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态折叠中间体的结构，并解决未解决的问题，即这些物种是阻碍有效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结合协调，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，组蛋白具有三个关键特征：1)作为核小体的蛋白质核心，其表征将阐明核小体的功能；2)它们体现了蛋白质序列简并性、高结构保守性和低序列相似性。简并性是预测方法的关键障碍，最好通过研究同源结构来解决；3)尽管存在保守折叠，但真核H2A-H2B异源二聚体和古生物hMfB和hPyA1同源二聚体通过不同的动力学机制折叠，从而可以研究单体和二聚体中间体如何促进快速结合和折叠。两个特定的目标将测试以下假设：1)更快的折叠是由动能中间体的人口实现的；中间产物的不稳定将减缓折叠速度。将确定单体和二聚体动力学中间体的结构，使用CD， FL， Cys保护和诱变来调节它们的稳定性，以确定它们的种群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，也会促进部分折叠物种的非通路寡聚化。这三种组蛋白的折叠效率，有或没有中间物折叠，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是双重的：定义一般规则，包括中间体的影响，对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，导致病理低聚物；并对核小体的组装和动力学进行详细的分子、热力学和动力学描述。这一建议的结果和长期目标具有医学相关性的两个方面。首先，许多人类疾病与蛋白质错误折叠有关，包括淀粉样蛋白原纤维。这些病理结构通常来自中间体。区域交换寡聚物，如组蛋白折叠，似乎特别容易受到病理性寡聚的影响。其次，组蛋白的稳定性和短暂填充物种决定了核小体的动力学和功能。DNA的核小体包装调节转录、复制和修复等过程。当这些过程因核小体功能、组装和动力学调节不当而出错时，就会导致疾病状态，尤其是癌症。

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.