The central theme of the principal investigator's NIH-supported research is the design, chemical synthesis, characterization, and collaborative application of new probes and reagents for studying biological systems. The target molecules are designed in response to the current needs and limitations of researchers and practitioners in the fields of biochemistry, molecular and cell biology, and medicine. The project involves a collaboration with two outstanding research groups at the U of O: Drs. C. Bustamante and O.H. Griffith, Chemistry department and Institute of Molecular Biology. Two sub-areas form the focus of this proposed five-year project. a) Novel substrates and inhibitors for biochemical and biophysical studies related to phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC enzymes are a receptor-controlled family that are centrally important in the amplification of cellular signaling processes. Additionally, bacterial PI-PLC catalyzes the cleavages of cell surface proteins attached to the membrane by way of a glycosylphosphatidylinsoitol (GPI) anchor. The GPI anchor cleaving activity is of importance to medicine as diagnostic tools for the analysis of proteins presented on the outer surface of cell membranes. Anchored proteins include activation antigens of the immune system, adhesion molecules, scrapie prion proteins and the carcinoembryonic antigen, a human tumor marker. The significance of our PI-PLC work lies in providing powerful new tools for studying the structure (inhibitors bound at the active site of the crystalline enzymes) and mechanism of action (i.e., novel chromogenic, fluorogenic or chemiluminescent substrates) of the PI-PLCs in their central role in cellular signal transduction. Links to cancer and Alzheimer's disease have also been reported for the PI-PLCs. b) Novel reagents for atomic force microscopy (AFM). The direct visualization of individual biomolecules in their native state in buffer is being achieved using the relatively new and powerful technique of AM. The resolution limit with biological specimens is typically 50-100 A and is limited by the sharpness of the tip. The sharpest tips available are carbon tips with a radius of curvature of about 100 A.
One aim i s to design and synthesize chemically well-defined tips that taper to a single atom, thus approaching the ultimate time of resolution. The investigators also address limitations in a second application of AFM, namely functional group imaging (FGI). FGI provides direct information about the different chemical groups at the surface and has important ramifications in fields as diverse as ligand-receptor interactions, adhesion, lubrication, and molecular machining, the latter with enzymes immobilized on the tip.
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