Protein-nucleic acid recognition probed by solid-state Nuclear Magnetic Resonance spectroscopy at fast Magic-Angle Spinning

• Applicant: Professor Dr. Thomas Wiegand
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry ; Physical Chemistry of Solids and Surfaces, Material Characterisation
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 455240421

Protein-nucleic acid interactions are involved in a variety of biological events ranging from the replication of genomic DNA to the synthesis of proteins. Noncovalent interactions such as hydrogen bonds, dispersion and electrostatic interactions are responsible for the molecular recognition of nucleic acids by proteins. This project focusses on the detection of such weak chemical interactions in non-crystalline protein-nucleic acid complexes. Solid-state Nuclear Magnetic Resonance (NMR) methods (the “NONCOV” approach) will be developed and implemented to probe such effects directly, particularly focussing on protons which are at the centre of protein-nucleic acid contacts. Proton NMR observables serve as sensitive reporters for the engagement of protons in such contacts and their measurement will be achieved by fast Magic-Angle spinning (MAS) experiments (employing MAS frequencies of more than 100 kHz) allowing for a sufficient reduction of the proton NMR linewidths. In combination with the experimental data, quantum-chemical calculations of NMR observables carried out on small protein-nucleic acid fragments will lead to an increase of the theoretical understanding of the response of an NMR observable to the strength of a specific noncovalent interaction. The final objective of NONCOV is to model the binding of nucleic acid to large protein assemblies. Therefore, hydrogen bonds between the protein and RNA/DNA phosphate groups will be identified in proton-detected 1H,31P correlation experiments. Besides restraints based on the direct responses of proton NMR observables (e.g. proton chemical-shift values and J-coupling constants) on protein-RNA/DNA binding events, distance restraints from paramagnetic NMR experiments will be extracted by attaching paramagnetic spin labels to nucleic acids and by investigating paramagnetic relaxation enhancements on the protein NMR spectra. This will allow us additionally to determine distance restraints by Electron Paramagnetic Resonance experiments, for example between a spin label bound to the nucleic acid and a tag attached to the protein. As a proof-of-principle, the NONCOV approach will be established for the bacterial DnaB helicase from Helicobacter pylori involved in unwinding double-stranded DNA during DNA replication. This will enable further insights into the conformational and dynamic changes occurring during DNA loading and translocation of such ring-shaped helicases. The NONCOV approach is transferrable to further biological and chemical applications in which noncovalent interactions are involved, e.g. in the context of phase separation phenomena or supramolecular chemistry.

• DFG Programme: Research Grants
• Major Instrumentation: 0.7mm Festkörper-NMR-Probenkopf
• Instrumentation Group: 1741 Festkörper-NMR-Spektrometer