STRUCTURAL BIOLOGY OF EF-HAND CALCIUM BINDING PROTEINS

EF-hand家族的Ca2+结合蛋白在Ca2+信号转导途径的许多方面都起着核心作用，从通道的打开和关闭的调节，到控制Ca2+信号的强度和持续时间，再到这些信号转导成生化和生物力学反应。超过1000个EF-hand蛋白基序已经从它们独特的序列特征中被鉴定出来。然而，尽管它们分布广泛，但人们对它们序列的哪些特征提供了它们的特定功能，或者用什么分子机制来区分它们的不同细胞靶标知之甚少。我们的研究计划的目的是确定EF-hand蛋白的生化和生物学功能的分子基础，以便我们更好地了解它们在健康和疾病中的作用。最终，我们将利用这些知识来改变或重新设计Ca2+依赖的细胞活性，用于医学和生物技术应用。当前提案的总体目标是回答关于细胞内Ca2+信号转导的潜在分子基础的两个关键问题：(i) EF-hand蛋白的序列如何指定其对Ca2+结合的响应？（ii）不同的EF-hand蛋白如何与特定的蛋白靶点相互作用和调节？第一个问题将通过使用calbindin D9k作为模型系统的一系列耦合诱变、生物物理和结构研究来解决。这项工作的一个关键目标将是创造一种新的钙结合蛋白-钙调蛋白杂交蛋白，钙结合蛋白调节蛋白。第二个问题将通过结合生物物理和结构研究来解决，以确定Ca2+诱导caltractin活性的机制和钙依赖性蛋白激酶的调节结构域。这些结果将把EF-hand蛋白的大量实验数据联系起来，揭示这些蛋白的功能机制，并有助于确定它们的生物活性。

The EF-hand family of Ca2+ binding proteins has a central role in many aspects of Ca2+ signal transduction pathways, from the regulation of the opening and closing of channels, to controlling the intensity and duration of Ca2+ signals, to the transduction of these signals into biochemical and biomechanical responses. Over 1000 EF-hand protein motifs have been identified from their unique sequence signatures. Yet despite their widespread distribution, very little is known about what features of their sequences provide them with their specific functions or what molecular mechanisms are used to distinguish among their diverse cellular targets. The objective of our research program is to define the molecular basis for the biochemical and biological function of EF-hand proteins, so that we may better understand their roles in health and disease. Ultimately, we will use this knowledge to alter or de novo design Ca2+-dependent cellular activities for medical and biotechnology applications. The overall objective of the current proposal is to answer two key questions regarding the underlying molecular basis for intracellular Ca2+ signal transduction: (i) How does the sequence of an EF-hand protein specify its response to the binding of Ca2+? (ii) How do different EF-hand proteins interact with and modulate specific protein targets? The first question will be addressed by a series of coupled mutagenesis, biophysical, and structural studies using calbindin D9k as a model system. A key goal in this work will be to create a new calbindin-calmodulin hybrid protein, calbindomodulin. The second question will be addressed by combined biophysical and structural studies seeking to determine the mechanisms of the Ca2+-induced activities of caltractin and the regulatory domain of the calcium dependent protein kinase. These results will link the large volume of experimental data available on EF-hand proteins, uncover the mechanisms by which these proteins function, and help to define their biological activities.