Biophysical Characterization Of Macromolecules

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A major application of the computational affinity- and kinetic rate distribution analysis previously developed for enhanced surface plasmon resonance biosensing was the characterization of antibodies against antigens derived from Japanese encephalitis virus, Dengue virus, vaccinia virus, and different anthrax proteins, all in collaboration with LID/NIAID. For most targets, several antibodies were characterized and their affinity compared in detail. A second application area of biosensing, in conjunction with analytical ultracentrifugation, was the study of the interferon receptor IFNAR-2 interaction with interferons alpha-2c and CM3, in collaboration with DIR/NIAID. Here, qualitative biosensor studies on the cooperativity of the two ligands and the discovery of the self-association of interferon alpha-2c added to the novel finding of the mutual influence of the interferons in their receptor binding. As an application of our techniques for the study of multi-protein interactions in solution by multi-signal sedimentation velocity analytical ultracentrifugation and by isothermal titration calorimetry, we have embarked with LCMB/NCI on the collaborative characterization of adaptor proteins essential for T-cell activation. Previously, collaborative studies indicated the role of multi-valent adaptor protein cross-linking in the formation of signaling particles in vivo. Using similar methodology, we have examined the ternary system of interactions between an SLP-76 fragment in different phosphorylation states, Nck, and Vav. The results suggest the formation of triple protein complexes of various stoichiometries. Analytical ultracentrifugation provides unique information on protein self-association and hetero-association. We have previously developed techniques for the computational deconvolution of diffusion to arrive at high-resolution sedimentation coefficient distributions, and have shown theoretically how these can be interpreted quantitatively in the framework of coupled migration and chemical reaction, representing a robust method for measuring macromolecular binding constants. This tool was applied in collaboration with LI/NIAID to the characterization of different cytomegalovirus glycoproteins (putative immunoevasins) and interaction with a NKG2D ligand. In a different context, this approach was used to study the role of self-association of glutamate receptor variants. Further, several additional collaborative projects with different IRP laboratories on the characterization of proteins are in a pilot stage, or were conducted using standard biophysical methodology.