3-DIMENSIONAL RECONSTRUCTION OF ALPHA-2-MACROGLOBULIN

ALPHA-2-巨球蛋白的三维重建

• 批准号: 3296613
• 负责人: JAMES K STOOPS
• 金额: $ 11.36万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3296613
• 关键词: acidity /alkalinity; chemical binding; chymotrypsin; conformation; electron microscopy; image processing; macroglobulins; methylamines; neutron diffraction; plasmin; protease inhibitor; protein structure; trypsin

Alpha2-Macroglobulin (alpha2M) is a major inhibitor of endoproteases found in the plasma vertebrates. Alpha2M is also involved in a wide variety of physiological processes such as binding and transport of hormones and ions, and in modulating the immune response through various mechanisms. Additionally, alpha2M has been implicated in the development of arterial thrombosis and thus may have a role in the development of atherosclerosis. Alpha2M is a high molecular weight glycoprotein (Mr = 725,000) composed of four identical subunits (Mr = 180,000) when reduced and denatured. The mechanism of protease inhibition is accompanied by a drastic change in the conformation of alpha2M resulting in an entrapment of the protease. Although several forms of alpha2M have been observed by electron microscopy, it has been difficult to relate these morphologic structures with the molecular mechanism of the conformational change. The large size of alpha2M and the ability to crystallize it has precluded any attempt to determine the structure by X-ray diffraction. High resolution low dose microscopy with computer imaging seems to be the most logical approach for resolving the three dimensional structure of alpha2M both in its native and protease-exposed forms. The determination of the three-dimensional structures of alpha2M will provide some insight for relating the molecular mechanism of the conformational change with the morphological structures. We propose to determine the three dimensional structure of alpha2M in its native form and after exposure to various proteases (trypsin, chymotrypsin, plasmin) by using a conical tilt series of images obtained by low dose electron microscopy of the protein fixed with a non-denaturing stain at a neutral pH like methylamine tungstate. We will develop a model from the reconstructed three-dimensional structures and carry out neutron scattering experiments to conform and/or refine the structures obtained from the three dimensional reconstructions. We will carry out timed experiments to determine whether the two forms observed after exposure to proteases are different projections of a same prototype or whether one is an intermediary which will transform itself into the other form. We will attempt to locate the protease binding site on alpha2M by using plasmin as a model protease which, because of its higher molecule weight, is likely to be better resolved by computer imaging.

Alpha2-巨球蛋白 (alpha2M) 是一种主要抑制剂 脊椎动物血浆中发现的内切蛋白酶。 Alpha2M 也是 参与多种生理过程，例如 激素和离子的结合和运输，以及调节 通过各种机制进行免疫反应。 此外，α2M 与动脉血栓形成有关 因此可能在动脉粥样硬化的发展中发挥作用。 Alpha2M 是一种高分子量糖蛋白 (Mr = 725,000) 当还原时由四个相同的亚基 (Mr = 180,000) 组成 变性了。 蛋白酶抑制的机制伴随着 α2M 构象的巨大变化导致 蛋白酶的包埋。 尽管 alpha2M 的多种形式 通过电子显微镜观察，很难 将这些形态结构与分子机制联系起来 的构象变化。 alpha2M 的大尺寸和 结晶的能力已经排除了任何确定的尝试 通过X射线衍射确定结构。 高分辨率低剂量 计算机成像显微镜似乎是最合乎逻辑的 解决alpha2M三维结构的方法 其天然形式和蛋白酶暴露形式。 决心 alpha2M 的三维结构将提供一些 关于构象分子机制的见解 随形态结构的变化。 我们建议确定 原始形式的 alpha2M 的三维结构和 接触各种蛋白酶（胰蛋白酶、胰凝乳蛋白酶、 纤溶酶）通过使用低浓度获得的圆锥形倾斜系列图像 用非变性固定的蛋白质的剂量电子显微镜 在中性 pH 值下染色，如甲胺钨酸盐。 我们将开发 来自重建的三维结构的模型和 进行中子散射实验以符合和/或改进 从三维重建获得的结构。 我们将进行定时实验来确定两者是否 接触蛋白酶后观察到的形式不同 同一原型的预测或是否是中间人 它将自身转变为另一种形式。 我们将尝试 使用纤溶酶定位 alpha2M 上的蛋白酶结合位点 一种模型蛋白酶，由于其分子量较高， 通过计算机成像可能会更好地解决。