Protein Stability, Folding/Unfolding, and Formation of D

蛋白质稳定性、折叠/解折叠和 D 的形成

• 批准号: 6680015
• 负责人: ANDREW F SHRAKE
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6680015
• 关键词: chemical stability; chemical standardization; dimer; disulfide bond; elastases; electron microscopy; glycoproteins; matrix assisted laser desorption ionization; model design /development; molecular site; physical model; protease inhibitor; protein denaturation; protein folding; protein structure function; structural biology

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.

总结：人α-1-蛋白酶抑制剂（A1-PI）是一种丝氨酸蛋白酶抑制剂，在体内抑制嗜中性粒细胞弹性蛋白酶的活性，在25 ℃下，在1.5 M Gu中经历胍-HCl（Gu）诱导的具有中间状态的双相解折叠。该中间状态似乎短暂稳定（超过1-2小时的时间），主要是形成单体和二聚体的中间体，进一步更缓慢地分解。 Marszal博士通过用缓冲液稀释来折叠这些较小的中间体，并观察到在折叠物质的混合物中，折叠的二聚体而不是折叠的单体进一步聚合。 她已通过MALDI-TOF测量证实该物质为二聚体，并显示其为二硫键连接（通过每个单体中的单个巯基）。 杜博士已经证实了这一点。80%的A1-PI单体具有游离巯基，并且二聚体没有表现出抑制活性。 重要的是，Marszal博士已经证明，纯化二聚体聚合产生的物质似乎是其倍数，即没有形成三聚体。 此外，具有与A1-PI的RCL相同的序列的反应中心环（RCL）肽（其通常靶向嗜中性粒细胞弹性蛋白酶的活性位点）与单体结合，但似乎不与二聚体结合，从而防止单体中间体而不是折叠二聚体的聚合。 先前已经报道了肽对单体聚合的抑制作用，并部分用于支持聚合模型，该模型涉及将一个A1-PI分子的RCL插入相邻分子上β-折叠的中心链之间，从而形成线性链（即，用于聚合的环-折叠模型）。 电子显微镜数据表明，从纯化的二聚体形成的聚合物是线性珠状结构，类似于从单体（通过在高温下加热）形成的那些和从来自具有分泌不良的突变体A1-PI的患者的肝细胞中的包涵体分离的那些。 二硫键连接的二聚体的结构限制和其形成线性聚合物的能力难以与所提出的环片模型相协调。 我们提出，形成的二硫键连接的二聚体可能涉及RCL埋葬，而不是插入，和二聚体的聚合物可能形成的β-折叠暴露在二聚体的外表面上的相互作用。 这里提出的模型，涉及二硫键连接的二聚体，是一种替代机制，因为在某些反应条件下，不同的聚合机制可能会竞争。