In the last reporting period we have continued and intensified our long-standing collaboration with the laboratory of Dr. Lawrence Samelson (NCI) on the study of multi-protein interactions in signaling particles after T-cell activation. Jointly we have succeeded in establishing the formation of a large multi-protein complex by analytical ultracentrifugation, using the strategies of multi-signal sedimentation velocity previously developed in our laboratory. A detailed examination of this protein complex can elucidate the role of cooperativity and avidity in signal transduction, and help establishing a physical mechanism or principles governing this critical event in the engagement of the immune system. Related, we have continued our long-standing collaboration with the laboratory of Dr. David Margulies (NIAID) on the characterization of interactions of proteins constituting the peptide loading complex, and TCR/MHC interactions that trigger T-cell activation. We have also continued our collaboration with Dr. Dan Sackett (NICHD) on the study of tubulin dimer dissociation. Tubulin self-assembles into microtubules, which are critical for the cellular homeostasis and for cell division, and therefore tubulin has long been a major target for cancer drugs. By studing the distinct self-association properties in tubulin species from different tissues and organisms, we will gain more detailed insight in the dynamics of microtubule assembly. Since the dimerization is very high affinity, these studies rely on the use of the fluorescence detection in sedimentation velocity. A third major focus has been the study of the early events in HIV-1 virus capsid formation, in collaboration with Dr. Alan Rein (NCI), with the goal to better understand protein interactions and protein/nucleic acid interactions and, ultimately, their potential disruption with therapeutic molecules. To this end, we have studied different variants of the Gag protein binding to short oligonucleotides, and found evidence of previously unknown interaction mechanisms providing cooperativity in the capsid assembly. This study is also taking advantage of fluorescence detection in sedimentation velocity, and we have embarked on developing a fluorescence detector with improved optical design to achieve higher sensitivity and precision, and to allow the measurements of faster sedimentation processes viral particles. A poorly understood aspect of protein behavior in the cellular environment is the effect of weak interactions at the high intracellular concentrations. In order to better characterize weak interactions we have implemented new sedimentation approaches to measure second virial coefficients. We have started to apply this collaboratively, to eye crystallins in collaboration with the laboratory of Dr. Graeme Wistow (NEI). Weak interactions are also critical in the behavior of therapeutic proteins in formulation; accordingly we engaged in the study of interactions of therapeutic monoclonal antibodies in formulation buffers in collaboration with Dr. Jai Pathak (NIAID/VCR); and to characterize trace aggregation in antibody formulations in collaboration with Dr. Kang Chen (FDA). Other collaborative applications of AUC include studies of coated gold nanoparticles and their interactions with different serum proteins with Dr. Alioscka Souza (University of Sao Paolo), and the study of chaperone interactions with the laboratory of Dr. Sue Wickner (NCI).
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