The major objectives are: (i) to understand the molecular basis of oxygen transport in human normal and abnormal erythrocytes; (ii) to understand the molecular and cellular basis of sickle cell anemia; and (iii) to use hemoglobin (Hb) as a model for investigating the effects of single-site structural modifications on the conformation and dynamics of proteins in solution. The principal biophysical technique used in the proposed research on human hemoglobins and erythrocytes is nuclear magnetic resonance (NMR) spectroscopy. High-resolution NMR (1H, 13C, 15N, and 31P nuclei), nuclear relaxation, nuclear Overhauser effect, 2-dimensional NMR, and modern solid-state NMR techniques are being used in our proposed research. In addition, the technique of semisynthetic reincorporation of specific 13C- (and/or 15N-) labeled amino acids to the amino- and carboxyl-terminal residues of both Alpha and Beta chains of human normal and abnormal hemoglobins including sickle cell hemoglobin (Hb S) will be used to prepare appropriate 13C- (and/or 15N-) labeled hemoglobins needed for our NMR studies. The proposed NMR experiments for the Hb molecule will be designed to gain information about the structural and dynamic properties of important amino acid residues involved in the cooperative oxygenation process and the Bohr effect, structures, dynamics, and properties of partially ligated species, the structural differences of the Hb molecule in the solution and crystalline states, the intermolecular contact sites in the Hb S polymer, and the binding sites of antisickling compounds to Hb S. The proposed research on sickle cell anemia will provide an opportunity to correlate cellular and molecular properties of Hb S erythrocytes and also new insights for a rational management of the disease including the design of suitable antisickling drugs. The proposed NMR experiments for the erythrocytes will be designed to gain information about the structures, properties, and dynamics of hemoglobins inside the intact red blood cells, intracellular environments and metabolism in normal and abnormal erythrocytes including sickle cell erythrocytes. An excellent way to correlate the structure-function relationships in proteins is to combine the information obtained from X-ray crystallography, NMR spectroscopy, and genetics. This proposed research is a demonstration of the power of this approach. A unique feature of this proposed research is to compare the structures and properties of human normal and abnormal hemoglobins in purified form in solution and in crystalline states as well as inside intact erythrocytes.
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