Acquisition of Biophysical Instrumentation

Request is made in this application for Biophysical Instrumentation for the characterization of proteins. The instruments are: a BIALite biosensor (Pharmacia), Isothermal Titration and Differential Scanning Calorimeter (MicroCal), Dynamic light scattering instrument (Protein Solutions, DLS), Stopped flow attachment (SLM, SF), and Image Analyzer (Appligene, IA). These state-of-the-art instruments allow characterization of the size, shape, and interactions of proteins in rigorous, thermodynamic ways not previously readily accessible. The group of instruments complement each other and represent a broad, unified approach to studying the physical chemical properties of interesting and important proteins. The Biosensor directly measures protein-protein or ligand-protein interactions in real-time. The Calorimeter measures the thermodynamics of ligand binding (ITC) and denaturation (DSC) of proteins. The DLS provides direct determination of the hydrodynamic radius of native proteins in solution. The SF attachment will enable fluorometric kinetic measurements in the msec region. The IA will facilitate quantitation of proteins on gels for purity assessment, interaction analysis, record keeping, and publications. The major focus of the group of four investigators applying for these instruments is the study of function related to structure in a diverse set of proteins. The requested instruments are needed by this group of senior and junior investigators to strengthen the physical chemical basis of the structural studies that are being done. The department currently contains a protein sequencing core facility, a molecular graphics computing center, and a spectroscopic laboratory (circular dichroism and flourescence). The five new instruments will complement these facilities and enable the goals of characterization of proteins and protein-protein interactions to be greatly enhanced with this modern technology. The four protein systems under study are: nerve growth factor (NGF), its receptors, and related neurotrophins; phosphofructokinase (PFK) from several species; yeast mitochondrial ATP synthase; and isocitrate dehydrogenase (IDH) with evolutionarily related dehydrogenases. All four of the investigators heavily utilize site specific mutagenesis. The characterization of the molecular weight of the native state (by DLS) and the polypeptide chain (with the assistance of IA) of mutant proteins is extremely important for understanding the affects of individual amino acid residues on function. Direct determination of the thermodynamics of ligand binding to PFK and IDH will be greatly facilitated with the ITC calorimeter. Direct studies of association of NGF with its receptors and the association of the subunits of the ATP synthase will be possible with the Biosensor. Characterization of the stability of native and mutant proteins in all four systems willbe possible with the DSC calorimeter and complement previous results of the stability of NGF from so~vent denaturation. Rapid kinetic studies of ligand binding to IDH, PFK, and the NGF receptor will be achievable with the SF attachment to the currently existing SLM fluorometer and may be critical in the studies of the catalytic site of PFK, IDH, and ATP synthase. No other access to instrumention of this type currently exists at FUHS. These instruments will also be made available to other investigators in the school and will probably be more broadly used than currently described.