STRUCTURE/ACTIVITY RELATIONS IN CALMODULIN

钙调蛋白的结构/活性关系

基本信息

批准号：
2331432
负责人：
ANTHONY J PERSECHINI
金额：
$ 11.8万
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 1998-04-30
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）都在所有卵形细胞中都发现了钥匙凭借其依赖Ca 2+依赖性事件的作用能够调节许多不同酶的活性目标。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-激酶和一氮氧化物合酶。我们的长期目标是定义结构对于体外结合和激活所必需的决定因素 CAM通过不同的靶酶。凸轮是哑铃形分子。这哑铃的裂片或“重量”每个由一对EF手组成 Ca2+结合域。尽管凸轮的裂片，每个都可以在凸轮目标复合体中执行唯一的功能。我们的假设是CAM之间的许多功能差异叶与独特的氨基酸决定因素有关。 IE。，在没有裂片的同源位置的决定因素在功能上可以互换。我们有三个基本的短期至中期目标。（1）识别包含独特的裂片中的位置结合和激活酶靶标的决定因素。（2）至确定与肽的正确结合相关的独特决定因素代表靶酶的孤立凸轮结合域。（3）到区分CAM和CAM直接相互作用的独特决定因素来自不同靶酶的凸轮结合域 CAM与目标中其他区域之间的相互作用。具体实验目的是：（1）合成改变的凸轮的合成，其中残基在裂片中的同源姿势已被用重组DNA技术。（2）主链的初步验证通过测量Ca2+结合亲和力来改变凸轮的构象每种蛋白质的化学计量法，并通过评估其CD光谱在存在和不存在Ca2+的情况下。蛋白质失败初始验证将不会进一步研究。（3）确定改变凸轮的能力结合和激活肌球蛋白轻链激酶，植物NAD激酶，钙调神经酶和一氧化氮合成酶活性。（4）确定每个改变的凸轮的晶体结构作为最终验证其预期的三维结构。（5）确定每个复合物之间的离解常数改变的凸轮和肽代表CAM结合域的 MLCK。（6）CAM和CAM之间的空间关系的表征（5）中的肽通过估算结合肽的位点之间的距离以及使用荧光能量转移的天然或改变凸轮上的那些技术。