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（由适用提供）：该项目的目的是定义可用于识别“变质”蛋白质的特征特征，这些特征，多肽，多肽在本机状态的无关结构之间相互互换。趋化因子家族的不寻常成员人淋巴结蛋白（LTN/XCL1）以〜1 s-1的速度进行了两种不同的天然状态结构之间的可逆重排。我们求解了每个物种的NMR结构，发现一个状态（LTN10）对应于配置的趋化因子折叠并激活XCR1，XCR1是淋巴结蛋白的特异性G蛋白偶联受体（GPCR），而另一个（LTN40）形式（LTN40）形式a dimeric a dimeric- sandwich具有高亲和力的glycosaminogminoglycanslycanslycanslycanslycans（Gag）（Gag）（Gag）（Gag）（Gag）（Gag）（Gag）。在物理溶液条件下，这两个组合物同样丰富。淋巴结蛋白提供了一个独特的机会来解决有关这种新蛋白质类别的基本问题：一个氨基酸序列如何简单地编码两个具有相等热力学稳定性的完全不同的天然状态结构，而变质蛋白是如何源于（（可能是）单个单倍祖先的？我们的机械假设是，在物理温度下，一种结构的“冷”变性为变质互连的潜力与另一个边缘稳定但不相关的结构的潜力。我们将追求三个旨在在其在趋化因子家族中的亲戚的背景下揭示LTN变质的起源的特定目的，而这些目标都没有表现出这种行为。首先，我们将通过使用NMR和荧光光谱绘制折叠能环境来监测尿素引起的每个构象状态的展开，从而定义冷变性在LTN变质中的作用。接下来，我们建议确定足以稳定非词法趋化因子中替代天然状态的结构元素。我们假设LTN序列中的“二重性代码”由稳定和破坏稳定的结构元素组成，这些元素可以调整每个状态折叠的自由能，以便它们在物理条件下同样填充。最后，我们将使用新的计算方法进行祖先基因重建来识别分离趋化因子家族的变质和非单态分支的进化节点。古代序列的实验定义的结构，热力学和功能谱（约100-4亿年历史）将使我们能够鉴定关键的氨基酸变化，从而导致结构性变质的出现。总的来说，拟议的研究将评估生理温度下冷变性的功能相关性，作为蛋白质变质的机制，并对变质蛋白进行首次进化分析。此外，该项目将为任何趋化因子（与许多人类疾病直接相关的蛋白质家族）提供第一个详细分析。