Our long range goals are to derive complete mechanistic descriptions of the functional properties of hemoglobins and myoglobins, using site-directed mutagenesis, conventional rapid mixing and equilibrium methods, ultrafast laser photolysis techniques, vibrational and NMR spectroscopies, X-ray crystallography, and various computational methods. Our strategy is to use mammalian myoglobin (Mb) as a simple prototype for identifying the roles of specific amino acids, structural motifs, and stereochemical effects in regulating O2 binding and protein stability. The resultant ideas will then be tested in the alpha and beta subunits of recombinant human hemoglobin and other heme proteins that function in O2 transport, storage, and sensing.
Six specific aims are proposed: (1) to test the distal electrostatic mechanism for ligand discrimination in myoglobin; (2) to examine proximal mechanisms for regulating O2, CO, and NO binding; (3) to map out specific pathways for ligand movement into and out of myoglobin; (4) to determine quantitatively the structural compromises between globin stability and physiological function; (5) to establish an allosteric kinetic mechanisms for O2 binding to recombinant human deoxyhemoglobins; and (6) to evaluate the different stereochemical factors that control ligand binding to the alpha and beta subunits of recombinant human hemoglobin. The significance of this work is four-fold. First, the mechanisms involved in regulation of O2 affinity, discrimination against CO binding, cooperativity, and inhibition of oxidation are applicable to all heme proteins. Second, our library of Mb and Hb mutants provides the testing ground for developing computational methods to predict heme protein structure and function. Third, recombinant myoglobin and hemoglobins are excellent model systems for understanding the evolutionary compromises between protein expression, stability, and function. Fourth, these basic biophysical studies provide rational strategies for designing efficient and safe hemoglobin-based 02 delivery pharmaceuticals.
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