Two-field TROSY NMR to reveal interactions of intrinsically disordered proteins and weakly folded states. Application to transcription regulation.

Objective

Interactions of biomolecules are essential for all molecular processes of life. Biomolecules interact dynamically, constantly changing their conformational ensembles to adapt to stimuli and carry out their functions. Protein interactions often involve intrinsically disordered regions (IDRs), which are challenging targets for traditional methods like X-ray crystallography and cryo-electron microscopy). Nuclear Magnetic Resonance (NMR) excels at capturing molecular dynamics experimentally and characterizing IDRs with atomic-resolution. However, interacting IDRs suffer from signal broadening from conformational exchange, particularly when the interface retains a degree of disorder, often preventing NMR analysis. We need new methods to investigate these interactions. To address this, we introduce two-field TROSY NMR, a new methodology that leverages a world-unique prototype NMR spectrometer with two magnetic centers. This approach combines the favorable characteristics of two nuclei fluorine-19 and carbon-13 at two fields (1.88 and 14.1 T): narrower and more intense signals at low field for fluorine-19, combined with signal detection at high field for optimal relaxation of carbon-13 nuclei (less sensitive to chemical exchange broadening), ensuring high resolution and sensitivity. This new ability to observe 13C19F pairs on aromatic side chains will allow us to determine conformational properties of weakly folded states and reveal subtle conformational changes in IDPs, crucial for understanding biological processes like transcription. We will apply this method to investigate the interactions between the hub RST domain from Radical-Induced Cell Death1 and three transcription factors partners, which explore the conformational disorder-order continuum. This project will be a breakthrough for NMR methodology and instrumentation, opening a new era for the investigation of weakly folded systems and provide fundamental new insights on the molecular processes of transcription.