In the last reporting period we have pursued a collaboration with Dr. David Margulies on the characterization of interactions of proteins constituting the peptide loading complex. This was continued and expanded. Specifically, we examined the stoichiometry and affinity of the interactions between tapasin, as well as TAPBPR (TAP binding protein-related) protein, with MHC class I molecules when loaded with different peptides. While largely employing analytical ultracentrifugation at the current state, this project may potentially evolve into a model application for global multi-method analysis of multi-protein interactions. In a separate project in collaboration with Dr. Mark Mayer, we have continued to study the homo- and hetero-oligomerization of different isoforms of glutamate receptor amino terminal domains. The quaternary structure of these receptors controls their ion gating properties. We have further developed the necessary methodology for studying high-affinity interactions with fluorescence-detected sedimentation velocity. In order to further broaden our experience with the practical application of fluorescence-detected sedimentation velocity, we have also carried out a collaborative study with Dr. Tanja Mittag on the self-association of SPOP protein. We have embarked on a collaboration with Dr. Patrizia Farci applying surface plasmon resonance biosensing to examine the interaction of anti-core antibodies isolated and cloned from livers of patients HBV-associated acute liver failure with their HBV core antigens. The determination of binding epitopes, affinities and kinetics of these antibodies against the homologous core and wild type may provide new insights into the role of these antibodies in the pathogenesis of ALF. Finally, continuing our long-term collaboration with Dr. Lawrence Samelson on the study of multi-protein interactions in signaling particles after T-cell activation, we have carried out initial experiments aimed at the assembly of four-component complexes of adapter proteins, and the study of their stoichiometries and affinities by multi-signal sedimentation velocity and calorimetric techniques.
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