Kinetic Intermediates in Folding of Histone Dimers

这项提议的总体目标是表征组蛋白动力学折叠中间体的结构和 解决悬而未决的问题，即这些物种是阻碍生产性折叠的陷阱还是手段 以加速折叠，如果是这样的话，是如何进行的。这一提议将把对蛋白质折叠的理解扩展到二聚体， 二级和三级结构的形成必须与适当的 多肽，同时避免不适当的结合导致聚集和纤颤。作为一种折叠 模型中，组蛋白有三个关键特征：1)作为核小体的蛋白质核心，它们的特性将 阐明核小体的功能；2)它们例证了蛋白质序列的简并性和高度结构保守性 序列相似性低。简并是预测方法的关键绊脚石，这是最好的 通过研究同源结构来探讨；以及3)尽管有保守的折叠，但真核细胞的H2A-H2B 异源二聚体和古HMFB和hPyA1同源二聚体通过不同的动力学机制折叠，允许 研究单体和二聚体中间体如何促进快速缔合和折叠。 两个特定的目标将检验以下假设：1)通过动力学种群实现更快的折叠 中间体；破坏中间体的稳定会减慢折叠速度。单体和二聚体的结构 动力学中间体将被确定，使用CD，FL，Cys保护和突变来调节它们的 以确定它们的种群是否倾向于快速折叠。2)加速的动力学中间体 折叠是有利的，即使在促进旁路齐聚的大分子拥挤的溶液中也是如此 部分折叠的物种。有折叠和无折叠的三种组蛋白的折叠效率 中间体，将在挤满高浓度惰性聚合物的溶液中进行检测。 这些研究的长期目标有两个：定义一般规则，包括 中间体，关于折叠不良的多肽如何有效识别其他大分子同时避免 不适当的关联，如与自身的关联，会导致病理性寡聚体；并发展出详细的 核小体组装和动力学的分子、热力学和动力学描述。这样做的结果 提案和长期目标有两个方面的医学相关性。首先，许多人类疾病 涉及蛋白质错误折叠，包括淀粉样纤维。这些病理结构通常是由 中间体。结构域互换的寡聚体，如组蛋白折叠，似乎特别容易受到 病理性寡聚。其次，稳定性和短暂繁殖的组蛋白物种决定了 核小体动力学和功能。DNA的核小体包装调节过程，如 转录、复制和修复。当这些过程由于核小体的错误调节而出错时 功能、装配和动力学、疾病状态，尤其是癌症。

The overall goal of this proposal is to characterize the structure of histone kinetic folding intermediates and address the unresolved issue of whether such species are traps that hinder productive folding OR are means to accelerate folding, and if so, how. This proposal will expand understanding of protein folding to dimers, where formation of secondary and tertiary structure must be coordinated with the appropriate association of polypeptides, while avoiding inappropriate association leading to aggregation and fibrillation. As a folding model, histones have three key features: 1) as the protein core of the nucleosome, their characterization will elucidate nucleosome function; 2) they exemplify protein sequence degeneracy¿high structureconservation with low sequence similarity. Degeneracy is a key stumbling block to prediction methods, that is best approached by studying homologous structures; and 3) despite a conserved fold, the eukaryotic H2A-H2B heterodimer and the archael hMfB and hPyA1 homodimers fold by different kinetic mechanisms, permitting study of how monomeric and dimeric intermediates contribute to rapid association and folding. Two specific aims will test the following hypotheses: 1) Faster folding is achieved by population of kinetic intermediates; destabilizing the intermediates will slow folding. The structure of monomeric and dimeric kinetic intermediates will be determined, using CD, FL, Cys protection and mutagenesis to modulate their stabilities to determine if their population favors rapid folding. 2) Kinetic intermediates which accelerate folding are favorable, even in macromolecularly crowded solutions that promote off-pathway oligomerization of partially folded species. The folding efficiency of the three histones, which fold with and without intermediates, will be examined in solutions crowded with high concentrations of inert polymers. The long term goals of the studies are two-fold: to define general rules, including the impact of intermediates, on how poorly folded polypeptides efficiently recognize other macromolecules while avoiding inappropriate associations, such as with self, that lead to pathological oligomers; and to develop a detailed molecular, thermodynamic and kinetic description of nucleosome assembly and dynamics. The results of this proposal and the long term goals have two aspects of medical relevance. First, many human diseases involve protein misfolding, including amyloid fibrils. These pathological structures typically arise from intermediates. Domain-swapped oligomers, like the histone fold, seem particularly susceptible to pathological oligomerization. Second, stability and transiently populated species of histones dictate nucleosome dynamics and function. Nucleosomal packaging of DMA regulates processes such as transcription, replication and repair. When these processes go awry because of misregulation of nucleosome function, assembly and dynamics, disease states result, particularly cancer.