STRUCTURAL KINETIC STUDIES BY NUCLEAR MAGNETIC RESONANCE

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DESCRIPTION: The conformations of human ADP ribosylation factor 1 (ARF1) are to be studied by solution state NMR. Field cycling pure quadruple resonance will be applied for the first time to proteins, to permit direct spectroscopy of Zn++, Ca++, Mg++ and other species in interaction with their surroundings. ARF 1 is a small G protein implicated in formation and regulation of non-clathrin coated vesicles. ARF 1 is a member of a large complex called coatamer found on the surface of these vesicles. The GTP form of ARF 1 is required for coatamer formation, and the GDP form promotes vesicle fusion. The ARF proteins have an extra N-terminal sequence, which is helical and modified by myristoylation at the N-terminus. It is hoped to understand the forms of ARF 1 that exist in the cell, GDP- or GTP-ligated, and how they interact with phospholipid through the myristoyl group. Initially, the non-myristoylated protein will be studied in the GDP and GTP forms, and also in those forms lacking the distinctive ARF N-terminal sequence. It is also hoped to study the myristoylated forms of the protein by solubilizing it with a minimum amount of detergent. These proteins, and many others, contain ions such as those mentioned above playing roles as structural elements, active catalytic centers, or messengers. There has been almost no study of these ions directly by spectroscopy when bound to proteins. Pure quadruple resonance can determine the non-symmetric electron charge distribution around an ion, and be used to infer the degree of covalent interaction involved in its binding. It is proposed to develop a form of this spectroscopy that will be sufficiently sensitive for studies of frozen protein solutions and single crystals, thereby providing a completely new way to study changes in the surroundings of these ions directly. It will be based on field-cycling NMR, in which a frozen sample will be pneumatically moved into and out of a 500 MHz magnet many times in a single experimental run. This method has been demonstrated previously in studies of metal alloys and small molecules. Many other kinds of problems might also be studied by the method, such as bond orientations of substrates containing deuterons or 17O, or the spectroscopy of boron-containing inhibitors bound to proteins.