Design of Alpha-Chains to Fully Neutralize HbS Polymerization

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The assembly of the quinary structure of involves the cooperative integrations of the noncovalent inter tetrameric interactions of multiple intermolecular contact sites, and there is a significant additive and/or synergistic effect between various sets of intermolecular contact sites. The earlier studies with human-nonhuman and nonhuman-nonhuman chimeric alpha-chains carrying a set of a sequence differences of the intermolecular contact have established the perturbation of such additive/synergistic interactions leads to the generation super-inhibitory alpha-chains. The set of linked- sequence differences of the contact sites present in pig alpha-chain and human-pig chimeric alpha-chain are enough to completely neutralize the Val-6(beta) dependent polymerization reaction. We hypothesize that we can identify these linked-contact site sequence differences and transplant these into human alpha-chain and endow with the polymerization neutralizing potential. We will increase the data base of polymerization inhibitory/neutralizing potential of linked-sequence differences of contact sites by assembling additional interspecies Beta-s hybrids (using alpha-chains of dog, cat and chicken, and the chimeric alpha-chains of these species) and identify multiple sets of linked sequence differences of the contact sites that are likely to neutralize the polymerization potential of the Beta-s-chains.These sequence differences will be grafted into human alpha-chain using modular construction approach that will involve chemical/enzymic ligation of four segments of alpha-globin with desired sequence differences as the primary approach, and site directed mutagenesis as the back up approach. If the transplanted sequences differences fail to endow the human alpha-chain the full polymerization inhibition and/or neutralization parent non-human or chimeric alpha-chain, these will be fine tuned by engineering additional contact site sequence differences. The linkage map of the interaction of contact sites the sequence differences of which neutralize the Beta-s polymerization potential, and functional complementation of these sequence differences from the cis and/or trans positions will be delineated by designing and assembling new alpha-chain constructs with subsets of the sequence differences.