The intent of this project is to develop new and to improve existing biophysical methodology for the characterization of biological macromolecules in solution, and to apply these methods collaboratively to the study of proteins and their interactions. Experimental techniques employed are analytical ultracentrifugation, static and dynamic light scattering, isothermal titration calorimetry, differential scanning calorimetry, circular dichroism spectroscopy, and surface plasmon resonance biosensing. ? ? For the study of reversible formation of multi-protein complexes in solution, we have previously developed a novel multi-signal analysis for sedimentation velocity analytical ultracentrifugation. It can permit the label-free detection of sedimentation coefficient distributions of currently up to three protein components, and thus measure the stoichiometry of multiple hydrodynamically separated protein complexes. We have gained further experience through the application of this approach to several collaborative projects, and have explored in more detail its potential for studying protein-nucleic acid interactions. Systems studied include adaptor protein complexes involved in signal transduction after T-cell activation, cell surface receptors interacting with the HIV envelope protein, and ribonuclease H. ? ? Complementary to this sedimentation analysis for determining the number and stoichiometry of multi-protein complexes, we have embarked on the development of computational approaches for the quantitation of energetics and detection of cooperativity in the assembly of multi-protein complexes by isothermal titration calorimetry. We have collaboratively tested this new approach in the study of adaptor protein complexes. The data analysis tools were made available in public domain software. ? ? We have further refined methods for sedimentation analysis accounting for the influence of reaction kinetics on the sedimentation behavior of interacting molecules. This includes mathematical methods for data subset analysis and models accounting for repulsive steric interactions during sedimentation. Different aspects of these new and previously developed ultracentifugation methods were applied collaboratively to the characterization of enzymes, detergent-solubilized membrane proteins, and soluble fragments of immunological cell surface proteins. ? ? In the area of optical biosensing, we have refined our mathematical analysis of functional heterogeneity of immobilized surface binding sites, a method that was applied in an extensive collaboration on the characterization of the affinities and kinetic properties of potential therapeutic antibodies. Further, we have continued on the development of recovery techniques aimed at the identification of unknown binding partners to surface-immobilized molecules. ? ? In order to complement our experimental tools for the characterization of size-distributions of biomolecules, we have formulated and collaboratively tested novel mathematical models accounting for the length-distribution of amyloid fibrils, and their evolution with time.
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