3-DIMENSIONAL RECONSTRUCTION OF ALPHA-2-MACROGLOBULIN

ALPHA-2-巨球蛋白的三维重建

• 批准号: 3296612
• 负责人: JEAN-PIERRE BRETAUDIERE
• 金额: $ 11.02万
• 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/3296612
• 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.

α 2-巨球蛋白（α 2 M）是α 2-巨球蛋白的主要抑制剂 在血浆脊椎动物中发现的内切蛋白酶。 Alpha 2 M也是 参与各种各样的生理过程， 结合和运输激素和离子，并在调节 免疫反应通过各种机制。 此外，Alpha 2 M 与动脉血栓形成有关， 因此可能在动脉粥样硬化的发展中起作用。 Alpha 2 M是一种高分子量糖蛋白（Mr = 725，000） 还原时由四个相同的亚基组成（Mr = 180，000）， 变性 蛋白酶抑制的机制是伴随着 alpha 2 M构象的急剧变化导致 蛋白酶的截留。 虽然alpha 2 M的几种形式 通过电子显微镜观察， 将这些形态结构与分子机制联系起来 的构象变化。 alpha 2 M的大尺寸和 结晶的能力排除了任何试图确定 通过X射线衍射确定了结构。 高分辨率低剂量 显微镜和计算机成像似乎是最合理的 α 2 M三维结构解析方法 以其天然形式和暴露于蛋白酶的形式。 测定 alpha 2 M的三维结构将提供一些 有关构象的分子机制的见解 随着形态结构的变化而变化。 我们建议确定 α 2 M的天然形式的三维结构， 在暴露于各种蛋白酶（胰蛋白酶，胰凝乳蛋白酶， 纤溶酶）通过使用锥形倾斜系列图像获得的低 用非变性蛋白质固定的蛋白质的剂量电子显微镜 在中性pH下染色，如钨酸甲胺。 我们将开发 - 来自重建的三维结构的模型，以及 进行中子散射实验， 从三维重建中获得的结构。 我们将进行定时实验，以确定这两个 暴露于蛋白酶后观察到的形式不同 同一原型的投影或是否为中介 它会转变成另一种形式 我们将尝试 通过使用纤溶酶定位α 2 M上的蛋白酶结合位点， 一种模型蛋白酶，由于其较高的分子量， 通过计算机成像可能会更好地解决。