Biophysics of Macromolecular Complexes

大分子复合物的生物物理学

基本信息

批准号：
7734073
负责人：
Gary Felsenfeld
金额：
$ 33.92万
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/7734073
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

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

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项目摘要

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,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).

染色质结构和体系结构。我们对天然染色质片段的生物物理特性和结构感兴趣。鸡叶叶酸受体和β-珠蛋白基因基因座是此类结构研究的理想选择，因为（i）该区域具有凝结和转录活性的染色质，并且（ii）该系统已通过基因调节进行了广泛的研究，使我们能够将整体染色质结构与转录相关联。可以用限制性酶HPAII从细胞核中释放出跨越开发的叶酸受体和β-珠蛋白基因的组成性凝结的染色质区域，跨越了开发调节的叶酸受体和β-珠蛋白基因的15.5 kbp。我们先前已经分析了该冷凝染色质片段的流体动力学特性，并表明它是扩展的棒。这提供了有关异染色质结构的见解，该结构散布在各种基因组中。这些研究中开发的生物物理和生物学工具使我们能够进一步扩大研究。使用红细胞前体细胞系（6C2），我们表明，β-珠蛋白基因基因座的16.2 kbp区域可以通过限制酶NHEI和XHOI从细胞核中释放出来。在这条特定的细胞系中，该染色质区域具有转录不活动的所有标志。我们已经分析了该兼性异染色质片段的流体动力学特性，并表明它也是扩展的棒。但是，与15.5 kbp组成性冷凝的染色质不同，该16.2 kbp染色质片段包含一个较小的组蛋白蛋白与核酸比率。因此，结构的相似性观察到了染色质结构在适应不同DNA接头长度中的灵活性。我们还研究了15.5和16.2 kbp染色质片段的性质，并没有发现任何突然构象变化的证据，表明这些从核中释放出来的染色质片段并未假定最近对某些重构结构描述的更紧凑的构象（Ghirlando和Felsenfeld，Felsenfeld，j）。我们进一步将β-珠蛋白基因簇分为一系列五个不同的染色质片段，使用限制性酶BAMHI，大小为2.1至8.0 kbp。我们表明，沉积系数与尺寸的依赖性与观察到的染色质观察到的延长棒类似。我们目前正在开发规程，以比较从6C2细胞获得的β-珠蛋白基因染色质片段与从10天老年和成年鸡肉红细胞释放的β-细胞获得的。由于β-珠蛋白基因被转录为10天老的红细胞，但在成人红细胞中不活跃，这些研究将使我们能够将本质和辅助异染色质的结构与转录活性和无活性染色质素的结构相关联。大分子组件。与分子生物学实验室以及其他实验室，蛋白质和蛋白质核酸组件的合作，已使用流体动力学方法来表征其形状，化学计量和相互作用的亲和力。这些研究扩展了生化和结构研究，并提供了重要的机械信息。最近发表的关于与James H. Hurley博士合作进行的有关完整酵母ESCRT-I异驱剂的工作提供了一个典型的例子。从酵母到人类，运输-I（ESCRT-I）复合物所需的内体分选复合物是保守的，并指导泛素化的跨膜蛋白的溶酶体降解，在人类中，人类免疫缺陷病毒（HIV）的萌芽。该复合物由VPS23，VPS28，VPS37和MVB12组成，我们表明这些蛋白质具有高亲和力，形成了伸长且单分散的1：1：1：1：1复合物。核心复合物的沉积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，以及对该络合物如何与内体膜相互作用的机械理解。大约25 nm的细长形状和尺寸表明，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，2007）。