Several types of nmr data depend on internuclear distances and the details of molecular motion. These include spin-lattice relaxation rates, spin-spin relaxation rates and nuclear Overhauser effects. A number of technical and interpretational impediments to the application of these experiments in protein systems can be overcome by introduction of fluorine atoms into the protein structure; this """"""""fluorine labeling"""""""" can be accomplished by covalent modification of amino acid residues or by biosynthetic incorporation of fluorine-containing amino acids into protein structures. In favorable circumstances, such nmr experiments may (1) identify the type(s) of amino acid residues near the fluorine nucleus, (2) provide indications of the distances between these amino acids and fluorine nuclei, and (3) produce estimates of the time scales for conformational motion near a fluorine nucleus. We propose to use fluorine nmr spectroscopy to elucidate details of protein structures, focusing on (1) serine proteases, including chymotrypsin, trypsin and elastase, (2) hemoglobin and cytochrome c of the rabbit and (3) carbonic anhydrase from rabbit. Serine proteases are essential in a wide variety of processes; extensive crystallographic data on these enzymes do not address the dynamical nature of their three-dimensional structures as related to catalytic action. We hope to examine the structural correlates of those factors which regulate the oxygen-binding ability of hemoglobin in solution. Cytochrome C will be examined to address the question of the extent of structural perturbation by fluorine substitution. The experimental methods which provide the samples of hemoglobin used in the proposed work should also provide sufficient samples of carbonic anhydrase for fluorine nmr experiments and studies of this enzyme, which is important in release of carbon dioxide from respiring organisms, will be carried out. In support of these studies, new nmr experiments for identification of amino acids which interact with fluorine nuclei in protein will be developed and analyzed theoretically. The proposed work will help define the scope and limitations of the fluorine nmr experiments discussed as well as provide new details about the structure and dynamics of the proteins studied.
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