The main objective of the ERC project Weakinteract is to develop new approaches using solid-state nuclear magnetic resonance (NMR) spectroscopy to understand complex phenomena in biology and chemistry. The project proposes to tackle several challenging (bio-)systems forming supramolecular assemblies at an unprecedented spatial resolution. So far, our work has been focused on:
. Consolidation of a laboratory for the isotope labeling of recombinant biological supramolecular assemblies: NMR spectroscopy is a powerful technique that is suffering for low sensitivity. During the first year of the Weakinteract project, we have set-up a laboratory fully dedicated to the isotopic labeling of complex biological assemblies. Starting from model systems, we have built the experimental procedures required for the isotope labeling, comprising bacterial expression, purification, self-assembly and various biophysical characterization (X-ray diffraction and electron microscopy).
. Development of a new solid-state NMR technique to detect aromatic resonances: Several supramolecular assemblies investigated in the Weakinteract project are rich in aromatic residues (Phenylalanine, Tryptophan, Tyrosine and Histidine). It is known that aromatic-aromatic interactions, i.e. pi-pi stacking, often form the molecular basis of the supramolecular interface between aromatic-rich subunits. We have developed a new solid-state NMR technique to efficiently detect 1H aromatic spins in the relevant solid (i.e. assembled) state. We developed a new solid-state NMR proton-detected three-dimensional experiment, in collaboration with Y. Nishiyama (JEOL, Japan) dedicated to the observation of protein proton side chain resonances in nano-liter volumes. The experiment takes advantage of very fast magic angle spinning and double quantum 13C-13C transfer to establish efficient (H)CCH correlations detected on side chain protons. The method is particularly efficient for detecting aromatic spins. We demonstrated our approach on a fibrillar assembly made by the prion domain HET-s.
. Development of NMR approaches to tackle structural characterization of amyloid fibril structure. We used approaches based on fast magic-angle spinning to achieve the direct detection of 1H resonance in the solid state. Access to 1H resonances is useful to speed up the resonance assignment process and detect meaningful 1H-1H proximities.
. Understanding the structural basis of the amyloid fold: Amyloids are proteins that can undergo a conformational change from a soluble, monomeric to an insoluble, polymeric state, defined by the formation of aggregates ranging from oligomers to protofilaments and fibrils. Several amyloid proteins have been associated with the propagation of neurodegenerative diseases. Recently, numerous amyloid proteins have been identified in mammalians, fungi, bacteria or plants as crucial molecular determinants in the execution of native and beneficial biological functions, these proteins are named “functional amyloids” (in contrast to pathological amyloids). Recently, the formation of high-order fibrillar amyloid assemblies has been discovered in several signalling pathways controlling immunity-related cell fate. The precise role of amyloids in cell fate pathways and the structural mechanisms related to the templating and propagation of amyloid-based assemblies in signal transduction is still poorly understood. We have been investigating, in collaboration with S. Saupe (CNRS Bordeaux) the molecular basis of functional amyloid cross-seeding at atomic resolution.
. Various functional amyloids have been investigated by NMR methods to deliver crucial information about their molecular conformation in the relevant assembled state.
. Structural studies of bacterial filaments by solid-state NMR: we achieved the production of samples suitable for structural studies, based on two approaches: (1) recombinant expression and purification of monomeric subunit proteins, later used to perform in vitro self-assembly of filaments. (2) Extraction of bacterial filaments from the cell. Our current analysis is based on NMR data, used in combination with cryo-electron microscopy to deliver hybrid structure of the complete filamentous structure.
