Design of Alpha-Chains to Fully Neutralize HbS Polymerization

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.