FROZEN-HYDRATED ELECTRON MICROSCOPY OF CAATPASE

CaATPase约占肌浆膜蛋白的80% 横纹肌网状结构(SR)。它的作用是从细胞中去除钙离子。 肌肉收缩后的肌浆，从而影响放松。 广泛的其他ATP的氨基酸序列的相似性- 依赖的阳离子泵定义了一个不同的家族，其中CaATPase是 研究最深入的成员。特别是，许多研究都有 试图将建立良好的反应周期的事件与 分子上的物理位置和低分辨率结构 由负染标本的电子显微镜制作而成。我们 建议在高分辨率下研究CaATPase的结构，主要是通过 冷冻水合电子显微镜。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 底物结合的物理位置，以及(3)表征构象 由化学反应循环驱动的变化，并导致 钙跨SR膜的运输。 三维重建。我们建议解决三个问题-- 通过研究薄三维空间实现高分辨率的空间结构 冷冻-水合状态下洗涤剂增溶CaATPase的结晶。 这些晶体产生4A分辨率的电子衍射，我们建议 用电子学的标准方法收集三维信息 结晶学。从由此产生的三维重建中，我们 应该能够描述跨膜α-的排列(1) 螺旋，(2)被认为构成细胞质柄的α-螺旋，以及 (3)胞质头部的3个主要区域。我们还建议解决 使用长螺旋管的中等分辨率(15A)的结构 CaATPase，这是由天然SR膜上的钒酸盐诱导的。 特定于场地的标签。我们建议在CaATPase上标记特定的站点， 然后被用来对枯萎的三个- 空间晶体或螺旋管。CAATPase将共价 标记有十一烷基金的络合物，将与CAATPase偶联 用特定部位的DIDS标签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来解决结构问题 不同的晶体形态，由稳定不同的条件产生 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，我们还将研究 使用笼状三磷酸腺苷和铬三磷酸腺苷进行磷酸化的结构后果。 笼状ATP将用于时间分辨研究，磷酸酶是 在快速冻结晶体后的短时间内(例如，0.25秒)被捕获 通过闪光释放三磷酸腺苷。或者，铬-三磷酸腺苷产生一种长的- 活(天)前和之后都将生成的磷酸酶 结晶。

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.