STRUCTURAL BIOLOGY OF EF-HAND CALCIUM BINDING PROTEINS

Ca 2+结合蛋白的EF-手家族在Ca 2+信号转导途径的许多方面具有中心作用，从调节通道的打开和关闭，到控制Ca 2+信号的强度和持续时间，再到将这些信号转导成生化和生物力学响应。 超过1000个EF-手蛋白基序已被确定其独特的序列特征。 然而，尽管它们分布广泛，但人们对它们的序列的哪些特征为其提供了特定功能或使用哪些分子机制来区分其不同的细胞靶点知之甚少。 我们的研究计划的目标是确定EF-手蛋白的生化和生物学功能的分子基础，以便我们可以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Ca 2+依赖的细胞活动，用于医学和生物技术应用。目前的建议的总体目标是回答两个关键问题的细胞内Ca 2+信号转导的潜在的分子基础：（i）EF-手蛋白的序列如何指定其响应的结合Ca 2+？(ii)不同的EF-手蛋白如何与特定的蛋白质靶点相互作用并调节它们？ 第一个问题将解决一系列的耦合诱变，生物物理，和结构的研究使用钙结合蛋白D9 K作为模型系统。 这项工作的一个关键目标是创造一种新的钙结合蛋白-钙调蛋白杂交蛋白，calbindomodulin。 第二个问题将通过结合生物物理和结构研究来解决，以确定钙离子诱导的caltractin和钙依赖性蛋白激酶的调节结构域的活动的机制。 这些结果将连接大量的EF-手蛋白的实验数据，揭示这些蛋白的功能机制，并帮助定义它们的生物活性。

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.