The central theme of this research project is the design, chemical synthesis, characterization and collaborative application of new probes and reagents for studying biological systems. Target molecules are designed in response to the current needs and limitations of researchers and practitioners in the fields of biochemistry, molecular biology, cell biology and medicine. Five sub-areas form the focus of this proposed five-year project. a) Novel photochemically triggered cross-linking reagents. Cross-linking reagents provide valuable structural and mechanistic information at the molecular level about near-neighbor interactions between two biomolecules and may, for example, provide a basis for understanding how complex biomolecular assemblies function. The development of a new class of fluorinated, cleavable, photochemically triggered cross-linking reagents expected to have superior properties is proposed. These molecules will be used in a collaborative study with Dr. Capaldi to study the mechanism of action of the important enzyme F(o)F(1)-ATP synthase. b) Photolabeling within the hydrophobic cavity of mutated T4-lysozyme. Photolabeling is one of the most fruitful ways to gain information about the protein binding site of small molecules. Dr. Matthews has very recently described a novel series of crystalline mutated T4-lysozymes having a well defined hydrophobic cavity that binds small molecules. The crystals provide a unique opportunity for studying the behavior of the new photolabeling reagents as they occupy a hydrophobic """"""""binding site."""""""" c) Substrates with new and unusual properties for biochemical and biophysical studies of phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC enzymes occupy a central role in cellular function, for example, the release of proteins anchored to the cell surface or the amplification of cellular signals such as the binding of a hormone molecule on the cell surface. To aid in collaborative biochemical and biophysical studies of the bacterial and mammalian enzymes with Dr. Griffith, novel substrates and inhibitors will be developed. d) Novel reagents designed to aid in the visualization of individual DNA molecules under water on an atomically flat surface by atomic force microscopy (AFM). The visualization of individual biomolecules by powerful imaging techniques is becoming a reality. However, only AFM appears to have, the potential to observe biomolecules in an aqueous environment in their native state. Dr. Bustamante has very recently published images of individual DNA molecules laying on a (atomically flat) mica surface under air or propanol-water. Imaging in water was not possible owing to motion of the DNA. Novel approaches toward pinning the DNA under water to the mica surface are proposed. e) A microbiosensor for inositol phosphate. Enzyme coupled field effect transistors (ENFETs) are undergoing rapid development as highly selective and sensitive microbiosensors with potential for intracellular applications. Dr. Wybourne and the principal investigator have recently immobilized enzymes on sub-micron structures fabricated with electron-beam lithography in Dr. Wybourne's microelectronics laboratory. The objective is to build an ENFET that will detect inositol phosphate, the hydrolysis product of the action PI-PLC on phosphadityl inositol.
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