FROZEN HYDRATED ELECTRON MICROSCOPY OF CA ATPASE

CA ATP酶的冷冻水合电子显微镜

• 批准号: 2732845
• 负责人: David L. Stokes
• 金额: $ 27.27万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2732845
• 关键词: affinity chromatography; affinity labeling; calcium flux; calcium transporting ATPase; charge coupled device camera; computer program /software; conformation; cryoscopy; crystallization; electron microscopy; enzyme structure; fluorescent dye /probe; gold; image processing; laboratory rabbit; maleimides; protein purification; sarcoplasmic reticulum; stoichiometry; structural biology; thapsigargin

DESCRIPTION providing ATP-dependent transport of various ions across a variety of cellular and subcellular membranes. These pumps are responsible for such important phenomena as the cell resting potential (Na+/K+-ATPase) and muscle relaxation (Ca2+-ATPase). The calcium pump (Ca2+-ATPase) has been an archetype for this family and has been characterized by every conceivable means, including kinetics, spectroscopy, site-directed mutagenesis and chemical modification. Our understanding of the molecular mechanism, however, is hindered by our ignorance of the molecular structure. This proposal aims to determine this structure by methods of electron crystallography employing frozen-hydrated crystals of Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum. In particular, two crystal forms are being studied. Thin, multilamellar crystals of purified, detergent-solubilized Ca2+-ATPase diffract to high resolution and a three-dimensional structure at 6 A resolution is proposed by modifying standard electron crystallographic methods developed for two-dimensional membrane proteins. Tubular crystals in the sarcoplasmic reticulum membrane have previously been used for a 14 A structure and the organization of the molecule will be further investigated by labelling Ca2+-ATPase with site-specific compounds and locating these labels in 3D reconstructions. The resolution of the structure from tubular crystals will also be improved by using improved facilities for electron microscopy and improved strategies for image analysis. Structures from these two crystal forms represent different conformational states of Ca2+-ATPase, corresponding to major intermediates in the reaction cycle. Thus, comparison of the resulting structures will help to understand the structural basis for coupling ATP hydrolysis to calcium transport. Given the homologies in amino acid sequence and similarities in reaction mechanisms, these conclusions will apply more broadly to other members of the family of P-type ion pumps (e.g., Na+/K+-ATPase, H+/K+-ATPase) and help develop a general mechanism for ATP-dependent ion transport. In the case of copper transport, deficiencies which lead either to Menkes or Wilson disease, a better understanding of this mechanism may eventually help in developing strategies for treatment.

描述提供各种离子跨膜的ATP依赖性运输， 各种细胞和亚细胞膜。 这些泵负责 细胞静息电位（Na+/K+-ATP酶） 肌松（Ca ~（2+）-ATP酶）。 钙泵（Ca 2 +-ATPase）具有 是这个家庭的原型， 可能的手段，包括动力学，光谱学，现场定向 诱变和化学修饰。 我们对分子的理解 然而，我们对分子结构的无知阻碍了这种机制的研究。 该建议旨在通过电子的方法来确定这种结构 晶体学，使用来自 骨骼肌肌浆网 特别是，两种晶体形式 正在被研究。 薄，多层晶体的纯化， 洗涤剂溶解的Ca ~（2+）-ATPase具有高分辨率， 通过修改，提出了分辨率为6 A的三维结构 标准电子晶体学方法开发的二维 膜蛋白 肌浆网膜中的管状晶体 以前曾用于14 A结构和组织 分子将进一步研究标记Ca 2 +-ATP酶与 位点特异性化合物并在3D重建中定位这些标签。 从管状晶体结构的分辨率也将得到提高 通过使用改进的电子显微镜设备和改进的策略， 用于图像分析。 这两种晶体形式的结构代表 Ca 2 +-ATP酶的不同构象状态，对应于主要的 反应循环中的中间体。 因此，比较结果 结构将有助于理解耦合ATP的结构基础 水解至钙转运。 考虑到氨基酸的同源性 序列和相似的反应机制，这些结论将 更广泛地应用于P型离子泵家族的其它成员（例如， Na+/K+-ATP酶，H+/K+-ATP酶），并帮助开发一种通用机制， ATP依赖性离子转运。 在铜运输方面， 导致门克斯病或威尔逊病， 这种机制最终可能有助于制定治疗策略。