Biophysics of Macromolecular Complexes

染色质结构和架构。 组蛋白在细胞核内将基因组DNA包装并浓缩成染色质。 蛋白质如CCCTC结合因子（CTCF）通过施加拓扑约束、介导远程基因组相互作用和参与转录事件，通过被动和主动机制帮助指导染色质高阶组织。 CTCF是一种高度保守的DNA结合蛋白，仅在双侧性动物中发现。 在人类中，该蛋白由11个锌指DNA结合结构域组成，两侧是保守的N-末端和C-末端尾，占蛋白质的约57%。 虽然锌指特异性识别DNA基序并与RNA相互作用，但N-和C-末端结构域的作用仍然主要未知。 这些末端与粘着蛋白相互作用，并帮助稳定描绘拓扑相关的染色质结构域的复合物。 但是，这不可能是他们唯一的作用。 我们已经表明，这些N-和C-末端结构域在溶液中本质上是无序的，许多核和DNA结合蛋白共有的属性。 目前的工作重点是确定结合到CTCF的N-和C-末端的蛋白质伴侣，并研究形成的复合物，以了解CTCF如何调节真核细胞核内的高阶基因组组织。 人类CTCF具有直系同源物CTCFL，主要与精子发生和一些癌症类型相关。 虽然CTCF和CTCFL具有高度保守的11个锌指DNA结合结构域，并识别相同的DNA基序，但它们的N-和C-末端显著不同，这表明这些蛋白质的不同作用来自它们的末端。 类似地，虽然在两侧性动物中和在进化过程中具有核心锌指DNA结合结构域是保守的，但CTCF在门中可能具有不同的末端。 我们有兴趣从选定的物种中表征CTCF的蛋白质伴侣，以进一步剖析蛋白质在组织基因组中所起的多重作用。 具有生物学意义的大分子组装体。 我们利用流体动力学方法，特别是沉降速度和平衡分析离心，来表征关键的生物组件，确定它们的形状和化学计量，并测量它们的相互作用亲和力。 与John Louis（LCP-NIDDK）合作，我们正在研究二聚体SARS-CoV-2主要蛋白酶（MPro）的成熟和过渡态类似物抑制剂的作用。 MPro作为二聚体发挥功能，是病毒复制和繁殖所不可或缺的。MPro作为单体形成，作为多蛋白链的一部分。 MPro二聚化是其释放和产生病毒复制和转录所必需的非结构蛋白所必需的。 口服COVID-19疗法最近获得了FDA的紧急使用授权，目标是主要的蛋白酶MPro，凸显了其重要性。 使用精心设计的单体形式的MPro（M）和比较其性质的野生型二聚体MPro（WT），我们表明，蛋白酶活性需要的酶的二聚体形式。 GC 376是一种过渡态类似物蛋白酶抑制剂，在兽医应用中用于治疗猫冠状病毒感染，与MPro（M）和MPRo（WT）相互作用。 有趣的是，MPro（M）以比MPro（WT）更高的亲和力结合GC 376。 此外，使用活性测定和流体动力学方法，我们表明GC 376以浓度依赖性方式促进MPro（M）二聚化。 GC 376不能恢复MPro（WT）的催化活性，这反映了单体MPro（M）设计的结构差异。 然而，数据表明，过渡态类似物的结合有利于酶的二聚体形式，增强催化活性。 这些观察结果可能为设计和鉴定更有效的蛋白酶抑制剂提供有价值的工具。

Chromatin structure and architecture. Histone proteins package and condense genomic DNA into chromatin within the cell nucleus. Proteins such as the CCCTC binding factor (CTCF) help direct chromatin higher-order organization through passive and active mechanisms by imposing topological constraints, mediating long-range genomic interactions, and participating in transcriptional events. CTCF is a highly conserved DNA binding protein found exclusively in bilaterians. In humans, the protein consists of an eleven zinc-finger DNA binding domain, flanked by conserved N-terminal and C-terminal tails constituting about 57 percent of the protein. While the zinc-fingers specifically recognize DNA motifs and interact with RNA, the roles of the N- and C-terminal domains remain primarily unknown. These termini interact with cohesin and help stabilize the complexes delineating topologically associated chromatin domains. However, it is unlikely that this is their sole role. We have shown that these N- and C-terminal domains are intrinsically disordered in solution, a property shared by many nuclear and DNA binding proteins. Current work focuses on identifying protein partners that bind to the N- and C-termini of CTCF and a study of the complexes formed to understand how CTCF regulates higher-order genome organization within the eukaryotic nucleus. Human CTCF has an ortholog CTCFL, primarily associated with spermatogenesis and some cancer types. While CTCF and CTCFL have a highly conserved eleven zinc-finger DNA binding domain and recognize identical DNA motifs, they differ significantly in their N- and C-termini suggesting that the diverse roles for these proteins arise from their termini. Similarly, while conserved among bilaterians and across evolution with a core zinc-finger DNA binding domain, CTCF may have divergent termini across phyla. We are interested in characterizing protein partners for CTCF from select species to dissect further the multiple roles that the protein plays in organizing the genome. Macromolecular assemblies of biological interest. We utilize hydrodynamic methods, particularly sedimentation velocity and equilibrium analytical centrifugation, to characterize critical biological assemblies, determine their shape and stoichiometry, and measure their interaction affinity. In collaboration with John Louis (LCP-NIDDK), we are studying the maturation of the dimeric SARS-CoV-2 main protease (MPro) and the effects of transition-state analog inhibitors. MPro functions as a dimer and is indispensable for viral replication and propagation. MPro is formed as a monomer, as part of a polyprotein chain. MPro dimerization is required for its release and the generation of non-structural proteins necessary for viral replication and transcription. Oral COVID-19 therapies recently granted emergency use authorization by the FDA target the main protease MPro, highlighting its importance. Using a carefully designed monomeric form of MPro(M) and comparing its properties to that of the wild-type dimeric MPro(WT), we show that protease activity requires the dimeric form of the enzyme. GC376, a transition-state analog protease inhibitor used in veterinary applications to treat feline coronavirus infections, interacts with MPro(M) and MPRo(WT). Interestingly, MPro(M) binds to GC376 with a higher affinity than MPro(WT). Furthermore, using activity assays and hydrodynamic methods, we show that GC376 promotes MPro(M) dimerization in a concentration-dependent manner. GC376 does not restore the catalytic activity observed for MPro(WT), reflecting structural differences in the monomeric MPro(M) design. However, data demonstrate that the binding of the transition state analog favors the dimer form of the enzyme, enhancing catalytic activity. These observations may provide a valuable tool for designing and identifying more effective protease inhibitors.