Energetic Insights into Protein Disorder and Function from the Scaffold IscU

描述（由申请人提供）：据信，相当大比例的蛋白质在没有明确结构的情况下存在并执行其生物学功能。内含子无序蛋白（IDP）缺乏刚性的三维折叠或含有大量缺乏结构的氨基酸。IDP功能的关键是它们能够对大量构象进行采样并与广泛的蛋白质伴侣相互作用。据预测，IDP在高级真核生物中非常普遍，其中高达一半的蛋白质完全是 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。虽然紊乱显然是蛋白质功能中大量使用的一种特征，但它对理论和实验表征提出了广泛的挑战，使我们对IDP的基本理解存在很大的知识缺口。本论文的目标是对一个蛋白质模型系统--大肠杆菌进行热力学和结构的基础研究。coli IscU，它利用无序和结构化构象作为其 功能IscU是铁-硫（Fe-S）簇生物合成和转移的支架蛋白， 在整个Fe-S簇组装和转移机制中，当它与几种蛋白质伴侣相互作用时，选择性地对其结构化或无序构象进行取样。具体目标1将应用由NMR、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。ISCU的独特之处在于，它在可观察到的温度下进行冷变性，而不需要化学物质的帮助来扰乱其能量景观。这种极其罕见的特性将使我们能够测试和回答仍然围绕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-蛋白质复合物的结构和能量学。该目标将试图测试在具体目标1中获得的IscU知识是否可以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我们如何使用SAXS建立IscU蛋白质-蛋白质复合物的分子模型的限制，并对IscU的蛋白质-蛋白质相互作用进行热力学研究。总之，这些研究将为IDP的结构和能量学及其与蛋白质伴侣的相互作用提供急需的基本见解。总体而言，拟议的研究将使申请人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NMR方法方面的培训。

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.