Protein Stability, Folding/Unfolding, and Formation of D

摘要：人α -1蛋白酶抑制剂（A1-PI）是一种丝氨酸蛋白酶抑制剂，在体内抑制中性粒细胞弹性酶的活性，在25℃下，胍- hcl （Gu）诱导双相展开，中间状态在1.5 M Gu下出现短暂稳定（1-2小时），主要形成单体和二聚体中间体，进一步聚合更慢。Marszal博士用缓冲液稀释了这些较小的中间体，并观察到在折叠物种的混合物中，折叠的二聚体，而不是折叠的单体，进一步聚合。她通过MALDI-TOF测量证实了该物种是二聚体，并表明它是二硫连接的（通过每个单体中的单个巯基）。杜博士已经确定，大约80%的A1-PI单体具有游离巯基，二聚体没有表现出抑制活性。重要的是，Marszal博士已经证明，由纯化二聚体聚合产生的物种似乎是它的倍数，即没有形成三聚体。此外，一个与A1-PI的RCL序列相同的反应性中心环（RCL）肽通常靶向中性粒细胞弹性酶的活性位点，与单体结合，但似乎不与二聚体结合，从而阻止了单体中间体的聚合，而不是折叠二聚体的聚合。先前已经报道过这种肽对单体聚合的抑制作用，并在一定程度上用于支持一种聚合模型，该模型涉及将一个A1-PI分子的RCL插入相邻分子的β -片的中心链之间，从而形成一条线性链（即聚合的环片模型）。电子显微镜数据表明，纯化二聚体形成的聚合物是线性的珠状结构，类似于单体（在高温下加热）形成的结构，也类似于从分泌不良的A1-PI突变患者肝细胞包体体中分离出来的结构。二硫键二聚体的结构限制及其形成线性聚合物的能力很难与所提出的环片模型相协调。我们提出二硫键二聚体的形成可能涉及RCL埋藏，而不是插入，二聚体的聚合物可能通过暴露在二聚体外表面的β片的相互作用形成。这里提出的模型，涉及二硫键二聚体，是一种替代机制，因为在一定的反应条件下，不同的聚合机制可能会竞争。

Summary: Human alpha-1-proteinase inhibitor (A1-PI), which is a serine protease inhibitor and which in vivo inhibits the activity of neutrophil elastase, undergoes guanidine-HCl (Gu) induced biphasic unfolding with an intermediate state in 1.5 M Gu at 25 deg C. The intermediate state appears transiently stable (over a 1-2 h period) primarily with the formation of monomeric and dimeric intermediates that further polymerize more slowly. Dr. Marszal has folded these smaller intermediates by dilution with buffer and observed that in the mixture of folded species, folded dimer, but not folded monomer, further polymerizes. She has confirmed that this species is dimer by MALDI-TOF measurements and shown that it is disulfide linked (through the single sulfhydryl in each monomer). Dr. Du has established that ca. 80% of the A1-PI monomer has free sulfhydryl and that the dimer exhibits no inhibitory activity. Importantly, Dr. Marszal has demonstrated that the species generated by polymerization of the purified dimer appear to be multiples of it, i.e. no trimer is formed. Furthermore, a reactive center loop (RCL) peptide, which has the same sequence at that of the RCL of A1-PI, which normally targets the active site of neutrophil elastase, binds to the monomer, but appears not to bind to the dimer, thereby preventing the polymerization of the monomeric intermediate but not of the folded dimer. The inhibition of polymerization of monomer by the peptide has been previously reported and used, in part, to support a model of polymerization involving the insertion of the RCL of one A1-PI molecule between the central strands of a beta-sheet on an adjacent molecule thereby forming a linear chain (i.e. a loop-sheet model for polymerization). Electron micrograph data demonstrate that polymers formed from purified dimer are linear bead-like structures that are similar to those formed from monomer (by heating at elevated temperature) and those isolated from inclusion bodies in hepatocytes from patients with a mutant A1-PI that is poorly secreted. The structural constraints of the disulfide linked dimer and its ability to form linear polymers are difficult to reconcile with the proposed loop-sheet model. We propose that formation of the disulfide linked dimer may involve RCL burial, not insertion, and that polymers of the dimer may form by interaction of beta-sheets exposed on the outer surfaces of the dimer. The model proposed here, involving disulfide linked dimer, is an alternate mechanism since under certain reaction conditions, different mechanisms of polymerization may compete.