Kinetic Intermediates in Folding of Histone Dimers

该提案的总体目标是表征组蛋白动力学折叠中间体的结构， 解决这些物种是阻碍生产性折叠的陷阱还是手段这一悬而未决的问题 来加速折叠，如果是的话，如何加速这一提议将扩大对蛋白质折叠到二聚体的理解， 其中二级和三级结构的形成必须与以下物质的适当结合相协调 多肽，同时避免导致聚集和原纤化的不适当缔合。诸如折叠 模型，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）它们证明蛋白质序列简并性、高度结构保守性 序列相似性低。简并性是预测方法的一个关键绊脚石， 通过研究同源结构接近;和3）尽管保守的折叠，真核H2 A-H2 B hMfB和hPyA 1同源二聚体通过不同的动力学机制折叠， 研究单体和二聚体中间体如何促进快速结合和折叠。 两个具体的目标将测试以下假设：1）更快的折叠是通过人口的动力学 中间体;使中间体不稳定将减缓折叠。单体和二聚体的结构 动力学中间体将使用CD、FL、Cys保护和诱变来确定，以调节它们的 以确定它们的种群是否有利于快速折叠。2)动力学中间体加速 折叠是有利的，即使在促进非途径寡聚化的大分子拥挤溶液中也是如此 部分折叠的物种。三种组蛋白的折叠效率，它们在有和没有 中间体，将在充满高浓度惰性聚合物的溶液中进行检查。 这些研究的长期目标有两个方面：确定一般规则，包括 中间体，关于折叠差的多肽如何有效地识别其他大分子，同时避免 不适当的协会，如与自我，导致病理性低聚物;并制定一个详细的 核小体组装和动力学的分子、热力学和动力学描述。的结果 建议和长期目标有两个方面的医疗相关性。首先，许多人类疾病 包括淀粉样纤维在内的蛋白质错误折叠。这些病理结构通常由以下原因引起： 中间体的结构域交换的低聚物，如组蛋白折叠，似乎特别容易受到影响。 病理性寡聚化第二，组蛋白的稳定性和瞬时分布决定了 核小体动力学和功能。DMA的核小体包装调节过程，例如 转录、复制和修复当这些过程因为核小体的错误调节而出错时 功能、组装和动力学，导致疾病状态，特别是癌症。

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.