Calmodulin Mediated Disorder-to-order Transitions: Calcineurin as a Model System

近年来，在许多重要的生物系统中发现了许多内在无序的蛋白质。内在无序的蛋白质是指完全或部分无结构的蛋白质。目前还不清楚为什么蛋白质紊乱如此普遍。提出了许多假设。在这个项目中测试的假设是，无序到有序的转变可以用来调节蛋白质的功能。钙调蛋白是一种非常丰富的、高度保守的钙结合蛋白，可以调节许多其他必需蛋白质的功能。最近有人提出，靶蛋白内的许多钙调素结合位点位于内在无序区域，其中一些在钙调素结合时经历无序到有序的转变。钙调蛋白是一种重要的，高度保守的丝氨酸/苏氨酸磷酸酶，当与钙调蛋白结合时被激活。当钙调素与钙调神经磷酸酶的调控区域结合时，就会引起构象变化，导致酶的激活。实验数据表明，在失活酶中，钙调蛋白的调控域是无序的，并且在钙调蛋白结合后，它经历了一个无序到有序的转变。本研究的目的是表征这种从无序到有序转变的物理性质。钙调蛋白被用作研究钙调素介导的调节生物活性的无序到有序转变的模型系统。这一目标将通过两个目标来实现：1)确定调节结构域作为一个孤立结构域经历钙调素介导的从无序到有序转变的能力；2)确定全长钙调磷酸酶在这种转变中所起的作用。将采用多种生物物理技术，包括圆二色性、荧光、核磁共振和分析性超离心。获得的数据将有助于构建钙调蛋白调节结构域无序到有序转变的模型，这反过来将作为未来研究钙调蛋白介导的其他蛋白质调节转变的基础。内在无序蛋白质领域的核心问题是为什么无序如此普遍？该项目将通过测试钙调素介导的无序到有序转换可以调节蛋白质功能的假设，对回答部分问题做出重大贡献。PI计划让一名博士后学者和几名本科生参与这个项目的各个方面。在可能的情况下，这些代表将包括来自代表性不足群体的代表。该博士后将接受蛋白质化学、分子生物学、光谱学和核磁共振光谱学方面的高级培训。本科学生将接受基本的实验室训练，包括蛋白质化学、分子生物学、光谱学、科学伦理以及数据分析和表达方面的训练。所有学员都将参与传播所产生的知识。项目负责人将把在这个项目中产生的知识纳入研究生水平的结构生物学和蛋白质课程。此外，数据将发表在同行评议的期刊上，并在科学会议和研究机构上发表。

In recent years many intrinsically disordered proteins have been identified in numerous biologically important systems. An intrinsically disordered protein is one that is entirely or partially unstructured. It is not clear is why protein disorder is so common. A number of hypotheses have been advanced. The hypothesis being tested in this project is that disorder-to-order transitions can be used to regulate protein function. Calmodulin is a very abundant, highly conserved calcium-binding protein known to regulate the functions of numerous essential other proteins. It was recently proposed that many of the calmodulin binding sites within target proteins are located in intrinsically disordered regions and that some of these undergo disorder-to-order transitions upon calmodulin binding. Calcineurin is an essential, highly-conserved serine/threonine phosphatase that is activated when bound by calmodulin. Upon binding of calmodulin to the regulatory domain of calcineurin, a conformational change is induced that results in activation of the enzyme. Experimental data suggests the regulatory domain of calcineurin is disordered in the inactive enzyme, and that it undergoes a disorder-to-order transition upon calmodulin binding. The goal of this research is to characterize the physical nature of this disorder-to-order transition. Calcineurin is being employed as a model system in studies of calmodulin-mediated disorder-to-order transitions that regulate biological activity. This goal will be achieved through two aims: i) determination of the ability of the regulatory domain to undergo a calmodulin mediated disorder-to-order transition as an isolated domain, and ii) determination of the role full length calcineurin plays in this transition. A variety of biophysical techniques will be employed, including circular dichroism, fluorescence, NMR, and analytical ultracentrifugation. The data obtained will enable construction of a model for the calcineurin regulatory domain disorder-to-order transition, which in turn will be used as the basis for future studies of other calmodulin mediated transitions involved in protein regulation.The central question in the field of intrinsically disordered proteins is why is disorder so common? This project will make a significant contribution towards answering part of that question through the testing of the hypothesis that calmodulin mediated disorder-to-order transitions can regulate protein function. The PI plans to have both a postdoctoral scholar and several undergraduate students work on aspects of this project. Where possible, these will include representatives from underrepresented groups. The postdoctoral scholar will receive advanced training in protein chemistry, molecular biology, spectroscopy and NMR spectrometry. The undergraduate students will receive basic laboratory training, training in protein chemistry, molecular biology, spectroscopy, scientific ethics, and in the analysis and presentation of data. All trainees will participate in the dissemination of knowledge generated. The PI will incorporate knowledge generated in this project into graduate-level courses on structural biology and proteins. In addition, data will be published in peer-reviewed journals, and presented at scientific meetings and research institutions.