Biophysics of Macromolecular Complexes

大分子复合物的生物物理学

• 批准号: 7593537
• 负责人: Gary Felsenfeld
• 金额: $ 36.46万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 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/7593537
• 关键词: Adult; Affinity; Architecture; Biochemical; Biological; Biological Process; Biophysics; Cell Line; Cell Nucleus; Cell physiology; Cells; Chickens; Chromatin; Chromatin Structure; Chromosome Structures; Collaborations; Complement; Complex; DNA; Data; Dependence; Dimensions; Elderly; Erythrocytes; Erythroid Progenitor Cells; Gene Cluster; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; HIV; Heterochromatin; Histones; Human; Integral Membrane Protein; Investigation; Ionic Strengths; Laboratories; Length; Linker DNA; Macromolecular Complexes; Mechanics; Membrane; Methods; Modeling; Molecular Biology; Molecular Biology, Other; Molecular Conformation; Nucleic Acids; Physical condensation; Play; Pliability; Property; Proteins; Protocols documentation; Publishing; Range; Role; Sedimentation process; Series; Shapes; Sorting - Cell Movement; Structure; System; Work; Yeasts; beta Globin; day; folate-binding protein; in vivo; insight; interest; macromolecular assembly; member; reconstitution; restriction enzyme; retinal rods; sedimentation coefficient; size; stoichiometry; tool

Chromatin structure and architecture. We are interested in the biophysical properties and structure of native chromatin fragments. The chicken folate receptor and beta-globin gene loci are ideal for such structural studies in that (i) the region possesses both condensed and transcriptionally active chromatin and (ii) the system has been extensively studied in terms of gene regulation, allowing us to relate the overall chromatin structure to transcription. The constitutively condensed chromatin region, spanning 15.5 Kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes, can be released from the cell nucleus with the restriction enzyme HpaII. We have previously analyzed the hydrodynamic properties of this condensed chromatin fragment and showed that it is an extended rod. This provides insights into the structure of heterochromatin, found interspersed within various genomes. The biophysical and biological tools developed in these studies have allowed us to further expand our investigation. Using an erythroid precursor cell line (6C2), we have shown that a 16.2 Kbp region of the beta-globin gene locus can be released from the nucleus with the restriction enzymes NheI and XhoI. In this particular cell line, this chromatin region possesses all the hallmarks of transcriptional inactivity. We have analyzed the hydrodynamic properties of this facultative heterochromatin fragment and showed that it is also an extended rod. Unlike the 15.5 Kbp constitutively condensed chromatin, however, this 16.2 Kbp chromatin fragment contains a smaller histone protein to nucleic acid ratio. Therefore, the similarity in structure observed highlights the flexibility of the chromatin structure in accommodating different DNA linker lengths. We have also studied the properties of the 15.5 and 16.2 Kbp chromatin fragments at higher ionic strengths and found no evidence of any abrupt conformational change, demonstrating that these chromatin fragments released from the nucleus did not assume the more compact conformations recently described for some reconstituted structures (Ghirlando and Felsenfeld, submitted to J. Mol. Biol.). We have further dissected the beta-globin gene cluster into a series of five distinct chromatin fragments ranging from 2.1 to 8.0 Kbp in size using the restriction enzyme BamHI. We showed that the dependence of the sedimentation coefficient with size is consistent with the extended rod like properties observed for chromatin. We are currently developing protocols to compare beta-globin genes chromatin fragments obtained from 6C2 cells with those released from 10-day old and adult chicken erythrocytes. As the beta-globin genes are transcribed in 10-day old erythrocytes but inactive in adult erythrocytes, these studies will allow us to relate the structures of constitutive and facultative heterochromatin with those of transcriptionally active and inactive chromatin. Macromolecular assemblies. In collaboration with members of the Laboratory of Molecular Biology, and other laboratories, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies extend the biochemical and structural investigations and provide important mechanistic information. A case in point is provided by the recently published work on the complete yeast ESCRT-I heterotetramer carried out in collaboration with Dr. James H. Hurley. The endosomal sorting complex required for transport-I (ESCRT-I) complex is conserved from yeast to humans and directs the lysosomal degradation of ubiquitinated transmembrane proteins and, in humans, the budding of the human immunodeficiency virus (HIV). The complex is composed of Vps23, Vps28, Vps37, and Mvb12 and we show that these proteins assemble with high affinity to form an elongated and monodisperse 1:1:1:1 complex. Sedimentation data for the core complex are consistent with the structural data, and hydrodynamic studies on extended ESCRT-I constructs allow for a modeling of the intact ESCRT-I complex and a mechanistic understanding of how the complex interacts with the endosomal membrane. The elongated shape and dimension of approximately 25 nm indicate that the ESCRT-I complex participates directly in regulating the mechanical aspects of cargo recruitment and membrane remodeling (Kostelansky et al., 2007).

染色质结构和架构。 我们对天然染色质片段的生物物理性质和结构感兴趣。 鸡叶酸受体和β-珠蛋白基因位点是理想的结构研究，因为（i）该区域具有浓缩和转录活性的染色质和（ii）该系统已被广泛研究的基因调控，使我们能够将整体染色质结构与转录。 组成性浓缩的染色质区域，跨越15.5 Kbp的DNA，两侧是发育调节的叶酸受体和β-珠蛋白基因，可以用限制性内切酶HpaII从细胞核释放。 我们先前已经分析了这个浓缩的染色质片段的流体动力学性质，并表明它是一个延伸的杆。 这提供了异染色质结构的见解，发现散布在各种基因组中。 在这些研究中开发的生物物理和生物工具使我们能够进一步扩大我们的调查。 使用红系前体细胞系（6C 2），我们已经表明，β-珠蛋白基因座的16.2 Kbp区域可以从细胞核中释放的限制酶NheI和XhoI。 在这个特殊的细胞系中，这个染色质区域具有转录不活跃的所有标志。 我们已经分析了这个兼性异染色质片段的流体动力学性质，并表明它也是一个延长杆。 然而，与15.5 Kbp的组成型浓缩染色质不同，这16.2 Kbp的染色质片段含有较小的组蛋白与核酸的比例。 因此，观察到的结构相似性突出了染色质结构在适应不同DNA接头长度方面的灵活性。 我们还研究了15.5和16.2Kbp染色质片段在较高离子强度下的性质，没有发现任何突然构象变化的证据，表明这些从细胞核释放的染色质片段没有呈现最近描述的一些重构结构的更紧凑的构象（GhirloveandFelsenfeld，提交给J. Mol. Chem. Biol.）。 我们进一步解剖了β-珠蛋白基因簇成一系列的5个不同的染色质片段，大小从2.1到8.0 Kbp的限制性内切酶BamHI。 我们发现，沉降系数与大小的依赖性是一致的染色质观察到的延长棒样属性。 我们目前正在开发的协议，比较β-珠蛋白基因染色质片段从6C 2细胞与10天大和成年鸡红细胞释放。 由于β-珠蛋白基因在10天大的红细胞中转录，但在成人红细胞中无活性，这些研究将使我们能够将组成性和兼性异染色质的结构与转录活性和无活性染色质的结构联系起来。 大分子组装体。 与分子生物学实验室和其他实验室的成员合作，蛋白质和蛋白质-核酸组装体的特征在于它们的形状，化学计量和相互作用的亲和力。 这些研究扩展了生物化学和结构研究，并提供了重要的机理信息。 最近发表的关于完整酵母ESCRT-Ⅰ异源四聚体的研究提供了一个恰当的例子。赫尔利 转运蛋白-I（ESCRT-I）复合物所需的内体分选复合物从酵母到人类都是保守的，并指导泛素化跨膜蛋白的溶酶体降解，以及人类免疫缺陷病毒（HIV）的出芽。 该复合物由Vps 23，Vps 28，Vps 37和Mvb 12组成，我们表明这些蛋白质以高亲和力组装形成细长的单分散1：1：1：1复合物。 核心复合物的沉降数据与结构数据一致，并且对扩展的ESCRT-I构建体的流体动力学研究允许对完整的ESCRT-I复合物进行建模，并对复合物如何与内体膜相互作用进行机械理解。 细长的形状和约25 nm的尺寸表明ESCRT-I复合物直接参与调节货物募集和膜重塑的机械方面（Kostelansky等人，2007年）。