Structural and energetic origins of metamorphic protein folding

变质蛋白质折叠的结构和能量起源

• 批准号: 8735210
• 负责人: Brian F Volkman
• 金额: $ 35.27万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-24 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735210
• 关键词: Address; Adopted; Affinity; Alzheimer' s Disease; Amino Acid Sequence; Amino Acids; Behavior; Biological; Biological Metamorphosis; Biological Process; Categories; Characteristics; Code; Computing Methodologies; DNA Sequence Rearrangement; Databases; Dependence; Development; Disease; Elements; Equilibrium; Exhibits; Family; Fluorescence Spectroscopy; Foundations; Free Energy; G-Protein-Coupled Receptors; Genes; Glycosaminoglycans; Goals; Human; Kinetics; Maps; Measurement; Measures; Methods; Molecular Conformation; Monitor; NMR Spectroscopy; Parkinsonian Disorders; Pattern; Peptide Sequence Determination; Phylogenetic Analysis; Physiological; Property; Protein Family; Proteins; Relative (related person); Role; Shapes; Solutions; Structure; Temperature; Testing; Thermodynamics; Urea; XCL1 gene; XCR1 gene; base; chemokine; design; human disease; in vivo; member; polypeptide; primary amyloidosis of light chain type; protein folding; protein function; protein misfolding; public health relevance; reconstruction; tool

DESCRIPTION (provided by applicant): The goal of this project is to define characteristic features that can be used to identify "metamorphic" proteins, polypeptides that interconvert between unrelated structures in the native state. Human lymphotactin (Ltn/XCL1), an unusual member of the chemokine family, undergoes a reversible rearrangement between two distinct native state structures at a rate of ~1 s-1. We solved the NMR structure of each species and found that one state (Ltn10) corresponds to the conserved chemokine fold and activates XCR1, the specific G protein-coupled receptor (GPCR) for lymphotactin, whereas the other (Ltn40) forms a dimeric ¿-sandwich with high affinity for glycosaminoglycans (GAG). The two conformations are equally abundant in physiological solution conditions. Lymphotactin provides a unique opportunity to address fundamental questions about this new category of proteins: How does one amino acid sequence simultaneously encode two entirely different native state structures with equal thermodynamic stability, and how did the metamorphic protein arise from a (presumably) single-fold ancestor? Our mechanistic hypothesis is that "cold" denaturation of one structure at physiological temperatures creates the potential for metamorphic interconversion with another marginally stable but unrelated structure. We will pursue three specific aims designed to reveal the origin of Ltn metamorphism in the context of its relatives in the chemokine family, none of which exhibit this behavior. First, we will define the role of cold denaturation in Ltn metamorphosis by mapping the folding energy landscape using NMR and fluorescence spectroscopy to monitor urea-induced unfolding of each conformational state. Next, we propose to identify the structural elements sufficient to stabilize an alternative native state in a non-metamorphic chemokine. We hypothesize that a "duality code" within the Ltn sequence is composed of both stabilizing and destabilizing structural elements that adjust the free energy of folding for each state so that they are equally populated under physiological conditions. Finally, we will use new computational methods for ancestral gene reconstruction to identify evolutionary nodes separating the metamorphic and non-metamorphic branches of the chemokine family. Experimentally defined structural, thermodynamic and functional profiles of ancient sequences (~100-400 million years old) will enable us to identify key amino acid changes leading to the emergence of structural metamorphism. Collectively, the proposed studies will assess the functional relevance of cold denaturation at physiological temperature as a mechanism for protein metamorphism and perform the first evolutionary analysis of a metamorphic protein. Additionally, this project will provide the first detailed analysis of thermodynamic stability for any chemokine, a protein family with direct relevance to many human diseases.

描述（由申请人提供）：该项目的目标是定义可用于识别“变态”蛋白质、在天然状态下不相关结构之间相互转化的多肽的特征，人淋巴趋化素（Ltn/XCL1）是一种不寻常的成员。趋化因子家族中的一种，以约 1 s-1 的速率在两种不同的天然状态结构之间进行可逆重排，我们解析了每个物种的 NMR 结构，并发现了一种状态。 (Ltn10) 对应于保守的趋化因子折叠并激活 XCR1，淋巴趋化素的特异性 G 蛋白偶联受体 (GPCR)，而另一个 (Ltn40) 形成二聚体 ¿ -对糖胺聚糖 (GAG) 具有高亲和力的三明治结构在生理溶液条件下同样丰富，为解决这一新类别蛋白质的基本问题提供了独特的机会：一个氨基酸序列如何同时编码两个完全不同的天然蛋白。具有相同热力学稳定性的状态结构，以及变质蛋白是如何从（可能的）单倍祖先产生的？我们的机制假设是一种结构在生理温度下的“冷”变性创造了潜力？我们将追求三个具体目标，旨在揭示 Ltn 趋化因子家族中其亲属的起源，其中没有一个表现出这种行为。接下来，我们建议识别足以稳定 Ltn 变态的结构元素，通过使用核磁共振和荧光光谱绘制折叠能量图来监测尿素诱导的展开。我们追求Ltn序列中的“二元性代码”由稳定和不稳定的结构元件组成，这些元件调整每个状态的折叠自由能，以便它们在生理条件下同等地分布。最后，我们将使用新的计算方法进行祖先基因重建，以识别区分趋化因子家族的变质和非变质分支的进化节点，实验定义了古代序列的结构、热力学和功能特征。 （约 100-4 亿年前）将使我们能够识别导致结构变质出现的关键氨基酸变化。总的来说，拟议的研究将评估生理温度下冷变性作为蛋白质变质机制的功能相关性并进行研究。此外，该项目还将首次对任何趋化因子（与许多人类疾病直接相关的蛋白质家族）的热力学稳定性进行详细分析。