This proposal describes the use of recombinant DNA (for single-site substitutions or for regional substitutions of larger segments and novel chemical modifications to study sickle hemoglobin (HbS) polymerization. The important regions of HbF, of current clinical interest and of HbA2, in inhibition of HbS polymerization and their relative strengths will be evaluated. Our recent finding of the 70-fold lower tetramer-dimer dissociation constant of HbF compared to HbS, HbA and HbA2 will be expanded. Studies will be continued on a hybrid HbA/F containing the amino acids of the a1gamma1 and alpha1gamma2 interfaces of HbF and the remaining sequence of the beta-chain. The mechanism by which the subunit interface properties are influenced by other parts of the sequence will be studied. A candidate is helix A (N-terminal amino acids 1-18) of the chain whose contribution both to subunit interface properties and to HbS polymerization will be evaluated. The a source of the proton uptake for both HbA and HbF dissociating to dimers will be investigated by mutagenesis of potential sites common to both Hbs. Dimer to monomer dissociation will continue to be studied with the aid of a recombinant Hb with a substitution in the alpa1beta1 interface. Inhibition of polymerization with recombinant HbS mutants will focus on obtaining quantitative information on the contributions of sites known to be at contact points between tetramers, either inter or intra-strand. We propose to establish which sites function independently and whether they are at lateral or axial contacts in order to identify them as important targets for anti-sickling agents. The second region that we propose to study is the central cavity where we will identify the most important sites in keeping HbS in the more open (sickling) conformation. A novel class of chemical anti-polymerization inhibitors directed at the central cavity will be synthesized based on familiar chemistry and tested to maintain the central cavity of HbS less open (non-sickling). Certain recombinant mutants with either selective or regional substitutions will be supplied to our collaborators for crystallographic studies (Rover), electron microscopy (Josephs) or kinetics of polymerization (Ferrone).
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