PROTEIN STABILITY AND FOLDING/UNFOLDING AND FORMATION OF PRIONS AND OTHER DELETER

蛋白质稳定性以及朊病毒和其他删除子的折叠/解折叠和形成

• 批准号: 6293806
• 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/6293806
• 关键词: anions; bromine; calorimetry; chemical kinetics; chemical stability; chlorine; iodine; ionic strengths; protease inhibitor; protein denaturation; protein folding; protein structure function; serum albumin; thermostability

Human alpha-1-proteinase inhibitor (A-1-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. Previously, we demonstrated that this intermediate state can consist of one or more folding intermediates. Size exclusion-HPLC in 1.5 M Gu shows that an equilibrium distribution of monomeric and polymeric intermediates forms over 2 h at 25 deg C. These polymeric intermediates are primarily dimeric, trimeric, and tetrameric with some higher polymers and are of a loop-sheet nature (i.e the reactive site loop, which normally targets the active site of elastase, is inserted between the central strands of a beta sheet region on another A-1-PI molecule). Folding by dilution from 1.5 M Gu gives levels of active monomer and inactive polymer that reflect those of the intermediates. Kinetic studies demonstrate that this equilibrium state is on the folding pathway and that folding from the fully unfolded state is under kinetic control thereby allowing the formation of the less stable but active monomer. Since starting in January, Dr. Marszal has discovered a much slower, higher order aggregation that occurs at 25 deg C over a period of days after the relatively rapid polymerization observed in 1.5 M Gu. This slower aggregation is observed both for the intermediates in 1.5 M Gu and for monomer and polymers formed from folding from 1.5 M Gu although the latter is slower. The polymer that forms elutes in the void volume of tandem TSK 3000 columns. Dr. Marszal has demonstrated that this high-order aggregate may be dissociated to monomer with high Gu. She is in the process of characterizing the nature of this high-order aggregate, which may not be of a loop-sheet nature. An interesting possibility is that the rapid polymerization in 1.5 M Gu may relate to the secretion defect in patients with mutant A-1-PI and the slower high-order aggregation to the formation of inclusion bodies of this mutant A-1-PI in the hepatocytes, the site of synthesis. In determining the specific inhibitory activity (potency) of samples of A-1-PI, Dr. Marszal utilized trypsin as the target protease, for which she accurately measured the concentration of the active enzyme with a chromogenic active site titrant. She has observed that at low trypsin and low A-1-PI concentrations, the binding of A-1-PI is not stoichiometric thereby indicating that the inhibition constant is lower than assumed. Thus, she plans to determine this constant in order to establish optimal conditions for determining the concentration of active A-1-PI thereby allowing an accurate redetermination of the active concentration of the CBER A-1-PI Reference Standard, which currently is estimated to have approximately 75% of its originally labeled potency. The stability of this standard will be monitored in terms of potency.

人α-1-蛋白酶抑制剂（A-1-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。 以前，我们证明了这种中间状态可以由一个或多个折叠中间体组成。 在1.5 M Gu中的尺寸排阻-HPLC显示在25 ℃下在2 h内形成单体和聚合物中间体的平衡分布。这些聚合物中间体主要是二聚体、三聚体和四聚体，具有一些更高的聚合物，并且具有环-片层性质（即，通常靶向弹性蛋白酶活性位点的反应性位点环插入另一个A-1-PI分子上β片层区域的中心链之间）。 通过从1.5 M Gu稀释折叠得到反映中间体的活性单体和非活性聚合物的水平。 动力学研究表明，这种平衡状态是在折叠途径上，并且从完全未折叠状态的折叠是在动力学控制下的，从而允许形成不太稳定但具有活性的单体。 自1月份开始，Marszal博士发现了一种更慢，更高阶的聚集，在1.5 M Gu中观察到相对快速的聚合后，在25 ℃下发生了一段时间。 对于1.5 M Gu中的中间体和从1.5 M Gu折叠形成的单体和聚合物都观察到这种较慢的聚集，尽管后者较慢。 形成的聚合物在串联TSK 3000柱的空隙体积中洗脱。 Marszal博士已经证明，这种高阶聚集体可以解离为具有高Gu的单体。 她正在描述这种高阶聚集体的性质，这种聚集体可能不具有回路表的性质。 一个有趣的可能性是，在1.5 M Gu中的快速聚合可能与突变A-1-PI患者的分泌缺陷有关，并且在肝细胞中，合成位点，这种突变A-1-PI的包涵体形成的较慢的高阶聚集有关。 在测定A-1-PI样品的特异性抑制活性（效价）时，Dr. Marszal使用胰蛋白酶作为靶蛋白酶，她使用显色活性位点滴定剂准确测定了活性酶的浓度。 她观察到，在低胰蛋白酶和低A-1-PI浓度下，A-1-PI的结合不是化学计量的，从而表明抑制常数低于假设的。 因此，她计划测定该常数，以建立测定活性A-1-PI浓度的最佳条件，从而准确重新测定CBER A-1-PI参比标准品的活性浓度，目前估计其效价约为其原始标记效价的75%。 将根据效价监测该标准品的稳定性。