Exploring thermodynamic and functional properties of a nucleotide binding protein intracellularly using high resolution fluorine NMR spectroscopy

• Applicant: Professor Dr. Michael Kovermann
• Subject Area: Biophysics ; Structural Biology
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 441495846

High-resolution NMR spectroscopy comprises a variety of tools to probe structural, dynamic and functional features of proteins. Most studies which have been conducted so far have been performed under dilute conditions („in vitro“) neglecting the natural environment proteins are exposed to. Therefore the present proposal aims to quantify the thermodynamic stability, intrinsic dynamics and ligand binding affinity of a protein under both, cell lysate and intracellular conditions. It is proposed to label the cold shock protein - which inherently possesses a nucleotide binding site - with fluorine and to subsequently study the protein in its native environment. Using this labeling strategy enables to blend out the large signal background arising from proton, nitrogen and carbon resonances efficiently. Two-dimensional heteronuclear NMR experiments using fluorine (representing the protein) and phosphorus (representing the ligand) as well as fluorine and carbon (representing isotopically labeled ligand) are plannend to be applied to determine the affinity between the nucleotide and the protein in cell lysate and in an intracellular context. Thus the proposed work aims in parallel to contribute to the open question if and how the general properties of a protein like structure, dynamics and function differ when comparing dilute with cellular conditions. The determination of the affinity between a protein and a ligand in a cellular context possesses in this context an ideal potential in order to expand the application of multidimensional NMR correlation experiments comprising fluorine resonances significantly.

• DFG Programme: Research Grants