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，“变质”蛋白质是指在自然状态下不相关结构之间相互转化的多肽。人淋巴动蛋白(LTN/XCL1)是趋化因子家族中的一个特殊成员，它以~1 S~(-1)的速率在两个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核磁共振结构，发现一个状态(Ltd N10)对应于保守的趋化因子折叠并激活淋巴肌动蛋白的特异性G蛋白偶联受体(GPCR)XCR1，而另一个状态(Ltd N40)形成与糖胺聚糖(GAG)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。淋巴肌动蛋白提供了一个独特的机会来解决关于这一新的蛋白质类别的基本问题：一个氨基酸序列如何同时编码两个具有相同热力学稳定性的完全不同的自然状态结构，以及变性蛋白质是如何从(可能的)单重祖先产生的？我们的机制假设是，一种结构在生理温度下的“冷”变性创造了与另一种边缘稳定但无关的结构进行变质相互转化的可能性。我们将追求三个特定的目标，旨在通过趋化因子家族中的亲属来揭示LTN变质的起源，这些家族中没有一个表现出这种行为。首先，我们将通过核磁共振和荧光光谱绘制折叠能量图谱来监测尿素诱导的每种构象状态的展开，从而确定冷变性在LTN变形中的作用。下一步，我们建议确定在非变性趋化因子中足以稳定替代自然状态的结构元件。我们假设LTN序列中的“对偶码”由稳定和不稳定的结构元件组成，这些结构元件调节每种状态的折叠自由能，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，以识别分隔趋化因子家族变质和非变质分支的进化节点。实验确定的古层序(距今约1亿-4亿年)的结构、热力学和功能剖面将使我们能够识别导致构造变质作用出现的关键氨基酸变化。总之，拟议的研究将评估生理温度下的冷变性作为蛋白质变性机制的功能相关性，并首次对变性蛋白质进行进化分析。此外，该项目将首次对趋化因子的热力学稳定性进行详细分析，趋化因子是一种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.