This project will address several fundamental issues relevant to structure-function relationships in monomeric hemoglobins and myoglobins. These proteins are of functional importance in the storage and transport of oxygen in a wide variety of organisms. They are the simplest proteins capable of reversible oxygenation and are models of the much larger and more complex vertebrate hemoglobins. The long term goal of the project is to use NMR methods to delineate the structural and dynamic differences between a series of monomeric hemoglobins which vary widely in the kinetics of their reactions with 02 and CO and to investigate the changes in structure and dynamics which accompany ligand binding. This research will provide completely new insights into the structural and dynamic factors, and the coupling between them, which control ligand binding reactions in hemoglobins. Detailed compairsons between myoglobin (Mb) and the very high affinity leghemoglobin (Lb) are of particular importance. The three- dimensional structure of LbCO in solution will be determined using state-of-the-art NMR method. Structural changes induced by binding of ligands (02, CO and isonitriles) to Mb, Lb, human Hb alpha-chains and Glycera dibranchiata monomeric hemoglobins will be determined. These studies will identify the static structural changes which may influence ligation kinetics. The 5- coordinate zinc protoporphyrin derivatives of these proteins will be used as diamagnetic analogues of the deoxy state. Amide proton exchange and 13C relaxation measurements on various complexes of Mb and Lb will be undertaken to identify functionally-important differences in protein dynamics and to determine the influence of the bound ligand on protein motions. Two-dimensional NMR spectroscopy will be used extensively. Structures will be calculated from nuclear Overhauser effect data and ring current shifts using both distance geometry and molecular dynamics methods. Amide proton exchange measurements will provide information on protein flexibility and side chain and backbone dynamics will be studies by 13C relaxation measurements.
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