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蛋白酶的活性，在25℃的1.5MGu中经历盐酸胍(Gu)诱导的两相展开，中间状态表现为瞬时稳定(在1-2小时内)，主要是形成单体和二聚中间体，进一步聚合速度较慢。Marszal博士通过用缓冲液稀释来折叠这些较小的中间体，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，并表明它是二硫键连接的(通过每个单体中的单个巯基)。杜博士已经证实，约80%的A1-PI单体含有游离基，二聚体没有抑制活性。重要的是，Marszal博士已经证明，由纯化的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，与A1-PI的RCL具有相同序列的反应中心环(RCL)肽与单体结合，但似乎不与二聚体结合，从而阻止单体中间体的聚合，但不阻止折叠的二聚体的聚合。先前已报道该肽对单体聚合的抑制，并部分地用于支持聚合模型，该聚合模型涉及将一个A1-PI分子的RCL插入到相邻分子上的β-折叠的中心链之间，从而形成线性链(即用于聚合的环状折叠模型)。电子显微镜数据表明，由纯化的二聚体形成的聚合物是线性珠状结构，类似于由单体(通过高温加热)形成的聚合物，以及从突变的A1-PI分泌不良的患者的肝细胞中分离出来的聚合物。二硫键连接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一致。我们认为，二硫键二聚体的形成可能涉及RCL的埋藏，而不是插入，二聚体的聚合物可能是通过暴露在二聚体外表面的β-折叠相互作用而形成的。由于在一定的反应条件下，不同的聚合机理可能相互竞争，所以本文提出的包含二硫键连接的二聚体的模型是另一种机理。

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.