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. Experimental techniques employed are analytical ultracentrifugation, static and dynamic light scattering, isothermal titration calorimetry, circular dichroism spectroscopy, and surface plasmon resonance biosensing. For analysis of macromolecular mixtures, a method to calculate a two-dimensional molar mass and molecular shape distribution has been developed. It is based on the global deconvolution of the hydrodynamic data from dynamic light scattering and sedimentation velocity, and thermodynamic data from sedimentation equilibrium. Also, we refined our previously developed methodology for measuring sedimentation coefficient distributions for the study of protein complexes, and global modeling methods for the study of protein self-association by sedimentation. We have published a tutorial review on sedimentation techniques in Protein Science. We have applied these sedimentation techniques to the study of several different protein interactions: We have recently shown that recombinant SIV gp140 forms a trimer complex similar to the envelope complex found on virions. However, HIV gp140 forms a heterogeneous set of oligomeric species in contrast to the virion envelope complex which is trimeric. We are collaboratively characterizing if specific regions of HIV gp140 or if the variation in the carbohydrate composition may promote trimer formation. In a different application, we have characterized by sedimentation and dynamic light scattering the size-distribution of an oligomeric CD4-Ig fusion protein that binds gp120 and inhibits viral entry. We have also characterized the oligomeric state and/or self-association properties of a recombination enzyme (junction resolvase) of vaccinia virus, the molecular chaperone gp57A of bacteriophage T4, and engineered leucine zipper peptides that have the capacity to form hydrogels. Several proteins were characterized with regard to their heterogeneous association, including E. coli and murine RNAse H1 interacting with hybrid DNA/RNA oligonucleotide, the adapter protein SLP-76 interacting with phopholipase C-gamma, and the binding of the antibody 14B7 to variants of anthrax protective antigen. Applying our methodology for the biophysical characterization of solution interactions, we have collaboratively embarked on the study of alpha synuclein, a protein that can form aggregates which are associated with Parkinsons disease. We have also examined and applied methodology for the characterization of protein lipid interactions.
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