Structural and energetic origins of metamorphic protein folding

描述（由申请人提供）：该项目的目标是定义可用于识别“变质”蛋白质的特征特征，多肽在天然状态下在不相关结构之间相互转化。人类淋巴趋化因子（Ltn/XCL1）是趋化因子家族中一个不寻常的成员，在两种不同的天然状态结构之间以~1 s-1的速率进行可逆重排。我们解决了每个物种的核磁共振结构，发现一个状态（Ltn10）对应于保守的趋化因子折叠并激活特异性G蛋白偶联受体（GPCR） XCR1，而另一个状态（Ltn40）形成对糖胺聚糖（GAG）具有高亲和力的二聚体¿-三明治。这两种构象在生理溶液条件下同样丰富。淋巴蛋白提供了一个独特的机会来解决关于这类新蛋白质的基本问题：一个氨基酸序列如何同时编码两个完全不同的具有相同热力学稳定性的天然状态结构，以及变形蛋白如何从（可能的）单一祖先产生？我们的机制假设是，一种结构在生理温度下的“冷”变性产生了与另一种略微稳定但不相关的结构的变质相互转化的潜力。我们将追求三个特定的目标，旨在揭示Ltn在趋化因子家族中亲属的背景下变质的起源，其中没有一个表现出这种行为。首先，我们将定义冷变性在Ltn变态中的作用，通过使用核磁共振和荧光光谱来绘制折叠能量景观，以监测尿素诱导的每个构象状态的展开。接下来，我们建议确定足以稳定非变质趋化因子的替代天然状态的结构元件。我们假设Ltn序列中的“对偶代码”由稳定和不稳定的结构元素组成，这些结构元素调节每个状态的折叠自由能，使它们在生理条件下均匀分布。最后，我们将使用新的计算方法进行祖先基因重建，以确定分离趋化因子家族的变质和非变质分支的进化节点。实验确定的古代序列（~1 -4亿年）的结构、热力学和功能特征将使我们能够确定导致结构变质作用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，并对变质蛋白进行首次进化分析。此外，该项目将首次详细分析任何趋化因子的热力学稳定性，趋化因子是与许多人类疾病直接相关的蛋白质家族。

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.