STRUCTURAL BIOLOGY OF EF-HAND CALCIUM BINDING PROTEINS

EF-Hand家族的钙结合蛋白在钙信号转导通路的许多方面发挥着核心作用，从调节通道的开放和关闭，到控制钙信号的强度和持续时间，再到将这些信号转化为生化和生物力学反应。从其独特的序列特征中已经识别了1000多个EF-Hand蛋白质基序。然而，尽管它们分布广泛，但人们对它们的序列特征提供了它们的特定功能，或者它们被用来区分不同的细胞靶标的分子机制知之甚少。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医疗和生物技术应用中依赖于钙离子的细胞活动。当前提案的总体目标是回答关于细胞内钙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钙结合的反应？(Ii)不同的EF-Hand蛋白如何与特定的蛋白质靶标相互作用和调节？第一个问题将通过一系列耦合的诱变、生物物理和结构研究来解决，该研究使用Calbindin D9k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，即钙调蛋白。第二个问题将通过生物物理和结构研究相结合的方式来解决，这些研究试图确定钙离子诱导的钙调蛋白活性的机制和钙依赖蛋白激酶的调节域。这些结果将联系关于EF-Hand蛋白质的大量实验数据，揭示这些蛋白质的功能机制，并有助于确定它们的生物学活性。

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.