Core 3: Structural Biology and Biophysics (SBB) Core

ABSTRACT The Structural Biology and Biophysics (SBB) Core will provide Program Project Investigators with high-qual- ity atomic resolution mapping and quantification of salient protein ligand binding sites using NMR and other biophysical techniques. The vast majority of proteins involved in DNA homology-directed repair and its regulatory

axis are comprised of significant stretches of intrinsically disordered (ID) residues that serve as ligand interac- tions sites. With class-leading expertise in the structural and biophysical characterization of ID proteins, the SBB Core will combine state-of-the-art NMR to map, at atomic resolution, the protein ligand interfaces and nucleic

acid binding sites, thus identifying critical amino acid residues mediating these interactions to guide the devel- opment of separation of function mutants. By their nature, intrinsically disordered protein ligand complexes are highly dynamic, transiently populated, and weakly associated, features that preclude high-resolution structure

determination by cryo-EM or X-ray crystallography. The approaches, techniques, and overall expertise available within the SBB Core circumvent these technical limitations to assist Program Project investigators in defining crucial ligand interaction interfaces at atomic resolution. Our biophysical and structural biology wherewithal will

furnish insights for understanding the structure-function relationship of salient protein-protein and protein-nucleic acid interactions. The SBB Core will work closely with the PBE Core in providing superior services to achieve the optimization of high-quality protein preparations and their characterization. Additionally, with available bio-

physical approaches, namely, isothermal calorimetry (ITC), surface plasmon resonance (SPR), microscale ther- mophoresis (MST), size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS), and mass photometry (MP), the SBB Core is well positioned to determine the kinetics and thermodynamics of

complex formation, determine subunit structures, and define complex stoichiometries of interactions germane to helping achieve the objectives of each of the three Research Projects.