Biophysics of Macromolecular Complexes

染色质结构和构筑。 细胞核内的DNA被包装成染色质，目前有多种模型描述了体外观察到的浓缩的30 nm染色质纤维的结构。然而，在体内缺乏这种结构的证据，除了成熟的鸟类红细胞等特殊细胞中，所有的染色质基本上都是不活跃的。我们感兴趣的是了解DNA在体内浓缩染色质中的组织，以及通过组织CTCF和粘附素等蛋白质对其高阶施加的拓扑限制。我们正在利用天然染色质片段开发高分辨率的染色体捕获构象分析，例如之前研究的浓缩异染色质，侧翼是发育调节的叶酸受体和β-珠蛋白基因。这些研究将使我们能够更好地了解体内染色质纤维的结构，从而为深入了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提供依据。 具有生物学意义的大分子组件。 利用流体力学方法对生物组装体的形状、化学计量比和相互作用的亲和力进行了表征。这些研究补充了目前的研究，Schmidt等人最近的工作证明了这一点。(2016)。在这项工作中，发展了一种简单的方法，利用电子顺磁共振(EPR)来确定溶液中蛋白质低聚物的数量。对HIV-1逆转录酶p66亚基的研究表明，甘油的存在会导致单体-二聚体平衡的改变。我们利用分析超速离心法表征了p66单体-二聚体的平衡，并确认了令人惊讶的EPR观察结果，证明了甘油的作用。分析超速离心法是用于所述流体力学研究的主要工具之一。在与NIH的同事和其他人的合作下，我们出版了一本描述该领域当前方法的教科书(Schuck等人，2016)。

Chromatin structure and architecture. DNA within the cell nucleus is packaged into chromatin and a variety of models currently describe the structure of the condensed 30 nm chromatin fiber observed in vitro. However, evidence for this structure in vivo is lacking, except in specialized cells such as mature avian erythrocytes in which all of the chromatin is essentially inactive. We are interested in understanding the organization of DNA within condensed chromatin in vivo, as well as the topological constraints imposed on its higher order by organizing proteins such as CTCF and cohesin. We are developing high resolution chromosome capture conformation assays utilizing native chromatin fragments, such as the previously studied condensed heterochromatin flanked by the developmentally regulated folate receptor and beta-globin genes. These studies will allow us to better understand the structure of the chromatin fiber in vivo, thus providing insight in the relations between chromatin structure and essential processes such as gene expression and DNA replication. Macromolecular assemblies of biological interest. Biological assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies complement current investigations, as evidenced by recent work by Schmidt et al. (2016). In this work, a simple method using electron paramagnetic resonance (EPR) is developed to determine the populations of protein oligomers in solution. Such studies on the p66 subunit of HIV-1 reverse transcriptase demonstrate that the presence of glycerol results in a shift of the monomer-dimer equilibrium. We utilized analytical ultracentrifugation to characterize the p66 monomer-dimer equilibrium and confirm the surprising EPR observations, demonstrating the effects of glycerol. Analytical ultracentrifugation is one of the primary tools used for the hydrodynamic studies described. In collaboration with colleagues from the NIH, and others, we have published a textbook describing current methodology in the field (Schuck et al., 2016).