Biophysics of Macromolecular Complexes

染色质结构和构筑。 我们对天然染色质片段的生物物理和结构特性很感兴趣。利用被广泛研究的鸡叶酸受体和β-珠蛋白基因座，我们先前已经表征了两个不同的染色质片段的流体动力学和大体结构特性。其中一个片段代表了一个结构紧凑的异染色质区域，该区域横跨15.5kbp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不活跃的β-珠蛋白基因位点释放的，跨越16.2kBP的DNA。尽管它们的组蛋白与核酸的比例不同，但我们已经证明这两个片段都采用了延长的杆状结构，这与为凝聚的30 nm染色质纤维提出的模型一致。已经提出了各种模型来描述浓缩的30 nm染色质纤维，每个模型都具有基本相同的核小体排列的不同的DNA路径。为了表征染色质中DNA的空间排列，我们目前正在利用体外模型系统和天然染色质片段开发高分辨率的染色体捕获构象分析。这些研究将使我们能够将适当的30 nm纤维模型分配给染色质，这反过来将提供更好的理解染色质结构和基本过程之间的关系，如基因表达和DNA复制。 大分子组装。 在与分子生物学实验室成员和其他人的合作下，利用流体力学方法对蛋白质和蛋白质-核酸组件的形状、化学计量和相互作用的亲和力进行了表征。这些研究扩展了目前的生化和结构研究，并提供了补充的机制信息，最近发表的关于酶I(EI)的研究就是例证，该研究是与G.Marius Clore博士合作进行的。 磷酸烯醇式丙酮酸：蔗糖磷酸转移酶系统是一种细菌信号转导途径，在该途径中，活跃的糖通过细胞膜转运到一系列的磷酸化级联反应中。最初的两个步骤是所有途径的分支所共有的，包括EI被磷酸烯醇式丙酮酸(PEP)自动磷酸化，然后从EI到组氨酸磷酸载体蛋白HPR的磷酸化转移。随后，磷酸基从HPR转移到糖专一性酶II，最终转移到即将到来的糖分子上。EI由一个与HPR结合的N端磷酸转移结构域(EIN)和一个包含PEP结合位点的C端二聚结构域(EIC)组成。我们表征了不同条件下EI的单体-二聚体平衡，并测定了在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，这些条件被选择用于溶液结构研究，因为在所使用的毫摩尔浓度下，蛋白质将主要是二聚体。这些结构研究表明，当HPR结合时，EIN结构域经历了大的铰链体旋转，从而为二聚体EI的催化循环提供了重要的机理信息。此外，结构数据与在表征EI自缔合期间确定的流体动力学参数是一致的(Schwieter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).