CRYSTALLIZATION OF A CALCIUM PUMP

钙泵的结晶

• 批准号: 3426152
• 负责人: HARLAN Tonie WRIGHT
• 金额: $ 3.69万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3426152
• 关键词: X ray crystallography; adenosine triphosphate; calcium transporting ATPase; chemical structure; crystallization; high performance liquid chromatography; laboratory rabbit; membrane permeability; monomer; muscle function; muscular dystrophy; protein sequence; sarcoplasmic reticulum; stoichiometry

The Ca-ATPase of fast twitch muscle sarcoplasmic reticulum is representative of a class of ion pumps involved in the homeostasis of the cations Na+, K+, H+ and Ca+2. The similarities in structures and kinetics of these ion pumps (Na/K-ATPase, H/K- ATPase and Ca-ATPase) have been interpreted to indicate that they have been derived from a common ancestral gene (1). Ca+2 is unique among these cations in that if functions as a messenger in response to hormones and transmitters. Loss of Ca-pump activity is associated with such diseases as Duchenne muscular dystrophy (2), Brody's disease (3) and experimental myotonia (4). The work proposed here has the specific aim of crystallizing rabbit sarcoplasmic reticulum Ca-ATPase with the long-term objective of obtaining detailed structural information through application of diffraction methods to such crystals. Ca-ATPase will be purified in the laboratory of Prof. F.N. Briggs using procedures developed in that laboratory (5), which yield homogeneous enzyme. This material is solubilized in the presence of detergents, and will be used in this form as the starting material for crystallization trials. Ca-ATPase exists in two stable conformational forms, and in different states of aggregation. For crystallization, those conditions known to favor the monomer will be used. Trails to optimize these conditions will be made by combining and exchanging detergents, as was done in the case of porin crystallization. Aggregation state will be monitored either by analytical HPLC or centrifugation in the presence of detergent. Mild succinylation and amidination of the Ca-ATPase will also be tried, since the former has been shown to reduce aggregation. The two conformational states: high (E1) and low (E2) calcium affinity, will be selected through binding of ligands known to stabilize one of the two states. The more stable, Ca-bound E1 form will be screened for crystallization first. Divalent transition metals and several different nucleotides will be tested for their effects on crystallization. The E2 state exists as a dimer in vesicles and is stabilized by vanadate. Vanadate- bound enzyme will be screened in crystallization trials, varying detergent, precipitant concentrations and pH. Crystallization trials will be done by the sitting drop or microdialysis methods. Since Ca-ATPase has roughly equal extent of cytoplasmically exposed and intrinsic membrane regions, small amphiphilic molecules such as the heptane-triols will be tested for their effects in promoting crystallization. Any crystals obtained by these methods will be surveyed for lattice order by x-ray diffraction still or precession photographs.

快速抽动肌肌浆网钙-三磷酸腺苷酶的研究 一类涉及动态平衡的离子泵代表 Na~+，K~+，H~+和Ca~+2的相似之处 这些离子泵(Na/K-ATPase、H/K-ATPase)的结构和动力学 ATPase和Ca-ATPase)被解释为表明 它们来自一个共同的祖先基因(1)。CA+2 在这些阳离子中是独一无二的，因为IF起着信使的作用 对荷尔蒙和递质的反应。钙泵的损失 活动与Duchenne肌肉疾病有关 营养不良2例，Brody病3例，实验性肌强直4例。 这里提出的工作有明确的目标，使之具体化 兔肌浆网钙-三磷酸腺苷酶的长期作用 目标是通过以下途径获取详细的结构信息 衍射法在这类晶体上的应用。CA-ATPase 将在F·N·布里格斯教授的实验室用 在该实验室开发的程序(5)，这些程序产生 同质酶。这种材料在存在的情况下溶解 洗涤剂，并将以这种形式作为起点 用于结晶试验的材料。CA-ATPase存在于两个 稳定的构象形式，并在不同的状态下 聚合。对于结晶来说，这些已知的条件有利于 将使用单体。对这些条件进行优化的尝试 将通过组合和交换洗涤剂制成，就像以前一样 在孔蛋白结晶的情况下进行。聚合状态将 通过分析性高效液相色谱或离心法进行监测 洗涤剂的存在。温和的琥珀酸化和氨化 CA-ATPase也将受到审判，因为前者已被证明 减少聚集。两种构象状态：高(E_1) 和低(E2)钙亲和力，将通过结合 已知的稳定两个状态之一的配体。越稳定， 首先对钙结合的E1型进行结晶筛选。 二价过渡金属和几种不同的核苷酸将 测试它们对结晶的影响。存在E2状态 作为囊泡中的二聚体，由钒酸盐稳定。钒酸盐- 结合酶将在结晶试验中筛选，不同 洗涤剂、沉淀剂浓度和pH值。结晶 试验将通过静坐滴注或微透析法进行。 由于Ca-ATPase在细胞质上具有大致相同的程度 裸露的和固有的膜区，小的两亲性 将对庚烷三醇等分子进行测试 促进结晶的效果。通过以下方式获得的任何晶体 这些方法将通过x射线检查其晶格有序程度。 衍射静止或进动照片。