Biophysics of Macromolecular Complexes

大分子复合物的生物物理学

• 批准号: 8148736
• 负责人: Gary Felsenfeld
• 金额: $ 29.32万
• 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/8148736
• 关键词: Biophysics; Macromolecular Complexes

Chromatin structure and architecture. We are interested in the biophysical and structural properties of native chromatin fragments. Using the extensively studied chicken folate receptor and beta-globin gene loci, we have previously characterized the hydrodynamic and gross structural properties of two distinct chromatin fragments. The one fragment represents a constitutively condensed heterochromatin region spanning 15.5 Kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes. The second fragment, which is released from the transcriptionally inactive beta-globin gene locus, spans 16.2 Kbp of DNA. Despite their different histone protein to nucleic acid ratio, we have shown that both fragments adopt extended rod like structures consistent with models proposed for the condensed 30 nm chromatin fiber. A variety of models have been proposed to describe the condensed 30 nm chromatin fiber, each having a topologically distinct DNA path for essentially the same arrangement of nucleosomes. In order to characterize the spatial arrangement of the DNA within chromatin we are currently developing high resolution chromosome capture conformation assays utilizing both in vitro model systems, as well as native chromatin fragments. These studies will allow us to assign the appropriate 30 nm fiber model to chromatin, which will in turn provide a better understanding of the relations between chromatin structure and essential processes such as gene expression and DNA replication. Macromolecular assemblies. In collaboration with members of the Laboratory of Molecular Biology, and others, 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 current biochemical and structural investigations and provide complementary mechanistic information as exemplified by recently published studies on Enzyme I (EI) carried out in collaboration with Dr. G. Marius Clore. The phosphoenolpyruvate:sugar phosphotransferase system is a bacterial signal transduction pathway in which active sugar transport across the cell membrane is coupled to a sequential phosphorylation cascade. The initial two steps are common to all branches of the pathway and involve the autophosphorylation of EI by phosphoenolpyruvate (PEP), followed by phosphoryl transfer from EI to the histidine phosphocarrier protein HPr. The phosphoryl group is subsequently transferred from HPr to the sugar specific enzyme II, and ultimately onto the incoming sugar molecule. EI consists of an N-terminal phosphoryl transfer domain (EIN) that binds HPr and a C-terminal dimerization domain (EIC) that contains the PEP binding site. We have characterized the monomer-dimer equilibria of EI under various conditions and determined an affinity of 0.8 micromolar in the presence of 100 mM sodium chloride and 4 mM magnesium chloride. These conditions were therefore chosen for solution structural studies as the protein will be predominantly dimeric at the millimolar concentrations used. These structural studies show that that the EIN domains of undergo large hinge body rotations when bound by HPr, thus providing important mechanistic information on the catalytic cycle of the dimeric EI. Furthermore, the structural data are consistent with hydrodynamic parameters determined during the characterization of the EI self-association (Schwieters et al., 2010).

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和结构特性感兴趣。 利用广泛研究的鸡叶酸受体和β-珠蛋白基因位点，我们之前已经表征了两个不同染色质片段的流体动力学和总体结构特性。 该片段代表了一个组成型浓缩的异染色质区域，横跨 15.5 Kbp 的 DNA，两侧是发育调节的叶酸受体和 β-珠蛋白基因。 第二个片段是从转录失活的 β-珠蛋白基因座释放的，跨越 16.2 Kbp 的 DNA。 尽管组蛋白与核酸的比例不同，但我们已经证明，这两个片段均采用延长的棒状结构，与针对浓缩的 30 nm 染色质纤维提出的模型一致。 人们提出了多种模型来描述浓缩的 30 nm 染色质纤维，每个模型都具有拓扑上不同的 DNA 路径，用于基本相同的核小体排列。 为了表征染色质内 DNA 的空间排列，我们目前正在利用体外模型系统以及天然染色质片段开发高分辨率染色体捕获构象测定。 这些研究将使我们能够为染色质分配适当的 30 nm 光纤模型，从而更好地理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。 高分子组装体。 与分子生物学实验室的成员和其他人合作，使用流体动力学方法对蛋白质和蛋白质-核酸组装体的形状、化学计量和相互作用的亲和力进行了表征。 这些研究扩展了当前的生化和结构研究，并提供了补充的机制信息，最近发表的与 G. Marius Clore 博士合作进行的酶 I (EI) 研究就是例证。 磷酸烯醇丙酮酸：糖磷酸转移酶系统是一种细菌信号转导途径，其中跨细胞膜的活性糖转运与连续的磷酸化级联偶联。 最初的两个步骤对于该途径的所有分支都是共同的，涉及 EI 通过磷酸烯醇丙酮酸 (PEP) 的自磷酸化，然后磷酰基从 EI 转移到组氨酸磷酸载体蛋白 HPr。 随后磷酰基从 HPr 转移至糖特异性酶 II，并最终转移至进入的糖分子上。 EI 由结合 HPr 的 N 端磷酰基转移结构域 (EIN) 和包含 PEP 结合位点的 C 端二聚化结构域 (EIC) 组成。 我们在各种条件下表征了 EI 的单体-二聚体平衡，并确定在 100 mM 氯化钠和 4 mM 氯化镁存在下的亲和力为 0.8 微摩尔。 因此，选择这些条件用于溶液结构研究，因为蛋白质在所使用的毫摩尔浓度下主要是二聚体。 这些结构研究表明，当与 HPr 结合时，EIN 结构域会发生较大的铰链体旋转，从而提供有关二聚体 EI 催化循环的重要机制信息。 此外，结构数据与 EI 自缔合表征过程中确定的流体动力学参数一致（Schwieters 等，2010）。