Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - Weakinteract (Weak interactions in self-organizations studied by NMR spectroscopy in the supramolecular solid-state)

Teaser

Self-assembly is a fundamental process by which individual subunits organize into ordered supramolecular entities, usually through weak interactions. A longstanding goal is to engineer synthetic self-organized structures, often inspired by protein assemblies found in the...

Summary

Self-assembly is a fundamental process by which individual subunits organize into ordered supramolecular entities, usually through weak interactions. A longstanding goal is to engineer synthetic self-organized structures, often inspired by protein assemblies found in the context of living cells, to design materials of high potentiality, e.g. drug delivery, scaffolding or electronic applications. There is a tremendous interest in physical chemistry to understand the role of weak interactions at the supramolecular interfaces. However, self-organizations usually form soft material, lacking crystalline order and at the same time exhibiting poor solubility. As a consequence, standard techniques for structural investigation such as X-ray crystallography or solution NMR usually fail or deliver only partial information, preventing an atomic-level understanding and therefore the design of new architectures.

The Weakinteract project aims at developing NMR spectroscopy in the relevant supramolecular solid-state for those non-crystalline and insoluble self-organizations. Weakinteract will exploit strategic isotope labeling, state-of-the-art solid-state NMR methods and integration of hybrid approaches to elucidate the assembly mechanisms, revealing the weak interactions at the supramolecular interfaces. One major aim of Weakinteract is to provide a robust approach dedicated to chemists, biophysicists and structural biologists in order to tackle weak interactions in the relevant assembled state, ultimately delivering atomic level structures and an understanding of the assembly process.

Work performed

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. 1H aromatic spins are ubiquitous in proteins and peptides and a very valuable source of structural information. 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.

. 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 structural architecture the protein HELLP from C. globosum, sharing homology to the RHIM motif found in the execution of necroptotic cell death in humans. We recombinantly produced and purified HELLP (215-278) amyloid fibrils, and first solid-state NMR data indicate a rigid amyloid core in b-strand conformation.

Final results

The Weakinteract project aims at investigating atomic structures, and assembly processes of sophisticated self-assemblies. As the current methodology is not always suitable for such complex assemblies, we develop and apply new solid-state NMR methods to capture structural and dynamic details at the atomic scale.

In particular, we have been so far involved in the development of state-of-the-art methods:

. Detection by solid-state NMR of aromatic proton resonances in protein assemblies using very-fast magic angle spinning probe. Our approach offers an efficient tool to monitor aromatic-aromatic interactions at the intermolecular interfaces.

. Assignment of proton resonances in fully protonated self-assemblies using solid-state NMR with ultra-fast magic angle spinning probe. The approach considerably decreases the quantity of sample required to perform atomic-level structural characterization and avoids deuteration.

Using several NMR methods developed in the project, we have been able to provide atomic information on the structural role of the amyloid fold during biological mechanisms involving programmed cell death. The network of weak interactions forming amyloid-amyloid interfaces is crucial, and we will next investigate at the highest resolution possible the impact of subtle changes in the interaction network on the structure-function interplay for amyloids.

Our ongoing research is focusing on the structural characterization of bacterial filaments by solid-state NMR, in order to decipher the role of weak interactions in the biological activities of the filaments.

Website & more info

More info: http://www.loquetlab.org/erc-project/.