XL-A Analytical Ultracentrifuge for the Analysis of Protein-Protein Interactions

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This is a proposal to acquire a Beckman Instruments Optima Series XL-A Analytical Ultracentrifuge equipped with UV scanner and Rayleigh optical systems. This instrument will replace 2 heavily used Beckman Model-E's in the Analytical Ultracentrifuge Research Laboratory (AURL) at Boston Biomedical Research Institute (BBRI). One of the Model-E's is currently equipped with a real-time video-based Rayleigh optical system, developed by the PI, that is being used heavily in sedimentation velocity and sedimentation equilibrium analyses. The rapid acquisition Rayleigh system has allowed the efficient application of the high precision time derivative method, also developed by Dr. Stafford, for sedimentation velocity analysis achieving a 100-1000 fold increase in the sensitivity of the ultracentrifuge optics. The Model-E's have become increasingly difficult to maintain and it is unlikely that Beckman Instruments will continue to support them after they introduce the Rayleigh optical system in late 1995. Many researchers at BBRI as well as many outside collaborators have benefited from the ultracentrifuge facility over the years. The continued viability of the AURL requires the acquisition of a new XL-A equipped with Rayleigh optics. The instrument will be shared by seven major research groups at BBRI and will be used in several on-going or planned outside collaborations. Some of these researchers have benefited from the facility in the past. Others are either currently or will in the future use the ultracentrifuge facility to investigate various protein-protein and protein-nucleic acid interactions. These include but are not limited to: P. and A. Wang, structure, molecular weight of Caldesmon and the affect of phosphorylation on its interactions with actin; T. Tao, structure and molecular weight of calponin; future studies will involve studies of the interaction of calponin with actin; T Tao, calcium dependence of interactions between troponin I and troponin C; R Lu, interactions of myosin regulatory and essential light chains with the cloned regulatory domain of the myosin Sl head; S.S. Lehrer, interactions of calponin and caldesmon with tropomyosin, and studies of the end-to-end polymerization properties of various cloned fragments of tropomyosin; H. Wohlrab, investigation of properties of the mitochondrial phosphate transport protein and interactions between it subunits; N. Sarkar, characterization of recombinant poly (A) polymerase I and poly(A) polymerase I and their interactions with primer RNA; Z. Grabarek, investigation of calcium dependent conformational changes in calmodulin and troponin-C and the calcium dependence of their interactions with target proteins; V. Raso, Targeting c-erbB-2 Epitopes with scFv Immunotoxins and Acid-Releasable Anti-Diphtheria Toxin (DT) Monoclonal Antibodies.