FROZEN-HYDRATED ELECTRON MICROSCOPY OF CA ATPASE

CA ATP酶的冷冻水合电子显微镜

• 批准号: 2080394
• 负责人: David L. Stokes
• 金额: $ 0.65万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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 1995-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2080394
• 关键词: X ray crystallography; affinity chromatography; calcium; calcium flux; calcium transporting ATPase; chromium; computer program /software; conformation; crystallization; electron microscopy; enzyme mechanism; enzyme structure; enzyme substrate; freezing; gold; high voltage electron microscopy; image processing; laboratory rabbit; liquid crystal; membrane transport proteins; molecular site; phase contrast microscopy; phosphoproteins; phosphorylation; protein purification; rare earth element; sarcoplasmic reticulum

CaATPase composes about 80% of the membrane protein of the sarcoplasmic reticulum (SR) of striated muscle. Its role is to remove Ca++ from the sarcoplasm following a muscle contraction, thereby affecting relaxation. Similarities in the amino acid sequence of a wide range of other ATP- dependent cation pumps have defined a distinct family, of which CaATPase is the most thoroughly studied member. In particular, many studies have attempted to link the events of the well-established reaction cycle to physical locations on the molecule and low resolution structures have been produced by electron microscopy of negatively stained specimens. We propose to study the structure of CaATPase at high resolution, primarily by frozen-hydrated electron microscopy. Through these studies, we aim (1) to reveal the secondary structure that composes the molecule, (2) to locate physical sites of substrate binding, and (3) to characterize conformational changes that are driven by the chemical reaction cycle and that result in the transport of calcium across the SR membrane. Three-Dimensional Reconstruction. We propose to solve the three- dimensional structure at high resolution by studying thin three-dimensional crystals of detergent-solubilized CaATPase in the frozen-hydrated state. These crystals produce electron diffraction to 4A resolution and we propose to collect three-dimensional information by standard methods of electron crystallography. From the resulting three-dimensional reconstruction, we should be able to describe the arrangement (1) of transmembrane alpha- helices, (2) of alpha-helices thought to compose the cytoplasmic stalk, and (3) the 3 main domains of the cytoplasmic head. We also propose to solve the structure at an intermediate resolution (15A) using long helical tubes of CaATPase, which are induced by vanadate within the native SR membrane. Site-Specific Labels. We propose to label specific sites on CaATPase that will then be utilized in three-dimensional reconstruction of wither three- dimensional crystals or helical tubes. CaATPase will be covalently labelled with an undeca-gold complex, which will be coupled to CaATPase either with the site-specific label DIDS or directly via a maleimide linkage. Conformational Changes. We propose to solve structures from three different crystal forms, produced by conditions that stabilize different conformational states; by comparing the structures, we hope to describe the structural basis for these different states. In addition, we will study the structural consequences of phosphorylation using caged-ATP and Cr-ATP. Caged-ATP will be used for time-resolved studies, the phosphoenzyme being trapped by rapid freezing of crystals a short time (e.g., 0.25s) after the release of ATP by a light flash. Alternatively, Cr-ATP produces a long- lived (days) phosphoenzyme that will be made both before and after crystallization.

caatp酶组成约80%的肌浆膜蛋白