STRUCTURAL CHARACTERIZATION OF THE NUCLEOSOME-CHD1 COMPLEX

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 CHD1是一种由ATP驱动的多结构域染色质重塑蛋白。虽然已知ATPase结构域通过打破组蛋白与DNA的接触来驱动DNA沿组蛋白核心移动，但DNA结合域也结合了核小体的侧翼区域。我们和其他人已经确定了单个组分的高分辨结构，但随着重塑周期的进展，核小体上的化学计量和准确定位尚不清楚。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体的SAXS包膜中，获得CHD1不同结构域相对于核小体的精确空间组织信息。 我们将获得核小体-CHD1复合体的几个变体及其单独成分的SAXS数据。我们已经纯化了野生型CHD1的均质样本和包含E.coliAraC蛋白序列特异性DNA结合域的嵌合构建体。我们还用含有AraC识别序列的DNA重组了核小体。我们已经证实在凝胶过滤柱和天然的聚丙烯酰胺凝胶上形成了1：2的核小体-CHD1复合体。我们还使用了生化功能分析来证明嵌合构建物具有酶活性。我们用SEC-MALS(尺寸排除层析-多角度光散射)进一步表征和确认了我们纯化的络合物的单分散性质。我们还获得了初步的SAXS数据，证实了我们样品的同质性。

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.