Final Report Summary - SECSYSNMR (Structural investigations of Bacterial Secretion by Solid-State NMR)
The structural organization of many subunits into large supramolecular objects is a ubiquitous strategy used in the fields of biology, chemistry and material research. It consists in oligomerizing (also called aggregation, multimerization, self-assembly, etc. depending on the experimental conditions) multiple small molecular components into ordered supramolecular entities. Upon the non-covalent assembly, individual subunits can gain in organization and stability, and the functional properties of the final system are dictated by the intact supramolecular structure rather than by its individual sub-components. In biology, these oligomeric systems contain many copies of small molecular subunits, ranging from peptides and proteins to DNA and RNA. The morphologies of the self-assemblies are diverse, however fibrils, filaments, tubes, capsids, pores, rings or large nanoparticles are commonly observed. Among the wide range of molecular assemblies observed in living cells, self-assemblies made of protein subunits are present everywhere and constitute complex functional entities in many biological events, such as in bacterial infections.
Understanding the processes that allow for the assembly of these biological systems and the knowledge of the high-resolution three-dimensional (3D) structures are key aspects for explaining their functional properties of the assembly in the cell. Protein assemblies present very challenging systems for high-resolution structural studies (i.e. at atomic resolution) due to their inherent tendency to rapidly form insoluble samples even at low concentration. The lack of solubility, as well as their large size, is a major obstacle for Nuclear Magnetic Resonance (NMR) spectroscopy performed in solution (a technique called solution NMR). Although a high degree of symmetry is usually observed for large protein assemblies, growing single crystals of these protein aggregates is very difficult, limiting the use of X-ray crystallography. In contrast, cryo-electron microscopy (cryo-EM) and solid-state NMR spectroscopy (ssNMR) are complementary techniques that have the potential to investigate the 3D structure and assembly mechanisms of complex biomolecular self-assemblies in their intact state.
The aim of this Career Integration Grant is to establish a state-of-the-art laboratory for structural investigations of bacterial protein assemblies by solid-state NMR techniques. It comprises the set-up of a biochemistry laboratory, dedicated to the production of isotopically protein samples, and their analysis by solid-state NMR. During the first period, we have achieved the set-up of protein sample production for NMR studies, leading to mg-quantities of isotope labeled protein subunits of relatively high purity. The experimental conditions for the assembly of the subunits into the final supramolecular system are critical for the correct subunit arrangement; we therefore optimized these conditions to obtain assemblies of homogeneous aspect. In that respect, we have reached independence concerning the biochemistry part of the project in the host institute. During the second part of the project, we have performed solid-state NMR spectroscopy to decipher the conformation of several protein assemblies to understand the structure-function relationship.
Understanding the processes that allow for the assembly of these biological systems and the knowledge of the high-resolution three-dimensional (3D) structures are key aspects for explaining their functional properties of the assembly in the cell. Protein assemblies present very challenging systems for high-resolution structural studies (i.e. at atomic resolution) due to their inherent tendency to rapidly form insoluble samples even at low concentration. The lack of solubility, as well as their large size, is a major obstacle for Nuclear Magnetic Resonance (NMR) spectroscopy performed in solution (a technique called solution NMR). Although a high degree of symmetry is usually observed for large protein assemblies, growing single crystals of these protein aggregates is very difficult, limiting the use of X-ray crystallography. In contrast, cryo-electron microscopy (cryo-EM) and solid-state NMR spectroscopy (ssNMR) are complementary techniques that have the potential to investigate the 3D structure and assembly mechanisms of complex biomolecular self-assemblies in their intact state.
The aim of this Career Integration Grant is to establish a state-of-the-art laboratory for structural investigations of bacterial protein assemblies by solid-state NMR techniques. It comprises the set-up of a biochemistry laboratory, dedicated to the production of isotopically protein samples, and their analysis by solid-state NMR. During the first period, we have achieved the set-up of protein sample production for NMR studies, leading to mg-quantities of isotope labeled protein subunits of relatively high purity. The experimental conditions for the assembly of the subunits into the final supramolecular system are critical for the correct subunit arrangement; we therefore optimized these conditions to obtain assemblies of homogeneous aspect. In that respect, we have reached independence concerning the biochemistry part of the project in the host institute. During the second part of the project, we have performed solid-state NMR spectroscopy to decipher the conformation of several protein assemblies to understand the structure-function relationship.