CALCIUM BINDING PROTEIN/TARGET INTERACTIONS

许多细胞内过程都是由细胞内蛋白质的瞬时变化来调节的。 Ca 2+离子浓度。 Ca ~（2+）的作用是由蛋白质介导的 特异性结合Ca 2+并呈现新的构象，然后 使它们能够与靶蛋白结合，包括一些酶， 影响其功能。 这个项目的目的是了解 两种最具特征的钙离子结合的分子机制 调节蛋白，钙调蛋白（CaM）和肌钙蛋白C（TnC），识别并 以Ca 2+依赖的方式调节它们的靶点。 Ca 2+和Ca ~（2+）诱导CaM和TnC的局部和整体构象变化 通过与选定的靶-全蛋白（磷酸二酯酶， 钙调神经磷酸酶、神经调节蛋白、肌钙蛋白I）和模拟 互动网站-将被表征。 人肝钙调素和钙调素的突变体 兔骨骼肌肌钙蛋白C的合成关键部位含有半胱氨酸残基。 将监测Cys侧链之间距离的变化 在用适合于荧光能量的发色团标记它们之后， 传输测量。 指定的功能意义 将通过评估CaM和TnC的构象转变来评估 突变体的靶向调节能力， 蛋白质片段被二硫键限制。 相互作用部位 与CaM和TnC上的靶分子的结合将详细表征， 定义特异性的决定因素。 突变体也将用于 评估某些疏水基团和离子基团在特定条件下的贡献， 位置的相互作用的强度和调节活动。 这些研究将有助于更好地了解 钙调素、肌钙蛋白C和其他钙结合蛋白介导的钙信号转导。 的 交互接口的详细描述和 特异性的决定因素对于识别 生理目标的其他成员的迅速增长的家庭 Ca 2+结合蛋白。

Many intracellular processes are regulated by a transient change in the concentration of Ca2+ ions. The effect of Ca2+ is mediated by proteins that bind Ca2+ specifically and assume a new conformation, which then enables them to bind to target proteins, including some enzymes, thereby affecting their function. The aim of this project is to understand molecular mechanisms by which the two best characterized Ca2+-binding regulatory proteins, calmodulin (CaM) and troponin C (TnC), recognize and modulate their targets in a Ca2+-dependent way. Local and global conformational changes induced in CaM and TnC by Ca2+ and by interactions with selected targets - whole proteins (phosphodiesterase, calcineurin, neuromodulin, troponin I) and peptides that mimic the interaction sites - will be characterized. Mutants of human liver CaM and rabbit skeletal TnC will by synthesized with Cys residues at key positions. Changes in distances between the Cys side chains will be monitored following their labeling with chromophores suitable for fluorescence energy transfer measurements. The functional significance of specified conformational transitions in CaM and TnC will be assessed by evaluating the target-modulating ability of mutants in which the movement of selected protein segments is restricted by a disulfide bond. Sites of interaction with target molecules on CaM and TnC will be characterized in detail with a view to defining determinants of specificity. Mutants will also be used to assess the contribution of some hydrophobic and ionic groups at specific positions to the strength of interaction and to the modulatory activity. These studies will contribute to a better understanding of the mechanism of Ca2+ signal transduction by CaM, TnC and other Ca2+-binding proteins. The detailed description o the interaction interfaces and definition of determinants of specificity will be important for identification of physiological targets for other members of the rapidly growing family of Ca2+-binding proteins.