Biophysics of Macromolecular Complexes
大分子复合物的生物物理学
基本信息
- 批准号:8939548
- 负责人:
- 金额:$ 33.36万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:AdoptedAffinityAmericanAnalytical BiochemistryArchitectureBindingBiochemicalBiologicalBiological AssayBiophysicsBirdsC-terminalCapsidCapsid ProteinsCell NucleusCell membraneCell physiologyCellsChemicalsChromatinChromatin FiberChromatin StructureChromosome StructuresChromosomesCleaved cellCollaborationsComplementCytoplasmDNADNA biosynthesisDimerizationDiseaseErythrocytesGAG GeneGene ExpressionGenesGeneticHIV-1HeterochromatinHumanIn VitroIndividualInternationalInvestigationJournalsLaboratoriesLengthMacromolecular ComplexesMethodsModelingMolecular BiologyMolecular ConformationNational Institute of Diabetes and Digestive and Kidney DiseasesNucleic AcidsNucleocapsidPeptide HydrolasesPeptidesPhysical condensationPlayPolyproteinsProcessPropertyProteinsRelative (related person)ResolutionRetroviridaeRoleShapesSiteSocietiesStructureUnited States National Institutes of HealthViralViral ProteinsVirionWorkZincanalytical ultracentrifugationbeta Globincohesindata collection methodologydimerflexibilityfolate-binding proteinimprovedin vivoinsightinterestmacromolecular assemblymonomerprotein complexstoichiometrytool
项目摘要
Chromatin structure and architecture.
DNA within the cell nucleus is packaged into chromatin and a variety of models currently describe the structure of the condensed 30 nm chromatin fiber observed in vitro. However, evidence for this structure in vivo is lacking, except in specialized cells such as mature avian erythrocytes in which all of the chromatin is essentially inactive. We are interested in understanding the organization of DNA within condensed chromatin in vivo, as well as the topological constraints imposed on its higher order by organizing proteins such as CTCF and cohesin. We are developing high resolution chromosome capture conformation assays utilizing native chromatin fragments, such as the previously studied condensed heterochromatin flanked by the developmentally regulated folate receptor and beta-globin genes. These studies will allow us to better understand the structure of the chromatin fiber in vivo, thus providing insight in the relations between chromatin structure and essential processes such as gene expression and DNA replication.
Macromolecular assemblies of biological interest.
Biological assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies complement current investigations, as evidenced by recent work carried out with the laboratory of Dr. Clore. GAG is the primary polyprotein involved in the assembly of the human HIV-1 retrovirus. It is expressed in the cytoplasm of the host cell and transported to the plasma membrane. In the course of the budding process, HIV-1 protease cleaves GAG, leading to the formation of the mature virion. Cleavage of the GAG polyprotein results in the formation of its constituent proteins, namely matrix, capsid, nucleocapsid and the intrinsically disordered p6. Matrix regulates the binding of GAG to the cell membrane and capsid assembles to form the viral capsid. Nucleocapsid binds the viral nucleic acids and p6 interacts with cellular and viral proteins. Structural, biochemical and biophysical studies on GAG and its components are expected to provide important insight into the mechanism of HIV-1 viral assembly and subsequent budding. Initial studies focused on the structure and dynamics of the full-length capsid protein, observed in the form of exchanging monomers and dimers. In the dimer form, the C-terminal domains responsible for dimerization adopt a single orientation. The relative orientations of the N- and C-terminal domains occupy a broad distribution of states that differ significantly for the monomer and dimer forms of the protein. Importantly, the orientations observed for this protein within the HIV-1 capsid assembly are only present in a small subpopulation of the dimer distribution of states (Deshmukh et al., Journal of the American Chemical Society, 2013). Subsequent studies considered the longer capsid-spacer peptide 1-nucleocapsid fragment of GAG. These studies demonstrate that both the capsid and the nucleocapsid retain their individual structure and tumble semi-independently of each other. The addition of nucleic acids, which bind to the nucleocapsid domain and fix the orientation of the two zinc knuckles, does not influence the structure of the capsid domain. However, access of the HIV-1 protease to the spacer peptide 1 nucleocapsid site is enhanced significantly, even though the flexible spacer peptide 1 remains unstructured in the presence of nucleic acids (Deshmukh et al., Angewandte Chemie International Edition English, 2014).
Analytical ultracentrifugation is one of the primary tools used for the above mentioned hydrodynamic studies. In collaboration with colleagues from the NIH, and others, we have further improved on the methodology for data collection. This ultimately leads to more accurate hydrodynamic parameters, important in particular for hydrodynamic modeling that routinely requires the highest accuracy (Ghirlando et al., Analytical Biochemistry, 2014; Zhao et al., Analytical Biochemistry, 2014).
染色质结构和架构。
细胞核内的DNA被包装成染色质,目前有多种模型描述了体外观察到的浓缩的30 nm染色质纤维的结构。 然而,这种结构在体内的证据是缺乏的,除了在专门的细胞,如成熟的禽红细胞,其中所有的染色质基本上是无活性的。 我们有兴趣了解凝聚的染色质内的DNA在体内的组织,以及组织蛋白质,如CTCF和cohesin的拓扑约束强加在其更高的顺序。 我们正在开发利用天然染色质片段的高分辨率染色体捕获构象分析,如先前研究的两侧发育调节叶酸受体和β-珠蛋白基因的浓缩异染色质。 这些研究将使我们能够更好地了解体内染色质纤维的结构,从而深入了解染色质结构与基因表达和DNA复制等基本过程之间的关系。
具有生物学意义的大分子组装体。
生物组装体的特征在于它们的形状,化学计量和亲和力的相互作用,使用流体动力学方法。 这些研究补充了当前的调查,最近与Clore博士实验室进行的工作证明了这一点。 GAG是参与人HIV-1逆转录病毒装配的主要多蛋白。 它在宿主细胞的细胞质中表达并转运至质膜。 在出芽过程中,HIV-1蛋白酶切割GAG,导致成熟病毒体的形成。 GAG多蛋白的切割导致其组成蛋白的形成,即基质、衣壳、核衣壳和固有无序的p6。 基质调节GAG与细胞膜的结合,衣壳组装形成病毒衣壳。 核衣壳结合病毒核酸,p6与细胞和病毒蛋白相互作用。 对GAG及其组分的结构、生物化学和生物物理学研究有望为HIV-1病毒组装和随后的出芽机制提供重要见解。 最初的研究集中在全长衣壳蛋白的结构和动力学上,以交换单体和二聚体的形式观察到。 在二聚体形式中,负责二聚化的C末端结构域采用单一取向。 N-和C-末端结构域的相对取向占据了广泛的状态分布,这些状态对于蛋白质的单体和二聚体形式显著不同。 重要的是,在HIV-1衣壳组装体内观察到的这种蛋白质的取向仅存在于二聚体状态分布的一个小的亚群中(Deshmukh等人,Journal of the American Chemical Society,2013)。 随后的研究考虑了GAG的较长的衣壳-间隔肽1-核衣壳片段。 这些研究表明,衣壳和核衣壳都保留了它们各自的结构,并且彼此半独立地翻滚。 添加核酸,其结合到核衣壳结构域并固定两个锌节的方向,不影响衣壳结构域的结构。 然而,HIV-1蛋白酶对间隔肽1核衣壳位点的接近显著增强,即使柔性间隔肽1在核酸存在下保持非结构化(Deshmukh等人,Angewandte Chemie International Edition English,2014)。
分析超离心法是用于上述流体动力学研究的主要工具之一。 通过与NIH和其他机构的同事合作,我们进一步改进了数据收集的方法。 这最终导致更准确的流体动力学参数,这对于通常需要最高精度的流体动力学建模特别重要(Ghirdham等人,Analytical Biochemistry,2014; Zhao等人,Analytical Biochemistry,2014)。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Gary Felsenfeld其他文献
Gary Felsenfeld的其他文献
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{{ truncateString('Gary Felsenfeld', 18)}}的其他基金
Organization and regulation of the human insulin locus
人胰岛素基因座的组织和调节
- 批准号:
8741425 - 财政年份:
- 资助金额:
$ 33.36万 - 项目类别:
Organization and regulation of the human insulin locus
人胰岛素基因座的组织和调节
- 批准号:
10006694 - 财政年份:
- 资助金额:
$ 33.36万 - 项目类别:
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