Biophysics of Macromolecular Complexes

染色质结构和架构。 我们感兴趣的是天然染色质片段的生物物理和结构特性。 使用广泛研究的鸡叶酸受体和β-珠蛋白基因位点，我们以前的特点是两个不同的染色质片段的流体动力学和总体结构特性。 一个片段代表了一个组成型浓缩的异染色质区域，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。 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。 尽管他们不同的组蛋白与核酸的比例，我们已经表明，这两个片段采用延长杆状结构与模型提出的凝聚30 nm染色质纤维一致。 已经提出了各种模型来描述浓缩的30 nm染色质纤维，每一个都具有基本上相同的核小体排列的拓扑学上不同的DNA路径。 为了表征染色质内DNA的空间排列，我们目前正在开发利用体外模型系统以及天然染色质片段的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纤维模型，这反过来又将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。 大分子组装体。 在与分子生物学实验室的成员和其他人的合作中，蛋白质和蛋白质-核酸组装体已经使用流体动力学方法在它们的形状、化学计量和相互作用亲和力方面进行了表征。 这些研究扩展了目前的生物化学和结构研究，并提供了补充机制信息，如最近发表的与G.马里斯·克洛 磷酸烯醇式丙酮酸：糖磷酸转移酶系统是一种细菌信号转导途径，其中糖跨细胞膜的主动运输与连续的磷酸化级联反应偶联。 最初的两个步骤是共同的所有分支的途径，并涉及EI的磷酸烯醇丙酮酸（PEP）的自磷酸化，然后从EI的磷酸转移到组氨酸磷酸载体蛋白HPr。 磷酰基随后从HPr转移到糖特异性酶II，并最终转移到进入的糖分子上。 EI由结合HPr的N-末端磷酰基转移结构域（EIN）和含有PEP结合位点的C-末端二聚化结构域（EIC）组成。 我们已经表征了EI在各种条件下的单体-二聚体平衡，并在100 mM氯化钠和4 mM氯化镁的存在下确定了0.8微摩尔的亲和力。 因此，选择这些条件进行溶液结构研究，因为蛋白质在所用的毫摩尔浓度下主要是二聚体。 这些结构的研究表明，EIN域的进行大铰链体旋转时，由HPr绑定，从而提供了重要的机械信息的催化循环的二聚体EI。 此外，结构数据与在EI自缔合表征期间确定的流体动力学参数一致（Schwieters等人，2010年）。

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).