRPs) form an incredibly diverse group of bioactive natural products estimated to millions of distinct sequences. These polycyclic compounds are composed of between 10 and 90 amino acids (aa), more than 10% of which being cysteines involved in disulfide bridges. These medium-sized molecules (often referred as miniproteins), have been identified as highly potent and selective binders of various receptors difficult to target with small molecules, making them attractive new pharmacophores. A growing number of DRPs recently entered the market or are currently in clinical trials highlighting their great therapeutic potential. If small DRPs (< 30 aa) can be synthesized by conventional solid phase peptide synthesis (SPPS), the production of longer ones represents a great synthetic challenge, and their chemical engineering towards a lead compound is even much more difficult. Most current efforts concentrate on solution-phase convergent synthesis, through chemoselective couplings of unprotected peptide segments by the native chemical ligation (NCL) reaction. But the throughput of these approaches is very low and still complicated by tedious purification and handling steps. This limits and slows down pharmacological studies and therapeutic or diagnostic applications. This project focuses on the development of technologies for the synthesis of DRPs with much higher throughput than existing methods. One key aspect for intensification of the current processes relies on the realization of the chemoselective assembly of the peptide segments on a solid support, in order to considerably lower the number of intermediate HPLC purification steps. The assembly of the miniproteins in the opposite direction compared to SPPS benefits from an additional “self-purification” feature allowing the use of unpurified peptide segments. We will evaluate several methodologies in parallel on a set of representative DRPs: (i) A method entirely based on NCL will be designed. This implies the use of a strategy to temporarily mask the reactivity of the thioester and prevent cyclisation or oligomerisation of the segments. This “one ligation approach” offers the advantage of using only two types of reaction conditions, for NCL and unmasking steps, which is particularly interesting for future automation of the process. A major bottleneck for applicability of NCL is the difficult synthesis of peptide thioesters. We recently developed a breakthrough methodology that will be used throughout the project. (ii) In order to minimize the number of synthetic steps by suppressing the need of masking groups, we will exploit a smart combination of three different chemoselective reactions used in alternation: NCL, strain-promoted azide/alkyne cycloaddition (SPAAC), and copper-catalyzed azide/alkyne cycloaddition (CuAAC). These reactions will be used for grafting the first segment on a solid support then peptide elongation. In the latter case, we will use our original peptidomimetic triazole ligation (PTL) approach exploiting the amide-mimicking nature of the CuAAC product, a triazole. (iii) Oxidative folding to regioselectively form the SS bonds can be a limiting factor for the production of DRPs: if most naturally occurring DRP can be folded under thermodynamic control, it implies the use of highly dilute conditions that complicate large scale production. We will take advantage of the solid support used for their syntheses for this folding step. An original approach based on fragments with preformed SS will also be implemented. Finally, the methods developed during this project will be applied to the high-throughput generation of a large number of highly diverse variants of one selected DRP, the muscarinic toxin 7, which is particularly promising from a therapeutic point of view. In particular, we will use an original combinatorial approach, which we expect to lead to the identification of several variants with unprecedented pharmacological profiles.

Members of the ATP-dependent chromatin remodeling complexes (remodelers), including the BAF complex, are recurrently mutated in human cancers. We recently reported a genome-wide investigation of remodeler function in embryonic stem (ES) cells (De Dieuleveult et al., Nature 2016). We detected extensive binding of remodelers to promoters and enhancer elements, and unexpectedly, to CTCF-occupied elements. CTCF is a DNA-binding protein that divides the genome into separated chromatin domains called ‘Regulatory Neighborhoods’. This 3D organization is thought to minimize illegitimate contacts between (onco-)genes and nearby unrelated enhancers, with recent data confirming that mutations in CTCF-sites are recurrent in human cancers. By combining the expertise of the three partners in the project, we will investigate the function of chromatin remodelers at CTCF-sites and determine how they contribute to organize 3D genome structure. Our 3D-reMODEL project has four objectives that will systematically elucidate the functional link between chromatin remodelers and CTCF in the structuring of mammalian 3D DNA domains and how perturbed collaboration between remodelers and CTCF may contribute to the cancerous phenotype: 1. We will describe how CTCF co-localizes with nine different remodelers in an orientation dependent manner at different types of DNA domains (TADs and various types of sub-TADs) in mouse ES cells. 2. Using ChIP-seq, we will dissect how the nine remodelers contribute to CTCF binding and Cohesin stabilization or vice-versa. 3. We will unravel the nature of CTCF-remodeler co-occupancy by testing for (direct) interactions between CTCF and remodelers. We will also characterize protein-protein interaction (PPI) networks of CTCF/Cohesin using co-immunoprecipitation and mass spectrometry approaches. 4. Using a combination of high resolution Hi-C and HiChIP approaches, we will analyze how loss-of-function of each remodeler affects the formation of different types of 3D DNA domains. In particular, we will monitor the consequence of Brg1 loss of function to determine if its function in regulating 3D DNA domains can predict part of its tumor suppressor function. We have performed preliminary experiments in which we mapped the ChIP-seq distribution of CTCF in ES cells depleted of either Chd4, Brg1 or Ep400 remodeler. We found that loss of each these remodeler alters CTCF binding with a specific pattern, suggesting a major function for this family of factors in the regulation of 3D chromatin domains. It will now be important to test how the other remodelers affect CTCF binding, if CTCF itself plays a role on the recruitment of remodelers and how changes in CTCF occupancy may cause changes in the 3D organization of the ES cell genome.