In addition to structure determination of isolated components and complexes, it is essential to determine the energetics and kinetics of the interactions, and changes in physical properties of the components on forming the complexes. To this end, a biophysics core laboratory has been established at BCC where researchers can measure functional properties of their purified systems. This is achieved using a variety of biophysical methods including calorimetry, optical spectroscopy such as fluorescence and circular dichroism, surface plasmon resonance, rapid reaction kinetics and hydrodynamic techniques.
Dr. J. Brad Chaires, director of the Biophysics Core Facility, has more than 25 years experience in biophysics, with particular recent emphasis on biological microcalorimetry. Dr. Nichola Garbett is the facility manager.
The dedicated Biophysics Core laboratory is conveniently located on the fourth floor of the James Graham Brown Cancer Center. The core occupies room 4H, a newly renovated 504 square foot laboratory. State-of-the-art biophysical instrumentation was purchased in Spring 2004, with installation completed in October 2004, when the core was opened to users. Instruments available include:
- MicroCal VP-ITC Calorimeter
- MicroCal VP-DSC Calorimeter with Pressure Perturbation Accessory
- MicroCal VP-Capillary DSC
- Calorimetric Sciences Corporation Nano II DSC
These instruments measure the heats of reactions directly, and may be used to determine complete thermodynamic profiles (free energy, enthalpy, entropy, heat capacity changes, compressibility) for binding interactions and conformational transitions. Such information may be used to define the molecular forces important for the reaction under study. The VP-Capillary DSC allows for high-throughput thermodynamic measurements. The instrument features a robotic liquid handling system that allows for 24 samples to be run per day. Only 13 such instruments are available in academic environments worldwide.
- Jasco J-810 Spectropolarimeter-Circular Dichroism (equipped with an auto-titrator, Peltier temperature control, and simultaneous fluorescence detection)
- Jasco FP-6500 spectrofluorometer with accessories
- Jasco V-550 double beam spectrophotometer with accessories
- On-line Instrument Systems, Inc. UV/Vis Diode Array Workstation.
These instruments allow measurements of absorbance, fluorescence, and optical activity of macromolecules and drugs that interact with the given target. Each spectroscopy provides a unique window into the reaction. These spectroscopies may be used to obtain thermodynamic profiles for binding reactions, conformational transitions, and denaturation reactions. Circular dichroism provides low-resolution structural information about protein and nucleic acid secondary structure, and is an invaluable adjunct to higher resolution NMR and crystallographic methods. They also can be used for early screening of new protein quality, as well as for drug-targeted binding screens.
- On-line Instrument Systems, Inc., Rapid Scanning Stopped-Flow Spectrophotometer System
- Biacore 2000 Surface Plasmon Resonance Instrument
These instruments allow the rates of reactions to be measure in the millisecond and beyond time scale. Reaction kinetics may be used to infer the detailed reaction mechanism for a binding interaction or conformational transition.
- Beckman-Coulter XL-A Analytical Ultracentrifuge
The analytical ultracentrifuge is a method firmly grounded in fundamental physical principles. The instrument may be used to determine molecular weights directly and unambiguously by sedimentation equilibrium. The sedimentation velocity method allows determination of hydrodynamic shapes and molecular dimensions, an invaluable adjunct to higher resolution NMR and crystallographic methods. Available modern software allows analytical ultracentrifugation to be used to study the thermodynamics of macromolecular interactions. This instrument was purchased with funds from an NCRR SIG.
- Wyatt Dawn Heleos light-scattering detector (and differential refractometer).
The multi angle static light scattering complements the analytical ultracentrifuge and can provide direct determination of molecular weights of assemblies and the radius of gyration. Collectively the facility provides a very diverse set of possibilities for characterizing systems of interest. The availability of the diversity of possible signals makes it possible to develop screening for interactions for almost any class of compounds.