Bacterial Protein Expression Core
Cell Biology and Imaging Core
EM Crystallography Core
EM Tomography Core
Eukaryotic Protein Expression Core
Fluorescence Spectroscopy Core
Protein Interactions Core
Protein NMR Spectroscopy Core
RNA Structure and Dynamics Core
Tissue EM Core
Virus Imaging Core
X-ray Crystallography Core
Protein Interactions Core
Director: Debbie Eckert, PhD
The Protein Interaction Core characterizes macromolecular biophysics and interaction energetics using a wide variety of state-of-the-art instrumentation and techniques that can analyze association and kinetic binding constants, enthalpies and entropies of binding, secondary structure, shape, molecular weight, stoichiometry, stability, and homogeneity.
Instrumentation and Capabilities
The facility houses two analytical ultracentrifugation (AUC) instruments used to study the hydrodynamic properties of proteins and macromolecular complexes: a Beckman XLA (scanning UV-Vis optics) and a Beckman XL-I (both UV-Vis and Rayleigh interference optics). Two types of experiments are being performed: 1) Sedimentation velocity, which measures hydrodynamic properties of macromolecules and complexes to provide information on shape, size and complex formation, and 2) Equilibrium sedimentation, which measures thermodynamic properties of macromolecules in solution, allowing accurate determination of masses, stoichiometries, and association constants for interacting systems. Protein equilibrium sedimentation experiments are typically monitored by absorbance at 280 nm (or at 230 nm when high sensitivity is required). The XLI Rayleigh interference optical system allows us to study complexes that contain UV-absorbing components (e.g. ATP), and is also the optical system of choice for sedimentation velocity experiments owing to its rapid acquisition times. Detailed methods and references for data analysis are contained in two recent Core publications (Schubert et al., Proc. Natl. Acad. Sci. USA (2010) 107:17951-6 and Bajorek et al., Nat. Struct. Mol. Biol. (2009) 16:754-62). We are currently upgrading our Beckman XLA and XLI AUC instruments, which will modernize their computer interfaces, increase their reliability and performance, and greatly prolong their usable lifetimes.
The facility operates the new MASS-1 real-time, label-free surface plasmon resonance-based analysis system from Sierra Sensors. With 16 sensor surfaces that can be independently addressed, the MASS-1 offers high-throughput sample analysis with in-line controls for up to 8 targets in order to characterize the affinities and kinetics of macromolecular interactions. Impressive signal/noise ratios are obtained from the high intensity laser light integrated with high speed optics, enabling detection of small or low concentration analytes.
Isothermal Titration Calorimetry
We operate an iTC200 Microcalorimeter from Microcal which allows us to measure enthalpies and rigorously determine all relevant thermodynamic parameters, and does not require immobilization of either the receptor or the ligand. A recent Core publication describes the use of our ITC (Zhai et al., J. Virol. (2011) 85:9222-6).
Our Aviv 410 CD Spectrometer allows us to analyze protein secondary structure and can monitor stability of proteins and protein complexes. A recent Core publication describes the use of CD for monitoring the stability of protein/protein interactions (Welch et al. J. Virol. (2010) 84:11235-44).
Other Instrumentation Available (Kay Lab, University of Utah, Dept. of Biochemistry)
Through our close connections with the Kay Lab, the Core also has ready access to instruments for peptide synthesis (PTI PS3 Automated Peptide Synthesizer), purification (Beckman semi-preparative HPLC, Agilent analytical HPLC with autosampler), and characterization (ABI 3000 triple-quad mass spectrometer).