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, static and 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 could be generalized to obtain weight-average frictional ratios of the sedimenting macromolecules. Also, we extended the previously developed methods for the hydrodynamic characterization of small molecules, charged macromolecules, and highly elongated non-ideally sedimenting macromolecules. These methods were applied to the characterization of the protein oligomeric state. Experimental model systems for the study of protein-surface binding were explored, and theoretical concepts for the description of extended self-association were developed. In surface plasmon resonance biosensing, we have further refined the previously developed small volume sample handling technique and applied it to the recovery of sample for mass spectrometry, as well as an assay for the sera reactivity of antibodies. All biophysical methods were collaboratively applied to the study of several proteins and their reversible interactions: We have characterized the oligomeric state of HIV and SIV recombinant and virion-derived envelope proteins gp120 and gp140, and a multimeric sCD4 fusion protein. We have extended previous studies of the structure-function relationship of the rotavirus nonstructural protein NSP2 to its temperature-sensitive variant tsE. Other proteins that have been studied include the T-cell receptor and its interactions with MHC molecules and superantigens, LY49A molecules, and G-proteins and its interactions. 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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