Supramolecular assemblies of proteins and peptides are ubiquitous in living cells and play key roles in many biological processes. Non-covalent assemblies of protein and peptide subunits form nanostructures such as fibrils, filaments, pores, nanotubes, etc. to execute crucial biological functions (transport across membranes, bacterial secretion, infection, prion propagation, etc.). Determine the atomic architecture of these nanostructures is a technological challenge.
The design of new architectures and especially the tuning of their properties is a formidable task that requires understanding and ultimately controlling the thermodynamic and kinetic parameters associated with the molecular assembly / disassembly process. Non-covalent weak interactions - e.g. van der Waals, Coulomb, hydrophobic interactions, hydrogen bonds or p-p stacking - are the relevant factors driving these processes. Revealing and understanding the interplay of these weak interactions at the highest possible detail is therefore of primary interest to manipulate and tune the supramolecular organization. This task is extremely challenging due to the inherent “soft” material character of most self-organized structures: (i) they usually do not exhibit 2D or 3D crystalline order and are therefore recalcitrant to X-ray diffraction methods; (ii) weak interactions, notably hydrophobic, drastically increase their insolubility making them unsuitable for solution Nuclear Magnetic Resonance (NMR). 3D models are usually computationally constructed by assuming that the intermolecular interfaces in the soft material are similar to the intermolecular packing observed in the single-crystal structure of the isolated subunit component; this assumption being difficulty validated experimentally. As a consequence, structures of these self-organizations solved at the atomic level are very scarce and the role of the weak interactions remains elusive. There exists thus an urgent necessity to establish alternative methods that overcome these limitations, providing a robust approach that captures the weak interactions driving the molecular assembly and the atomic architecture of those nanostructures. Ideally, new approaches should: (i) provide parameters at the atomic level; (ii) not require crystallinity or solubility and not be limited by any mesoscopic size range; (iii) be performed in the relevant supramolecular state of the soft material.
We aim at establishing a robust NMR methodology to decipher weak interactions in the context of nanostructures formed by protein / peptide / small molecule non-covalent assembly.
Our strategy is based on several approaches:
. development of strategic isotopic labeling schemes, especially to uncover intermolecular (i.e. subunit-subunit) interactions.
. solid-state NMR experiments to detect intramolecular and intermolecular proximities, with emphasis on the proton-detected approches based on ultra-fast magic-angle spinning NMR.
. combinaison on solid-state NMR-based and other biophysical data (from xray diffraction, electron microscopy) to establish atomic 3D models
- Assembly and characterization by solid-state NMR of a new prion protein:
We have developed a protocol for the production, purification and assembly of isotopically labeled samples of a new prion protein involved in programmed cell death. Using solid-state NMR, we have characterized its architecture in its relevant fibrillar form. These results have been published in PNAS in 2016. The technical details of the production and purification protocol have been submitted for publication.
-Assembly and characterization by solid-state NMR of a new prion protein:
We have produced several samples based on different isotope labeling schemes, in order to collect distance restraints based on solid-state NMR. We aim at solving the 3D structure at atomic resolution.
1. Daskalov et al., PNAS 2016
2. Habenstein et al., Biophys Chem. 2016
3. Habenstein et al., Methods Mol. Biol. Sous presse
Large molecular assemblies are ubiquitous in living cells and play key roles in many biological processes. Indeed, multiple copies of protein subunits can organize into large macromolecular nanostructures, in shapes of fibrils, filaments, pores, capsids, etc. Understanding the mechanisms responsible for the molecular assembly of such systems is of primary interest in biology in order to gain insights into their crucial functions. Inspired by these remarkable architectures, supramolecular chemists and material scientists aim at designing synthetic self-assemblies with a broad spectrum of applications, ranging from regenerative medecine to drug delivery. The design of new nanostructures and the tuning of their functionalities require a detailed description and understanding of the weak interactions driving the molecular assembly process.
For self-assembled nanostructures involved in cellular processes or engineered by supramolecular chemistry, the 3D structure determination is hampered by several technical challenges: (i) the assemblies usually lack crystalline order required to perform X-ray crystallography and (ii) the high molecular weight prohibits fast molecular tumbling, which restricts the use of solution Nuclear Magnetic Resonance (NMR). So far, hybrid approaches combining high-resolution structures of the isolated subunits and the molecular envelope obtained from electron microscopy have led to few low- to medium-resolution models of such assemblies. However, this approach lacks the experimental determination of the crucial subunit-subunit interfaces, which can lead to inaccuracies since the subunits can adopt different conformations in isolation compared to their relevant assembled states.
We have recently proposed a new approach based on modern solid-state NMR techniques to solve atomic structures of complex self-assembled nanostructures, demonstrated by an atomic model of a bacterial filament (Loquet et al., Nature 2012). This breakthrough forms the genuine basis of the NanoSSNMR project. We now envision disentangling more complex supramolecular self-organizations, either involved in synthetic or cellular processes. The NanoSSNMR proposal will exploit state-of-the-art solid-state NMR methods and strategic isotope labeling and integrate hybrid approaches to elucidate the assembly mechanisms, revealing the atomic structures of two complex self-assembled nanostructures.
Monsieur Antoine Loquet (Chimie et Biologie des Membranes et des Nanoobjets)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Université de Bordeaux Chimie et Biologie des Membranes et des Nanoobjets
Help of the ANR 299,125 euros
Beginning and duration of the scientific project: September 2014 - 48 Months