Kinetic Intermediates in Folding of Histone Dimers

描述（由申请人提供）：本提案的总体目标是表征组蛋白动力学折叠中间体的结构，并解决尚未解决的问题，即这些物质是阻碍生产性折叠的陷阱还是加速折叠的手段，如果是，如何。这一建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多肽的适当缔合相协调，同时避免导致聚集和原纤化的不适当缔合。组蛋白作为一种折叠模型，具有三个关键特征：1）作为核小体的核心蛋白，其特征将有助于阐明核小体的功能; 2）其特征是蛋白质序列简并性、高度保守性和低序列相似性。简并性是预测方法的一个关键绊脚石，最好通过研究同源结构来解决; 3）尽管有保守的折叠，但真核H2 A-H2 B异源二聚体和古细菌hMfB和hPyA 1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有助于快速缔合和折叠。两个具体的目标将测试以下假设：1）更快的折叠是通过动力学中间体的群体实现的;使中间体不稳定将减缓折叠。将确定单体和二聚体动力学中间体的结构，使用CD、FL、Cys保护和诱变来调节其稳定性，以确定其群体是否有利于快速折叠。2)加速折叠的动力学中间体是有利的，即使在大分子拥挤的溶液中，促进部分折叠物种的非途径寡聚化。三个组蛋白的折叠效率，折叠和没有中间体，将在充满高浓度的惰性聚合物的溶液中进行检查。这些研究的长期目标是双重的：定义一般规则，包括中间体的影响，关于折叠不良的多肽如何有效地识别其他大分子，同时避免不适当的关联，如与自身，导致病理性寡聚体;并开发详细的分子，热力学和动力学描述核小体组装和动力学。该建议的结果和长期目标具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。这些病理结构通常由中间体产生。结构域交换的寡聚体，如组蛋白折叠，似乎特别容易发生病理性寡聚化。第二，组蛋白的稳定性和瞬时分布决定了核小体的动力学和功能。DNA的核小体包装调节诸如转录、复制和修复的过程。当这些过程由于核小体功能、组装和动力学的失调而出错时，就会导致疾病状态，特别是癌症。

DESCRIPTION (provided by applicant): 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 structure conservation 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 DNA 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.