Carbohydrates are highly important biomolecules in living organisms, with by far the huge molecular diversity. Their structural deciphering represents a challenging task reflecting a strategic priority of the Glycosciences. In most cases, their actions are achieved by a more or less specific protein recognition. These last ones are able to decode molecular information within a given carbohydrate sequence, by matching non-covalent interaction points. As carbohydrates synthesis is not directly encoded by the genome, the approaches to release, extract and purify them from biological media are tedious and amounts available are very limited, complicating their characterization. To overcome such bottlenecks, we aim to design carbohydrate and protein chips coupled to mass spectrometry, to elucidate interacting sequences within carbohydrate-ligand complexes.

Protein transport across membranes involves complex translocation machineries. Most proteins and toxins exploit the endocytosis machinery and intracellular sorting to gain access to target cell compartments, while few others, such as the adenylate cyclase CyaA toxin, carry their own translocation apparatus. The CyaA toxin is a key virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. Whooping cough is a very contagious vaccine-preventable disease that can have dramatic consequences for persons at risk, like children. Indeed, pertussis is responsible for about 200,000 deaths per year, and most deaths occur in young infants who are either not or incompletely vaccinated. CyaA plays an important role in the early stages of respiratory tract colonization by B. pertussis. The CyaA toxin carries its own translocation machinery, and it is the only known toxin reported to-date that is able to invade cells by a direct translocation of its adenylate cyclase catalytic domain across the plasma membrane, from the extracellular environment to the cytosol of eukaryotic target cells. Once translocated, the catalytic domain is activated by host calmodulin binding and produces supraphysiological levels of cAMP that alter cell physiology, leading ultimately to cell death and subversion of host defense. The CyaA translocation process across the cell plasma membrane remains, however, largely unknown. The objective of this project is to address several key issues regarding the structure and function of CyaA. Currently, the high-resolution three-dimensional structure of the full-length CyaA toxin is unknown, as well as its structural organization and stoichiometry once inserted into membranes, both before and after translocation of the catalytic domain across the plasma membrane. Moreover, the molecular mechanism and the forces involved in the catalytic domain translocation across the plasma membrane have not yet been investigated. Finally, the effect of molecular crowding on the catalytic domain translocation across the plasma membrane and its subsequent folding upon calmodulin-binding, leading to enzymatic activation, are unknown. Taken together, our project aims to solve the structure of CyaA in solution and inserted into membrane, and to characterize its translocation process, using a combination of cutting-edge technologies. From an applied perspective, this knowledge will be instrumental in improving CyaA as antigen delivery vector against cancer cells and bacterial infections, and as well as protective antigen for vaccination against whooping cough.

BattAllox aims at targeting multiple electron transfer and tuneable redox properties for enhanced energy storage systems. Using bioinspired design principles and molecular engineering, this project focuses on interfacing redox-active isoalloxazine and alloxazine units with coordination chemistry to deliver highly tuneable and versatile redox systems. Redox behaviour at the molecular and higher levels will be studied on small-molecule organic units, organometallic complexes and Coordination Polymers. These redox species will be the basis of a new class of robust multi-electron transfer materials, for electrode materials, for example, and will be incorporated in redox-flow batteries. This multi-scale and multidisciplinary approach bring together molecular design, electrochemical studies, coordination networks and redox-flow batteries.

The discovery of intrinsically disordered proteins and regions (IDPRs) challenges our understanding of the physical chemistry of biological mechanisms. IDPRs increase the reach of biomolecular systems to project far and engage in multiple interactions, moving efficiently over nanometer distances. Yet, we still miss methods to investigate the geometry and timescales of these nanometer motions with high resolution. We will develop an integrative experimental and computational framework to characterize nanometer motions in IDPRs at atomic resolution, exploiting synergies between paramagnetic NMR, electron paramagnetic resonance (EPR) and molecular dynamics (MD) simulations. A series of methodological innovations will be pursued in each of these fields: (1) we will tackle a key limitation that currently prohibits the quantitative interpretation of NMR relaxation effects due to the interaction with electrons by quantifying them over a broad range of the most relevant magnetic fields with a unique sample shuttle apparatus combined with high magnetic fields. (2) We will reduce the current flaws in molecular dynamics force fields for IDPs by direct improvement of the force fields and selection of MD trajectories based on experimental constraints with a new protocol. (3) The complementary information provided by paramagnetic NMR and EPR will allow us to carry an original quantitative analysis with MD simulations leading to an unprecedented description of the kinetics and conformational pathways of nanometer motions in IDPs with atomic resolution. This methodology will be developed on the IDPRs from key proteins in the non-homologous end joining (NHEJ) pathway, a process essential for the repair of DNA double-strand breaks and adaptive immunity: the long disordered region of the enzyme Artemis and the IDPRs of the scaffolding and regulation proteins XLF and XRCC4. The consortium brings together specialists in NMR methodology and instrumentation, IDP NMR, molecular dynamics simulations, protein EPR, and the biology of NHEJ. The NANO-DISPRO project will provide new tools to investigate the kinetic and thermodynamic principles that underlie the function of IDPRs.

Fluorescence imaging has become a widespread mean of observing biological processes thanks to the development of new instrumental techniques and smart fluorescent probes. Biphotonic microscopy using and infrared excitation source has made it possible to perform in vivo imaging in physiological conditions. This technique is particularly used in neurosciences to monitor brain activity of small animals using fluorescence imaging. In this project, we aim to develop Dual-InPut (DIP) red-emitting fluorescent Ca2+ indicators optimized for two-photon excitation to perform in vivo calcium imaging. Dual-input probes are fluorophores with dual sensitivity towards two independent external factors. As such they operate a two-input AND logic gate function whose output is the fluorescence emission and they enable complex and high contrast imaging experiments. The DIP probes envisioned in this project will combine a protein sensitive fluorogen with a calcium ionophore: the fluorophore emission will thus be doubly conditioned to the binding of the target protein and to the detection of Ca2+. This will allow using fluorogenic targeting of protein tags to perform functional calcium imaging in specific cell types or subcellular organelles expressing the corresponding fusion protein, without having to wash the excess dye. The optimization of the two-photon brightness and the dual activation of the fluorescence will enable sophisticated calcium imaging in deep layers of rodents’ brain with controlled localization, high contrast and high temporal resolution. Thanks to the versatile genetic targeting strategy, the DIP probes will be suited to study the fine mechanisms of calcium regulation at the subcellular scale as well as to evidence neuronal heterogeneity by targeting specific neuronal networks. By replacing the calcium ionophore with other sensing groups, the DIP probes design will then be extended to perform wash-free localized two-photon imaging of various biological analytes.