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：
Gary Felsenfeld
金额：
$ 33.36万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
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：
https://reporter.nih.gov/project-details/8939548
关键词：
Adopted Affinity American Analytical Biochemistry Architecture Binding Biochemical Biological Biological Assay Biophysics Birds C-terminal Capsid Capsid Proteins Cell Nucleus Cell membrane Cell physiology Cells Chemicals Chromatin Chromatin Fiber Chromatin Structure Chromosome Structures Chromosomes Cleaved cell Collaborations Complement Cytoplasm DNA DNA biosynthesis Dimerization Disease Erythrocytes GAG Gene Gene Expression Genes Genetic HIV-1 Heterochromatin Human In Vitro Individual International Investigation Journals Laboratories Length Macromolecular Complexes Methods Modeling Molecular Biology Molecular Conformation National Institute of Diabetes and Digestive and Kidney Diseases Nucleic Acids Nucleocapsid Peptide Hydrolases Peptides Physical condensation Play Polyproteins Process Property Proteins Relative (related person)Resolution Retroviridae Role Shapes Site Societies Structure United States National Institutes of Health Viral Viral Proteins Virion Work Zinc analytical ultracentrifugation beta Globin cohesin data collection methodology dimer flexibility folate-binding protein improved in vivo insight interest macromolecular assembly monomer protein complex stoichiometry tool

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).

染色质结构和体系结构。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 30 nm 染色质纤维的结构。然而，体内这种结构的证据缺乏，除了特殊的细胞，例如成熟的禽类红细胞，其中所有染色质基本上都是不活跃的。我们有兴趣了解体内浓缩染色质内 DNA 的组织，以及通过组织 CTCF 和粘连蛋白等蛋白质对其高阶结构施加的拓扑约束。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测定，例如先前研究的两侧是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。使用流体动力学方法对生物组装体的形状、化学计量和相互作用亲和力进行了表征。这些研究补充了当前的研究，克洛尔博士实验室最近进行的工作证明了这一点。 GAG 是参与人类 HIV-1 逆转录病毒组装的主要多蛋白。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 蛋白酶裂解 GAG，导致成熟病毒体的形成。 GAG 多蛋白的裂解导致其组成蛋白的形成，即基质、衣壳、核衣壳和本质上无序的 p6。基质调节 GAG 与细胞膜的结合和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 及其成分的结构、生物化学和生物物理研究有望为 HIV-1 病毒组装和随后出芽的机制提供重要见解。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。在二聚体形式中，负责二聚化的 C 末端结构域采用单一方向。 N 端和 C 端结构域的相对方向占据了广泛的状态分布，对于蛋白质的单体和二聚体形式而言，这些状态存在显着差异。重要的是，在 HIV-1 衣壳组装中观察到的这种蛋白质的方向仅存在于二聚体状态分布的一小部分亚群中（Deshmukh 等人，美国化学会杂志，2013）。随后的研究考虑了 GAG 的较长衣壳间隔肽 1-核衣壳片段。这些研究表明，衣壳和核衣壳都保留了各自的结构，并且彼此半独立地翻滚。添加与核衣壳结构域结合并固定两个锌指节的方向的核酸不会影响衣壳结构域的结构。然而，即使柔性间隔肽 1 在核酸存在下仍然是非结构化的，HIV-1 蛋白酶对间隔肽 1 核衣壳位点的访问也会显着增强（Deshmukh 等人，Angewandte Chemie International Edition English, 2014）。分析超速离心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方法。这最终会带来更准确的流体动力学参数，对于通常需要最高精度的流体动力学建模尤其重要（Ghirlando 等人，Analytical Biochemistry，2014；Zhao 等人，Analytical Biochemistry，2014）。