STRUCTURE/ACTIVITY RELATIONS IN CALMODULIN

钙调蛋白的结构/活性关系

基本信息

批准号：
2143713
负责人：
ANTHONY J PERSECHINI
金额：
$ 10.86万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-02-01 至 1998-01-31
项目状态：
已结题

项目摘要

Calmodulin (CaM) is found in all eucaryotic cells where it plays a key role in orchestrating Ca 2+-dependent events in the cell by virtue of its ability to modulate the activities of a number of different enzyme targets. Among these are several protein kinases, phosphodiesterase, adenylate cyclase, inositol 1,4,5-trisphosphate 3-kinase, and nitric oxide synthase. Our long-term goal is to define the structural determinants that are necessary for in vitro binding and activation of different target enzymes by CaM. CaM is a dumbbell shaped molecule. The lobes or "weights" of the dumbbell each consist of a pair of EF hand Ca2+-binding domains. In spite of structural similarities between the lobes of CaM, each can perform unique functions in CaM-target complexes. It is our hypothesis that many functional differences between the CaM lobes are associated with unique amino acid determinants; i.e., determinants at homologous positions in the lobes that are not functionally interchangeable. We have three basic short- to medium-term goals. (1) To identify positions in the lobes containing unique determinants for binding and activation of enzyme targets. (2) To identify unique determinants associated with correct binding of peptides representing the isolated CaM-binding domains of target enzymes. (3) To distinguish unique determinants for direct interactions between CaM and the CaM-binding domain in different target enzymes from those for interactions between CaM and other regions in the targets. The specific experimental aims are: (1) Synthesis of altered CaMs in which residues at homologous positions in the lobes have been swapped for one another using recombinant DNA techniques. (2) Initial verification of the main-chain conformations of altered CaMs by measuring the Ca2+-binding affinities and stoichiometries for each protein and by evaluating their CD spectra in the presence and absence of Ca2+. Proteins failing initial verification will not be studied further. (3) Determination of the abilities of altered CaMs to bind and activate myosin light chain kinase, plant NAD kinase, calcineurin, and nitric oxide synthetase activities. (4) Determination of the crystal structures for each altered CaM as final verification of its expected three dimensional structure. (5) Determination of a dissociation constant for the complex between each of the altered CaMs and a peptide representing the CaM-binding domain of MLCK. (6) Characterization of the spatial relationship between CaM and the peptide in (5) by estimating distances between sites on bound peptide and those on native or altered CaMs using fluorescence energy transfer techniques.

钙调蛋白 (CaM) 存在于所有真核细胞中，在其中发挥着关键作用凭借其在细胞中协调 Ca 2+ 依赖性事件的作用调节多种不同酶活性的能力目标。其中有几种蛋白激酶、磷酸二酯酶、腺苷酸环化酶、肌醇 1,4,5-三磷酸 3-激酶和硝酸氧化物合酶。我们的长期目标是定义结构体外结合和激活所必需的决定因素 CaM 不同的靶酶。 CaM 是一种哑铃形分子。这哑铃的叶瓣或“配重”各由一对 EF 手组成 Ca2+ 结合域。尽管两者之间结构相似 CaM 叶，每个叶都可以在 CaM 目标复合物中执行独特的功能。我们假设 CaM 之间存在许多功能差异叶与独特的氨基酸决定簇相关； IE。，叶中同源位置的决定因素不是功能上可以互换。我们有三个基本的中短期目标目标。 (1) 识别包含唯一的波瓣中的位置酶靶点结合和激活的决定因素。 (2) 至识别与肽正确结合相关的独特决定因素代表目标酶的分离的 CaM 结合域。 (3) 至区分 CaM 和之间直接相互作用的独特决定因素不同靶酶中的 CaM 结合域 CaM 与目标中其他区域之间的相互作用。具体的实验目的是：（1）合成改变的CaM，其中残基位于叶中的同源位置已使用以下方法相互交换重组DNA技术。 (2) 主链初步验证通过测量 Ca2+ 结合亲和力来改变 CaM 的构象每种蛋白质的化学计量和评估其圆二色光谱在存在和不存在 Ca2+ 的情况下。蛋白质最初失败验证不再进一步研究。 (3) 的确定改变的 CaM 结合和激活肌球蛋白轻链激酶的能力，植物 NAD 激酶、钙调神经磷酸酶和一氧化氮合成酶活性。 (4) 确定每个改变的 CaM 的最终晶体结构验证其预期的三维结构。 (5) 确定每个之间的复合物的解离常数改变的 CaM 和代表 CaM 结合域的肽 MLCK。 (6) CaM与空间关系的表征通过估计结合肽上位点之间的距离来计算（5）中的肽以及使用荧光能量转移的天然或改变的 CaM 上的细胞技术。