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, circular dichroism spectroscopy, and surface plasmon resonance biosensing. In order to improve the analysis of protein size-distribution and interactions in free solution, methods based on the global analysis of analytical ultracentrifugation and dynamic light scattering have been developed. We have improved the sensitivity and resolution of macromolecular species, enabling the characterization of assembly intermediates in self-associating protein systems. In order to increase the precision of the frictional coefficient measured by sedimentation velocity, we have further developed the theory (and implemented for the data analysis) of pressure effects in ultracentrifugation. The goal is to permit comparison of experimental frictional coefficients with theoretical hydrodynamic calculations based on available crystal structures of proteins, and to thereby identify flexible protein domains and to determine more precisely the shapes of protein complexes in solution. For thermodynamic analyses of protein interactions, new global sedimentation equilibrium analyses with soft mass conservation constraints were developed which allow a more reliable characterization of the stoichiometry and association constants of reversibly interacting proteins, and permit the estimation of the fraction of incompetent protein species. For the measurement of the kinetics and thermodynamics of protein interactions, we have made significant progress in the interpretation of experimental surface binding kinetic data. By applying two-dimensional data deconvolution and regularization techniques previously developed for hydrodynamic studies to the analysis of protein surface binding, we have achieved, for the first time, a general characterization of the distribution of kinetic and thermodynamic binding constants of heterogeneous populations of surface sites. This is important for the analysis of surface plasmon resonance experiments, such as those conducted in many laboratories with Biacore instrumentation. We have applied all techniques described above to several collaborative studies of protein interactions: The new sedimentation velocity and hydrodynamic methods were used in the characterization of a multi-protein complex composed of signalling proteins of the adaptor protein LAT, the study of the assembly of tubulin rings in the presence of cryoptophycin, the self-association of the dendritic cell specific ICAM-3-grabbing nonintegrin binding receptor (DC-SIGN), the study of MHC engagement of LY49 receptors, the study of the self-association of the molecular chaperone gp57A of bacteriophage T4, and the study of a hexameric D1D2IgG fusion protein with CD16. We have collaboratively studied by sedimentation equilibrium the self-association of ribonucleases, and of the transcriptional regulator IRP1 and the iron-sulfur cluster binding protein Nfu. We have continued our efforts to collaboratively apply our biophysical methodology to the characterization of the filamentation process of alpha synuclein, which is associated with Parkinsons disease. Finally, thermodynamic, kinetic and hydrodynamic studies were conducted collaboratively on the interactions of the antibody 14B7 to variants of anthrax protective antigen.
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