Design of Alpha-Chains to Fully Neutralize HbS Polymerization

五元结构的组装涉及多个分子间接触位点的非共价四聚体相互作用的协同整合，不同组的分子间接触位点之间存在显著的加性和/或协同效应。早期对人类-非人类和非人类-非人类嵌合α -链的研究表明，这种加性/协同作用的扰动导致了超抑制性α -链的产生。猪α -链和人-猪嵌合α -链中存在的一组接触位点的连锁序列差异足以完全中和Val-6（β）依赖性聚合反应。我们假设我们可以识别这些连接-接触位点序列差异，并将其移植到人类α链中，并赋予聚合中和电位。我们将通过组装额外的种间β -s杂交体（使用狗、猫和鸡的α链以及这些物种的嵌合α链）来增加接触位点连锁序列差异的聚合抑制/中和电位数据库，并确定可能中和β -s链聚合电位的多组接触位点连锁序列差异。这些序列差异将使用模块化构建方法嫁接到人类α -链中，该方法将涉及将α -珠蛋白的四个片段以所需的序列差异作为主要方法进行化学/酶连接，并将位点定向诱变作为备用方法。如果移植的序列差异不能赋予人类α链完全的聚合抑制和/或中和亲本非人类或嵌合α链，这些将通过工程额外的接触进行微调

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.