Our staff maintains and operates a battery of sophisticated solution-based biophysical instrumentation. Among the analytical methods available to intramural researchers are: analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), dynamic light scattering (DLS) and circular dichroism spectroscopy (CD). Whether used separately or in concert, these instruments are powerful tools to properly characterize individual proteins and to study protein-protein interactions. In collaboration with Dr. Huang Zhao, we utilized sedimentation velocity analytical ultracentrifugation (SV-AUC) to examine Pseudomonas putida ferric-pseudobactin, PupR, wild type and several of its mutants as to their conformational states at various concentrations. Our results will help understand this conserved class of sigma regulators and the mechanisms involved in intracellular iron regulation of gram-negative bacteria. Also in collaboration with Dr. Zhao and with Dr. Farci (NIAID), we continue with SPR binding studies between the homologous and wild type HBV-core antigen produced from HBV -ALF (Hepatitis B virus-associated acute liver failure) and germline anti-core IgM and IgG antibodies that may play a major role in the pathogenesis of HBV-associated ALF. In collaboration with Dr. Robert Schwartz (NCI) Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) and Surface Plasmon Resonance (SPR) techniques will examine the mechanism of small molecule disruptors of CD47 signaling which results in protecting cells and tissues from a variety of insults. These experiments will provide the means to examine SIRPA-Fc and other small molecule candidates. In order to verify the formation of large molecular complexes between an HIV gp140 trimer and 1-3 molecules of CD4-Ig, we have, in collaboration with Dr. Paolo Lusso (NIAID) begun Analytical Ultracentrifugation experiments to characterize the individual proteins and to examine the protein combinations as to the relative quantity of each in experiments involving the mixtures. Dr Yamada (NIDCR)has recently found Fibulin-7-C, an extracellular matrix protein, and its fragment to have anti-angiogenic properties through its interaction with vascular endothelial growth factor receptor 2 (VEGFR2). Utilizing Analytical Ultracentrifugation, we are examining the binding of Fibulin-7-C and VEGF then Fibulin-7-C and VEGFR2 to confirm their interactions.
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