The purposed of this Shared Instrumentation grant proposal is to purchase a Biosensor BIAcore 2000 instrument for a group of seven investigators in the Department of Vascular Biology at The Scripps Research Institute. These investigators are studying protease and protease inhibitor structure and function in the vascular system, integrin structure and function, apolipoprotein structure and function, pro-hormone processing, and gene regulation. The areas of investigation of this group of investigators of this group of investigators are relevant to both physiological and pathological processes which include thrombolysis, atherosclerosis, myocardial infarction, obesity, cancer, hypertension, neuronal function, development and wound healing. These studies emphasize protein-protein and protein-DNA interactions. Since several of these investigations are studying either the same or related molecules, we will able to draw on the experience of other members of the users group in designing experiments to be performed on the BIAcore. The BIAcore 2000 instrument will allow us to directly observe and measure the binding affinities and association/dissociation kinetics of molecular interactions in real time, thus greatly accelerating the process of identifying and characterizing both protein-protein and protein-DNA interactions. The BIAcore instrument contains an immobilized ligand on a sensor chip and can monitor the adsorption of unlabeled proteins in free solution to the chip by means of surface plasmon resonance detection by changes in the refractive index in the vicinity of the surface of the chip. These changes are directly proportional to the change in adsorbed mass, enabling characterization of biomolecular interactions. The bound material can also be eluted and recovered from the sensor chip, thus allowing identification of previously unknown molecular interactions. This instrument can also be used to screen phage display libraries which express scFv sequences, as well as to characterize known protein-protein or protein-DNA interactions. The BIAcore 2000 will be used to support ongoing NIH-funded projects of the major users, and will substantially enhance the productivity of these research efforts.
