Energetic Insights into Protein Disorder and Function from the Scaffold IscU

从支架 IscU 深入了解蛋白质无序和功能

• 批准号: 8716395
• 负责人: Jameson R Bothe
• 金额: $ 1.95万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2014-08-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716395
• 关键词: Amino Acids; Anabolism; Binding; Biological Models; Biological Process; Cardiovascular Diseases; Chemicals; Complex; Data; Development; Diabetes Mellitus; Differential Scanning Calorimetry; Disease; Equilibrium; Escherichia coli; Escherichia coli Proteins; Eukaryota; Future; Goals; Heating; Investigation; Iron; Knowledge; Left; Libraries; Malignant Neoplasms; Maps; Measures; Methodology; Methods; Modeling; Molecular Conformation; Molecular Models; NMR Spectroscopy; Neurodegenerative Disorders; Organism; Paint; Plague; Play; Property; Protein Binding; Proteins; Research; Roentgen Rays; Role; Sampling; Scaffolding Protein; Signaling Protein; Societies; Stretching; Structure; Sulfur; System; Temperature; Testing; Thermodynamics; Training; burden of illness; cold temperature; conformational conversion; flexibility; insight; molecular modeling; mutant; protein complex; protein expression; protein function; protein protein interaction; protein purification; protein structure; public health relevance; scaffold; trait

DESCRIPTION (provided by applicant): It is believed that a significant percentage of proteins exist and carry out their biological function without a well- defined structure. Intrinsically disordered proteins (IDPs) lack a rigid three-dimensional fold or contain large stretches of amino acids devoid of structure. Key to the function of IDPs is their ability to sample a vast array of conformations and to interact with a wide range of protein partners. It is predicted that IDPs are highly prevalent in high order eukaryotic organisms where up to half of their proteins are entirely disordered or contain large stretches of amino acids that are disordered. Further, IDPs are believed to play significant roles in several diseases including cancer, cardiovascular disease, diabetes, and neurodegenerative diseases. While disorder is clearly a trait abundantly used in protein function, it has posed extensive challenges to theoretical and experimental characterization leaving a large knowledge gap in our basic understanding of IDPs. The goal of this proposal is to carry out fundamental thermodynamic and structural studies on a model protein system, E. coli IscU, which utilizes disordered and structured conformations as part of its function. IscU is the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis and transfer and selectively samples its structured or disordered conformation as it interacts with several protein partners throughout the Fe-S cluster assembly and transfer mechanism. Specific Aim 1 will apply a biophysical toolkit composed of NMR, small- angle X-ray scattering (SAXS), and differential scanning calorimetry to map the structural energetic landscape of IscU. Particularly unique to IscU is that it undergoes cold denaturation at observable temperatures without the aid of chemicals that perturb its energetic landscape. This extremely rare trait will allow us to test and answer structural and thermodynamic questions that still revolve around cold denaturation. Specific Aim 2 will focus on characterizing the structure and energetics of IscU protein-protein complexes. This aim will seek to test if the knowledge of IscU obtained in Specific Aim 1 can be applied in a predictive manner to complicated protein-protein interactions. We will develop methods to test and determine the limits of how well we can build molecular models of IscU's protein-protein complexes using SAXS and carry out thermodynamic studies on IscU's protein-protein interactions. Together, these studies will provide much needed fundamental insights into both the structure and energetics of IDPs and their interactions with protein-partners. Overall, the proposed studies will expose the applicant to the field of protein research and provide training in protein expression and purification, SAXS, and protein NMR methods.

描述(由申请人提供)：人们相信，在没有明确结构的情况下，有相当大比例的蛋白质存在并发挥其生物学功能。固有无序蛋白(IDPs)缺乏刚性的三维折叠或含有大量缺乏结构的氨基酸。国内流离失所者发挥作用的关键是他们有能力对大量的构象进行采样，并与广泛的蛋白质伙伴相互作用。据预测，IDPs在高级真核生物中高度流行，在那里，多达一半的蛋白质是完全 无序的或含有大量无序的氨基酸。此外，国内流离失所者被认为在包括癌症、心血管疾病、糖尿病和神经退行性疾病在内的几种疾病中发挥重要作用。虽然无序性显然是蛋白质功能中广泛使用的一个特征，但它给理论和实验表征带来了广泛的挑战，给我们对国内流离失所者的基本理解留下了巨大的知识鸿沟。这项建议的目标是对一个模型蛋白质系统进行基本的热力学和结构研究，该系统利用无序和结构化的构象作为其 功能。IscU是铁-硫(Fe-S)簇生物合成和转移的骨架蛋白。 在Fe-S簇组装和转移机制中，当它与几个蛋白质伙伴相互作用时，选择性地采样其结构或无序构象。具体目标1将应用由核磁共振、小角X射线散射(SAXS)和差示扫描量热法组成的生物物理工具包来绘制ISCU的结构能量景观图。ISCU特别独特的是，它在可观察到的温度下经历冷变性，而不需要化学物质的帮助来扰乱其充满活力的景观。这一极其罕见的特性将使我们能够测试和回答仍然围绕着冷变性的结构和热力学问题。具体目标2将侧重于表征ISCU蛋白质-蛋白质复合体的结构和能量。这一目标将寻求测试在特定目标1中获得的ISCU知识是否能够以预测的方式应用于复杂的蛋白质-蛋白质相互作用。我们将开发方法来测试和确定使用SAXS建立ISCU蛋白质-蛋白质复合体的分子模型的限度，并对ISCU的蛋白质-蛋白质相互作用进行热力学研究。总之，这些研究将对国内流离失所者的结构和能量以及他们与蛋白质伙伴的相互作用提供亟需的基本见解。总体而言，拟议的研究将使申请者接触蛋白质研究领域，并提供蛋白质表达和纯化、SAXS和蛋白质核磁共振方法方面的培训。