STRUCTURAL CHARACTERIZATION OF THE NUCLEOSOME-CHD1 COMPLEX

核小体-CHD1 复合物的结构表征

• 批准号: 8363549
• 负责人: GREGORY DEAN BOWMAN
• 金额: $ 0.57万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363549
• 关键词: ATPase Domain; Binding; Biochemical; Biological Assay; Complex; DNA; DNA Binding Domain; Data; Docking; Escherichia coli Proteins; Funding; Gel Chromatography; Grant; Histones; Individual; Macromolecular Complexes; Molecular Sieve Chromatography; Mono-S; Motion; National Center for Research Resources; Nature; Nucleosomes; Principal Investigator; Proteins; Research; Research Infrastructure; Resolution; Resources; Sampling; Source; Structure; United States National Institutes of Health; Variant; chromatin remodeling; cost; light scattering; polyacrylamide gels; reconstitution; stoichiometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. CHD1 is an ATP-driven multi-domain chromatin-remodeling protein. While it is known that an ATPase domain drives the motion of DNA along the histone-core by breaking histone-DNA contacts, the DNA-binding domain also binds the flanking regions of the nucleosome. We and others have determined the high resolution structures of the individual components but the stoichiometry and exact orientation on the nucleosome as the remodeling cycle progresses is yet to be elucidated. We hope to obtain precise information about the spatial organization of the different domains of CHD1 with respect to the nucleosome by docking high-resolution structures for the individual components into SAXS envelopes for the whole complex. We will obtain SAXS data for several variants of the nucleosome-CHD1 complex, and their individual components. We have purified homogeneous samples of wild-type CHD1 and chimeric constructs containing a sequence specific DNA-binding domain from the E.coli protein AraC. We have also reconstituted nucleosomes with DNA containing a strategically placed recognition sequence for AraC. We have confirmed formation of a 1:2 nucleosome-CHD1 complex on a gel-filtration column and a native polyacrylamide gel. We have also used biochemical functional assay to show that the chimeric constructs are enzymatically competent. We have further characterized and confirmed the mono-disperse nature of the complexes we have purified using SEC-MALS (Size Exclusion Chromatography - Multiple Angle Light Scattering). We have also obtained preliminary SAXS data that corroborates the homogeneity of our samples.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 CHD1是一种ATP驱动的多结构域染色质重塑蛋白。 虽然已知ATP酶结构域通过破坏组蛋白-DNA接触来驱动DNA沿着组蛋白-核心运动，但DNA结合结构域也结合核小体的侧翼区域。我们和其他人已经确定了高分辨率的结构的各个组成部分，但核小体的化学计量和确切的方向，作为重塑周期的进展还有待阐明。 我们希望通过将单个组分的高分辨率结构对接到整个复合物的SAXS信封中，获得关于CHD1不同结构域相对于核小体的空间组织的精确信息。 我们将获得核小体-CHD1复合物的几种变体及其单个组分的SAXS数据。 我们已经纯化了野生型CHD1和嵌合构建体的同源样品，所述嵌合构建体含有来自大肠杆菌蛋白AraC的序列特异性DNA结合结构域。 我们还用含有AraC识别序列的DNA重建了核小体。我们已经证实了凝胶过滤柱和天然聚丙烯酰胺凝胶上1：2核小体-CHD1复合物的形成。我们还使用生化功能测定来显示嵌合构建体是酶活性的。 我们已经进一步表征并证实了我们已经使用SEC-MALS（尺寸排阻色谱-多角度光散射）纯化的复合物的单分散性质。 我们还获得了初步的SAXS数据，证实了我们的样品的均匀性。