Overview

The goal of the Biophysical Core Facility is to provide structural biology support for UofL Health – Brown Cancer Center (BCC) scientists, with particular emphasis on the characterization of biophysical properties of biomolecules (including proteins, peptides, lipids, and polynucleotides) and their interactions with binding partners or novel drug candidates.

• The Biophysical Core provides all biophysical instrumentation necessary for detailed thermodynamic and hydrodynamic properties of biological macromolecules, macromolecular association reactions, and ligand-macromolecule interactions. Biophysical characterization of biomolecules provides information about DNA conformation, secondary / tertiary structures of proteins and their ability to dimerize or multimerize, whereas the characterization of macromolecular interactions allows for the calculation of K_a (binding affinity/association constant), K_d (dissociation constant), binding stoichiometry, and binding energetics (free energy, enthalpy, and entropy).
• The Biophysical Core provides a competitive advantage to drug development teams by integrating thermodynamics into the drug design and development process, which facilitates an understanding of the key molecular forces that govern drug interactions.
• The Biophysical Core Facility works very closely with the Molecular Modeling Facility, Protein Expression Facility and Medicinal Chemistry Facility.  Integration of these BCC cores provides a strong drug discovery platform for BCC members. 

Instrumentation

The Biophysical Core Facility houses multiple biophysical techniques providing rigorous characterization of the properties of biomolecules and their interacting partners.

a.  Rapid screening of biomolecule target-ligand interactions

• Differential scanning fluorimetry (DSF)
• Microscale thermophoresis (MST)
  • DSF and MST provide complementary technologies to determine the in vitro affinity of potential ligands for a given biomolecule target and preferably the relative affinity (selectivity) when presented with alternative related targets.  Biomolecules and identified ligands can then receive more rigorous characterization using a range of hydrodynamic, calorimetric, and spectroscopic techniques.

 b.  Hydrodynamic properties of biomolecules

• Analytical ultracentrifugation (AUC)
  • AUC measures the movement of biomolecules in high centrifugal fields.  This technique yields information about the size, shape, and interactions of biomolecules in solution, providing complementary information to higher resolution methods (e.g., NMR, X-ray crystallography).

 c.  Calorimetric characterization of biomolecule stability and binding interactions

• Isothermal titration calorimetry (ITC)
  • ITC directly measures the microcalorie-scale heat released or absorbed during a biomolecular binding event.  This technique provides a complete thermodynamic profile of binding (binding free energy, enthalpy, and entropy), as well as the binding affinity and binding stoichiometry.
• Differential scanning calorimetry (DSC)
  • DSC directly measures the microcalorie-scale heat released or absorbed during the temperature induced unfolding of biomolecules.  This technique provides a complete thermodynamic profile of biomolecule unfolding (unfolding free energy, enthalpy, and entropy), as well as indirect binding thermodynamics.

 d.  Spectroscopic characterization of biomolecule conformation and binding interactions

• Circular dichroism (CD)
  • CD measures the optical activity of biomolecules and their interacting partners.  This technique provides information about biomolecule conformation (e.g., secondary / tertiary structures of proteins, DNA conformation) and the conformational environment of interacting ligands.  Additional information can be obtained about biomolecule conformational changes related to batch, formulation (e.g., solvents, cosolutes, pH), temperature (unfolding / folding) and binding.
• Fluorescence spectroscopy
  • Fluorescence spectroscopy measures the fluorescence properties of biomolecules and interacting partners, typically monitoring intrinsic protein fluorescence (e.g., Trp, Tyr) or using extrinsic fluorescence probes.  This technique provides an alternative signal (fluorescence) to monitor the conformation of biomolecules and interacting partners that is complementary to other spectroscopic techniques (circular dichroism, absorbance spectroscopy).
• Absorbance spectroscopy
  • Absorbance spectroscopy measures the absorbance properties of biomolecules and interacting partners, typically using the intrinsic absorbance signal of the biomolecule (e.g., protein Trp / Tyr; DNA bases).  This technique provides an alternative signal (absorbance) to monitor the conformation of biomolecules and interacting partners that is complementary to other spectroscopic techniques (circular dichroism, fluorescence spectroscopy).

For more information, fee structure and cancer center member subsidies, please contact:
Nichola Garbett: nichola.garbett@louisville.edu