This proposed research addresses the inhibitory mechanisms of HbS polymerization by HbF, mechanisms of high oxygen affinity of HbF in red blood cells, and the mechanism of assembly of alpha and gamma chains of hemoglobin to form fetal hemoglobin using a variety of biochemical and biophysical methods. Using the results from these studies, the investigators hope to design novel HbF anti-sickling variants for eventual use in gene therapy in sickle cell disease.
In Specific Aim 1, amino acid residues in HbF will be identified which are critical for FS hybrid formation. Preliminary X-ray analyses of copolymers indicates that Val-beta 6 in HbS communicates with the acceptor pocket in the adjacent molecule and that Thr-beta 87 plays an important role through a bridge of interfacial water which strengthens the hydrophobic interaction. Further studies involving beta and gamma 87 are proposed.
In Specific Aim 2, studies will be done to characterize oxygen binding and polymerization properties of Hb F and S mixtures. The goal of these studies is to confirm differential oxygen binding of F mutants with 2,3-DPG effects similar to those of HbA. In preliminary studies, such mutants have been produced (Hb F gamma G1V, E5P, S143H). Studies in this aim will further characterize these mutants, including X-ray analyses, oxygen binding studies and copolymerization measurements.
In Specific Aim 3, studies will be undertaken to characterize the assembly of alpha and gamma chains to form HbF as well as the effect of mutant gamma chains on the assembly of alpha and beta-S chains. Mutants are proposed which will lead to the understanding of the residues which are critical to the understanding of the control of rates of these assemblies, with the overall goal to increase the efficiency of HSF hybrid hemoglobin. Specific locations for these mutants will be at the chain interfaces, but also gamma chain surface charge.
In Specific Aim 4, the investigators propose to use the results of the first three aims to design new and novel HbF variants which will have optimal properties of oxygen affinity and coassembly with beta-S chains to form HbFS hybrids. The ultimate goal of this aim is to specify the optimal gene product for gene therapy in patients with sickle cell anemia. In that regard, the investigators also point out that efficient expression is essential.
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