FROZEN-HYDRATED ELECTRON MICROSCOPY OF CAATPASE

过氧化氢酶的冷冻水合电子显微镜

• 批准号: 3161431
• 负责人: David L. Stokes
• 金额: $ 16.64万
• 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 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3161431
• 关键词: X ray crystallography; adenosinetriphosphatase; affinity chromatography; calcium; calcium flux; 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酶约占肌浆细胞膜蛋白的80%， 横纹肌的网状结构。 它的作用是将Ca++从 肌肉收缩后的肌浆，从而影响放松。 在氨基酸序列的相似性，广泛的其他ATP- 依赖性阳离子泵定义了一个独特的家族，其中CaATP酶是 研究得最透彻的成员。 特别是，许多研究 试图将已确立的反应周期的事件与 分子上的物理位置和低分辨率结构已经被 通过负染色标本的电子显微镜检查产生。 我们 我建议在高分辨率下研究CaATP酶的结构，主要是通过 冷冻水化电子显微镜 通过这些研究，我们的目标是（1） 揭示组成分子的二级结构，（2）定位 底物结合的物理位点，和（3）表征构象 由化学反应循环驱动的变化， 钙离子通过SR膜的转运。 三维重建。 我们建议解决这三个- 通过研究薄的三维结构， 冷冻水合状态的洗涤剂溶解的CaATPase晶体。 这些晶体产生电子衍射到4A分辨率，我们建议 通过电子的标准方法收集三维信息， 结晶学 从得到的三维重建，我们 应该能够描述跨膜α- 螺旋，（2）α-螺旋被认为构成细胞质柄，和 (3)细胞质头部的3个主要结构域。 我们还建议解决 使用长螺旋管在中间分辨率（15 A）下结构 的CaATP酶，这是由钒酸诱导的天然SR膜内。 站点特定标签。 我们建议标记CaATPase上的特定位点， 然后将用于枯萎的三维重建， 三维晶体或螺旋管。 CaATP酶将以共价键 用十一碳-金复合物标记，其将与CaATPase偶联 用位点特异性标记DIDS或直接通过马来酰亚胺 联动 构象变化。 我们建议从三个方面解决结构问题 不同的晶体形式，由稳定不同的 构象状态;通过比较结构，我们希望描述 这些不同国家的结构基础。 此外，我们将研究 使用笼状ATP和Cr-ATP磷酸化的结构后果。 笼状ATP将用于时间分辨研究，磷酸酶是 通过短时间快速冷冻晶体而捕获（例如，0.25s）之后， 通过闪光释放ATP。 另外，Cr-ATP产生长- 活（天）磷酸酶，将作出之前和之后， 晶化