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点的非共价四聚体间相互作用的协同整合，并且在不同的分子间接触点之间存在显著的相加和/或协同效应。早期对携带一组分子间接触序列差异的人-非人和非人-非人嵌合α链的研究已经证实，这种相加/协同作用的扰动导致了超抑制α链的产生。猪α链和人-猪嵌合α链中存在的一组接触点的连接序列差异足以完全中和Val-6(β)依赖的聚合反应。我们假设我们可以识别这些连接接触位点的序列差异，并将这些差异移植到人的阿尔法链中，并赋予聚合中和潜力。我们将通过组装额外的跨物种β-S杂交体(使用狗、猫和鸡的阿尔法链，以及这些物种的嵌合阿尔法链)来增加接触位点连锁序列差异的聚合抑制/中和潜力的数据库，并确定可能中和β-S链聚合潜力的多组接触位点的连锁序列差异。这些序列差异将通过模块化构建方法嫁接到人类阿尔法链中，该方法将以所需序列差异的四段α-珠蛋白的化学/酶连接为主要方法，并以定点突变为备用方法。如果移植的序列差异不能赋予人的阿尔法链完全的聚合抑制和/或中和亲本非人或嵌合的阿尔法链，这些将通过工程的额外接触来微调 站点序列差异。通过设计和组装带有子集的新的阿尔法链结构，将描绘其序列差异中和β-S聚合势的接触位点相互作用的连锁图，以及这些序列差异与顺式和/或反式的功能互补 序列差异。

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.