Protein Stability and Folding/Unfolding and Formation of Prions and Other Delet

蛋白质稳定性以及朊病毒和其他缺失的折叠/解折叠和形成

• 批准号: 6433595
• 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/6433595
• 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 (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 1.4 M Gu. Previously, we demonstrated that in 1.4 M Gu over 1 h at 23 deg C an apparent equilibrium forms among a monomeric and polymeric intermediates. More recently, with highly purified A1-PI, Dr. Marszal has shown that the apparent equilibrium involves a monomeric and a dimeric intermediate only and that the folded dimer slowly polymerizes to form high molecular weight polymers, which appear to be multimers of dimer. In the presence of folded monomer, this dimer polymerizes with no apparent change in concentration of monomer thereby indicating that dimer is, in fact, is the polymerizing unit. A long proposed model of polymerization hypothesizes the insertion of the flexible, reactive site loop of A1-PI into a beta sheet of a second molecule and so on, i.e. loop-sheet (or head-to-tail) polymerization of monomer, to form linear polymers. To explain our observations, we now propose that the dimer is a symmetrical, head-to-head dimer, possibly of a reciprocal loop-sheet nature, that polymerizes to form linear polymers by tail-to-tail contact presumably of identical hydrophobic patches (possibly beta sheet structures) on the surface of the dimer. The loop-sheet model of polymerization was proposed partly on the basis of studies of thermally induced polymerization. Dr. Marszal has preliminary, limited proteolysis data on Gu-induced and heat-induced dimers that indicate different structures for these two dimers thereby suggesting that the mechanisms of formation may be different also. Since starting as a Visiting Associate in August 2000, Dr. Du has begun a program to establish the potency and stability of the CBER reference standard for A1-PI, which was manufactured under contract in 1988 but which has never been utilized since the potency is somewhat lower than the labeled value.

人α-1-蛋白水解酶抑制物(A1-PI)是一种丝氨酸蛋白酶抑制物，体内抑制中性粒细胞弹性蛋白酶活性，在1.4MGu中经历了盐酸胍(Gu)诱导的双相展开。以前，我们证明了在1.4MGu中，在23℃，1h以上，单体和聚合物中间体之间形成了表观平衡。最近，Marszal博士用高纯度的A1-PI证明了表观平衡只涉及单体和二聚中间体，折叠的二聚体缓慢聚合形成高分子量聚合物，似乎是二聚体的多聚体。在折叠单体的存在下，这种二聚体在单体浓度没有明显变化的情况下聚合，从而表明二聚体实际上是聚合单元。长期以来提出的聚合模型假设A1-PI的柔性活性中心环插入第二个分子的β片层，等等，即单体的环片层(或头到尾)聚合，以形成线形聚合物。为了解释我们的观察，我们现在提出二聚体是对称的，头对头的二聚体，可能是相互环片性质的，通过尾部到尾部的接触聚合形成线性聚合物，推测是在二聚体表面上相同的疏水斑块(可能是β片层结构)。在对热诱导聚合研究的基础上，提出了部分环片聚合模型。Marszal博士有关于Gu诱导的和热诱导的二聚体的有限的初步蛋白质分解数据，这些数据表明这两个二聚体的结构不同，从而表明形成机制也可能不同。自从2000年8月开始担任客座助理以来，杜博士开始了一项计划，以建立A1-PI的CBER参考标准的效力和稳定性，该参考标准于1988年按合同制造，但由于效力略低于标签值而从未使用过。