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

钙调蛋白介导的无序到有序的转变：钙调神经磷酸酶作为模型系统

• 批准号: 0843551
• 负责人: Trevor Creamer
• 金额: $ 64.92万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843551&HistoricalAwards=false
• 关键词: Calmodulin; Mediated; Disorder; order; Transitions

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.

近年来，在许多重要的生物学系统中已经鉴定出许多本质上无序的蛋白质。本质上无序的蛋白质是完全或部分非结构化的蛋白质。目前尚不清楚为什么蛋白质紊乱如此常见。已经提出了一些假设。该项目正在测试的假设是无序到有序的转变可用于调节蛋白质功能。钙调蛋白是一种非常丰富、高度保守的钙结合蛋白，已知可调节许多重要的其他蛋白质的功能。最近有人提出，靶蛋白内的许多钙调蛋白结合位点位于本质上无序的区域，并且其中一些在钙调蛋白结合后经历无序到有序的转变。钙调神经磷酸酶是一种必需的、高度保守的丝氨酸/苏氨酸磷酸酶，与钙调蛋白结合时被激活。当钙调蛋白与钙调神经磷酸酶的调节结构域结合时，会诱导构象变化，从而导致酶的激活。实验数据表明，钙调神经磷酸酶的调节结构域在无活性的酶中是无序的，并且在钙调蛋白结合后经历无序到有序的转变。这项研究的目标是表征这种无序到有序转变的物理性质。钙调神经磷酸酶被用作钙调蛋白介导的调节生物活性的无序到有序转变的研究中的模型系统。这一目标将通过两个目标来实现：i) 确定调节结构域作为分离结构域经历钙调蛋白介导的无序到有序转变的能力，以及 ii) 确定全长钙调神经磷酸酶在这一转变中所起的作用。将采用多种生物物理技术，包括圆二色性、荧光、核磁共振和分析超速离心。获得的数据将能够构建钙调磷酸酶调节域无序到有序转变的模型，该模型反过来将用作未来研究涉及蛋白质调节的其他钙调蛋白介导的转变的基础。本质无序蛋白质领域的中心问题是为什么无序如此常见？该项目将通过测试钙调蛋白介导的无序到有序的转变可以调节蛋白质功能的假设，为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该项目的各个方面的工作。如果可能，这些人将包括代表性不足群体的代表。博士后学者将接受蛋白质化学、分子生物学、光谱学和核磁共振波谱测定方面的高级培训。本科生将接受基础实验室培训、蛋白质化学、分子生物学、光谱学、科学伦理以及数据分析和呈现方面的培训。所有学员都将参与所产生的知识的传播。 PI 将把该项目中产生的知识纳入结构生物学和蛋白质的研究生课程中。此外，数据将在同行评审期刊上发表，并在科学会议和研究机构上展示。