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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 CHD1是ATP驱动的多域染色质复制蛋白。 虽然众所周知，ATPase结构域通过打破组蛋白DNA接触来驱动DNA沿组蛋白核的运动，但DNA结合结构域也结合了核小体的侧翼区域。我们和其他人已经确定了各个组件的高分辨率结构，但是随着重塑循环的进行，核小体的化学计量和精确方向尚未阐明。 我们希望通过将单个组件的高分辨率结构停靠到整个复合物中的萨克斯信封中，以获取有关CHD1不同域的空间组织的精确信息。 我们将获得核小体CHD1复合物及其单个成分的几种变体的SAXS数据。 我们已经纯化了野生型CHD1的均匀样品和嵌合构建体，该构建体包含来自大肠杆菌蛋白ARAC的序列特异性DNA结合结构域。 我们还使用含有策略性放置的ARAC识别序列的DNA重构核小体。我们已经确认形成了凝胶滤成柱和天然聚丙烯酰胺凝胶上的1：2核小体-CHD1复合物。我们还使用生化功能测定法表明嵌合构建体具有酶促的能力。 我们进一步表征并确认了使用SEC-MALS（尺寸排除色谱 - 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，这些数据证实了样本的同质性。