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“变性”蛋白质的特征，即在天然状态下不相关结构之间相互转化的多肽。人类趋化因子家族中的一个特殊成员Ltn/XCL 1在两种不同的天然状态结构之间以约1 s-1的速率发生可逆重排。我们解决了每个物种的NMR结构，并发现一个状态（Ltn 10）对应于保守的趋化因子折叠并激活XCR 1，XCR 1是针对趋化因子的特异性G蛋白偶联受体（GPCR），而另一个状态（Ltn 40）形成了对糖胺聚糖（GAG）具有高亲和力的二聚体夹心。这两种构象在生理溶液条件下同样丰富。光变性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等热力学稳定性的天然状态结构，以及变性蛋白质如何从（大概）单倍祖先产生？我们的机械假说是，“冷”变性的一种结构在生理温度下产生的潜在变质相互转换与另一个边缘稳定，但无关的结构。我们将追求三个特定的目标，旨在揭示LTN变质的起源，在其亲属的趋化因子家族的背景下，没有一个表现出这种行为。首先，我们将定义冷变性LTN变态的作用，通过映射的折叠能量景观，使用NMR和荧光光谱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性趋化因子中的替代天然状态的结构元素。我们假设Ltn序列中的“二元代码”由稳定和不稳定的结构元素组成，这些结构元素调整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重建祖先基因，以确定分离的趋化因子家族的变性和非变性分支的进化节点。实验确定的古代序列（~ 1亿-4亿年）的结构，热力学和功能概况将使我们能够确定导致结构变质出现的关键氨基酸变化。总的来说，拟议的研究将评估在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，该项目将提供任何趋化因子的热力学稳定性的第一个详细分析，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.