Advanced NMR methods to investigate complex biological systems
- Authors
- Publication Date
- Jul 08, 2020
- Source
- HAL-Descartes
- Keywords
- Language
- English
- License
- Unknown
- External links
Abstract
Determining the structures, dynamics and interactions between diverse biomolecules are essential information for the understanding of their diverse biological functions. Nuclear magnetic resonance spectroscopy is a unique tool to investigate molecular conformation and motions over a large range of timescales. In this thesis, we present our work of investigating complex biomolecular system using advanced NMR methods.We used high-resolution NMR relaxometry to identify interactions between metabolites and macromolecules in complex biological samples. The method is based on the measurement and analysis of longitudinal relaxation rates over three orders of magnetic field with high-resolution detection. This approach was first developed and validated on a model sample, then applied to biological fluids relevant for metabolomics studies: human blood plasma and serum. The size dispersion of macromolecules in human blood was estimated with Fast Field Cycling NMR. Identified metabolites revealed different features in high-resolution relaxation dispersion profiles. The interactions of some metabolites with macromolecules were characterized. Furthermore, we show that relaxation dispersion profiles are sensitive to competitive binding between small molecules. In parallel, we implemented different isotope labeling methods and NMR strategies to study two different proteins. Full length human XLF protein was analyzed by high-resolution NMR. Its intrinsically disordered region was assigned and its interaction with DNA was characterized for the first time by NMR. We also explored challenging selective isotope labeling on S. cerevisiae polymerase η.
