The intent of this project is to develop new and improve existing methodology for the characterization of biological macromolecules, and to apply these methods collaboratively to the study of macromolecules and their interactions. Techniques employed are analytical ultracentrifugation, dynamic light scattering, isothermal titration calorimetry, and surface plasmon resonance biosensing. In analytical ultracentrifugation, further methodological advances have been made in the modeling of macromolecular size-distributions by direct boundary analysis and Lamm equation modeling. This proved to result in relative high-resolution size-distributions, which were highly useful for the analysis of protein oligomeric states. Also, we continued the development of methods for the hydrodynamic characterization of small molecules and charged macromolecules by sedimentation velocity methods. We have written a review on sedimentation equilibrium ultracentrifugation for the study of molecular interactions. For optical biosensing, we have further improved a method that allows a significant reduction of the required sample volume. These methods were collaboratively applied to the characterization of several proteins and their reversible interactions. One major focus were the oligomeric state of viral proteins, including the HIV envelope proteins gp120 and gp140, the self-association properties herpes simplex major capsid protein VP5, and the structure/function relationship of the nonstructural protein NSP2 of rotavirus. We have continued our study of G-protein subunits by surface plasmon resonance biosensing. We have started the characterization of several other proteins, including the rotavirus nonstructural protein NSP5, SPE4, TAF, calretinin, tubulin, MHC molecules, T-cell receptors, and the superantigen SPE-C. The characterization of many of these systems has been completed, and several collaborative publications are in press, submitted, and in preparation.
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