Core Three Narrative
Identification of the structure-function relationships of intrinsically disordered proteins is central to understanding their biological function in homology-directed repair (HDR) of DNA double-strand breaks. The Structural Biology and Biophysics Core will provide atomic resolution mapping of critical protein-protein and protein-nucleic acid interactions of complexes important for HDR. The insights provided by the Core will assist Program Project Investigators in dissecting the mechanisms of 53BP1 regulation of 5’ strand resection and the formation of RAD51-ssDNA nucleoprotein fragments, processes important for HDR.
Aim 1: Conduct NMR analysis of proteins with nucleic acids and/or protein ligands to map functional domains and binding sites.
Aim 2: Biophysically defines protein-ligand subunit structures and affinities for protein and nucleic acid binding partners.
Abstract
The Structural Biology and Biophysics (SBB) Core will provide Program Project Investigators with high-quality 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 interaction 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 development 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 biophysical approaches, namely, isothermal calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (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.
Facility Details, Core Three
NMR Spectroscopy: Two NMR instruments are located in the Biomolecular NMR Spectroscopy Core Facility at UTHSCSA, which is contiguous to the Core laboratory. Each instrument is equipped for biomolecular NMR applications with four independent RF channels, triple-axis pulsed field gradients, deuterium decoupling capability, a variable temperature controller, and high sensitivity cryogenically cooled 1H/13C/15N probes. The facility is run by a dedicated PhD-level manager who is available to assist in NMR pulse sequence optimization, data acquisition, maintenance and repair, and user training. Instrumentation includes (i) Bruker AVI 500 MHz and (ii) Bruker NEO 700 MHz with multichannel simultaneous send and receive capabilities. The spectrometers are each equipped with four independent RF channels, triple-axis pulsed field gradients (depending on the probe), deuterium decoupling capability, a variable temperature controller, and high-sensitivity cryogenically cooled 1H/13C/15N probes (1.7 mm and 5 mm TCI on the 500 and a 5 mm TCI on the 700). The 500 MHz NMR spectrometer is equipped with an NMR SampleJet that accepts 1.7-mm tubes in 96-well plate format. A Gilson 513 liquid handler is available for the automated preparation of NMR samples in 1.7mm tubes.
Macromolecular Interactions: The laboratory is equipped with a TA Instruments micro Isothermal Titration Calorimeter (ITC); a DynaPro NanoStar Dynamic Light Scattering instrument (Wyatt Technology) equipped with a dedicated Static Light Scattering (SLS) detector; Biotek Cytation 5 microplate spectrophotometer and fluorimeter with imaging capabilities; and has unrestricted access to a Wyatt DAWN 8 MALS in line with a Wyatt Optilab refractive index detector coupled to and Agilent 1260 Infinity II preparative HPLC system (Wyatt Technology); a Nanotemper Monolith Automated microscale thermophoresis (MST); a Refeyn TwoMP Mass Photometer (MP); and a Biacore T200 SPR.
Computer Resources: Operating systems and applications are maintained at the most current stable version. Dedicated computational resources (Libich Lab) include a Linux workstation (20 core, 32 GB RAM, 4 TB HDD) and 3 dual processor (8 cores each) MacPro workstations dedicated to NMR data processing and other computationally intensive calculations. Three additional Linux workstations are available in the Biomolecular NMR Core facility and are dedicated to processing and analysis of NMR data. The NMR Core maintains a dedicated sub-LAN for networked data storage and backup, and long-term data storage is available on the GCCRI shared network drives (petabyte capacity).
Protein Expression: Two Innova S44i temperature-controlled shaking incubators (16 L capacity) are available exclusively, with an additional (24 L) of shared temperature-controlled (heating/cooling) incubator space available as needed, 3 Beckman preparative centrifuges (Avanti J-20, rotors 15mL to 1L /tube capacity), 2 Fisherbrand Sonicators with microchips, and an Avestin-C3 cell homogenizer. Milli-Q grade laboratory water is available.
Protein Purification: Two Bio-Rad NGC Discovery FPLCs, three Bio-Rad BioLogic low-pressure chromatography systems, and a Waters HPLC equipped with a photodiode array detector are available, and the laboratory is fully equipped for protein purification, including 3 bench top preparative centrifuges (15 mL, 50 mL tube and 96-well plate rotors), pH meter, Implen Nanophotometer, NanoDrop 1000, two Eppendorph thermocyclers, Savant speed-vac, Alpha Innotech gel imager, Agilent diode array spectrophotometer.
Cell Culture: Tissue culture space assigned to the PI is available in the Greehey CCRI, includes one shared six-foot BSC, shaking incubators connected to N2 supply control O2 levels, a six-station BioTek Precision XS robot multiwell dispensing system, Amaxa nucleofection (96-well) system, five shared Essen IncuCytes (within an incubator with oxygen suppression capability via nitrogen control).
Microscopy: The core has exclusive use of a Zeiss Axiovert 200M Fluorescent microscope equipped with DIC optics and video capture. Additional microscopes are available in the GCCRI, including a Nikon spinning disk confocal and tissue culture/dissecting instruments. The Optical Imaging Facility is located in the building adjacent to GCCRI and maintains super-resolution and multiple confocal microscopes with computational resources and other appropriate support equipment.
