FROZEN HYDRATED ELECTRON MICROSCOPY OF CA ATPASE

CA ATP酶的冷冻水合电子显微镜

• 批准号: 2442815
• 负责人: David L. Stokes
• 金额: $ 26.22万
• 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/2442815
• 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atp依赖的各种离子通过a的运输