Structural Studies of Large Dynamic Complexes

这项Mira提案中描述的研究的主要总体目标是阐明结构和 大分子及其络合物的动力学特性，通常包含两个良好的- 结构化和非结构化的动态或展开区域，使用固态方法的组合，例如 结晶学或低温电子显微镜和溶液方法，如核磁共振，小角X射线散射和荧光。两大体系 已经在我们实验室研究了好几年了。核转录因子κB家族的成员被持有 在细胞质中，它们的结合抑制物(IκB)处于不活跃状态，直到细胞接收到外部信号。 核因子κB通过IκBα的磷酸化和泛素化激活，然后将其作为蛋白酶体的靶标 降解，释放核因子κB转位到细胞核内。在经典的负反馈机制中， 除了信号特异性应激反应基因外，核因子κB还上调IκBα的转录：新合成 IκBα在动力学上促进了核因子κB从dna上的解离，我们称之为分子剥离。一个 在汽提过程中形成了瞬时的三元络合物中间体，并在一个令人兴奋的新观察中， 最近的研究表明，静息的NFκB-IκBα复合体在细胞质中的稳定性被增强。 与特定的长非编码RNA(LncRNA)相互作用，形成稳定的三元复合体 类似于在分子剥离过程中在细胞核中形成的瞬时的NFκB-IκBα-DNA复合体。我们 利用多种生物物理技术对这种核因子κB-IκBα-RNA复合体的结构进行表征， 包括核磁共振，冷冻电子显微镜，与加布里埃尔·兰德博士合作，以及小角X射线 散射，与John Tainer博士合作，并将探索 核因子κB、IκBα与DNA的二元和三元络合物，以及三元NFκB-IκBα 特别是甲基标记的蛋白质。虽然人们对伴侣和共同伴侣的了解很多 Hsp90与其客户之间相互作用的结构基础尚不清楚。这个 根本的问题是，我们仍然不了解客户蛋白质在结合到 Hsp90分子伴侣，而我们对客户蛋白在Hsp90分子上的位置只有粗略的了解 联系。我们提出了一种创新的方法来制备客户蛋白-Hsp90复合体，通过重组 真核细胞的伴侣级联，但在使用 细菌细胞提取物。我们将制备雌激素受体配体结合域与Hsp90的复合物， 采用文献中已经使用的方法来证明在 哺乳动物细胞提取物。我们的无细胞方案将包括使用一系列单独的细菌细胞提取物 含有根据文献报道所需的过度表达的、折叠的辅助伴侣形成 与Hsp90形成稳定的复合体。这种无单元格系统的优点是，它可以调整到 通过改变组成细胞提取物的相对数量来形成复合体。

The major overall aim of the research described in this MIRA proposal is to elucidate the structural and dynamic characteristics of large macromolecules and their complexes, which generally contain both well- structured and less-structured dynamic or unfolded regions, using a combination of solid-state methods such as crystallography or cryo-EM and solution methods such as NMR, SAXS and fluorescence. Two major systems have been under study in our lab for several years. Members of the NFκB family of transcription factors are held in the cytoplasm in an inactive state by their bound inhibitors (IκBs) until the cell receives an external signal. NFκB is activated by phosphorylation and ubiquitination of the IκBα, which is then targeted for proteasomal degradation, releasing the NFκB to translocate into the nucleus. In a classical negative feedback mechanism, NFκB upregulates transcription of IκBα in addition to signal-specific stress-response genes: newly-synthesized IκBα kinetically enhances NFκB dissociation from the DNA in a process we have termed molecular stripping. A transient ternary complex intermediate is formed during the stripping process and in an exciting new observation, it was recently shown that the stability of the resting NFκB-IκBα complex in the cytoplasm is enhanced by interaction with a specific long non-coding RNA (lncRNA), which appears to form a stable ternary complex analogous to the transient NFκB-IκBα-DNA complex formed in the nucleus during molecular stripping. We propose the structural characterization of this NFκB-IκBα-RNA complex using a variety of biophysical techniques, including NMR, cryo-electron microscopy, in collaboration with Dr. Gabriel Lander, and small-angle X-ray scattering, in collaboration with Dr. John Tainer, and will probe the structural and dynamic differences between the binary and ternary complexes of NFκB, IκBα and DNA, and the ternary NFκB-IκBα-RNA complex using specifically methyl-labeled proteins. Although a great deal is known about chaperone and co-chaperone structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The fundamental problem is that we still do not understand the physical state of the client protein when it is bound to the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes contact. We propose an innovative method of preparation of a client protein-Hsp90 complex, by reconstituting the chaperone cascade of the eukaryotic cell, but in the context of a cell-free expression system employing bacterial cell extracts. We will prepare the complex of the estrogen receptor ligand-binding domain and Hsp90, adapting methods that have been used in the literature to demonstrate the presence of this interaction in mammalian cell extracts. Our cell-free protocol will include the use of a range of separate bacterial cell extracts containing the over-expressed, folded co-chaperones required according to literature reports for the formation of a stable complex with Hsp90. The advantage of this cell-free system is that it can be tuned to the optimization of complex formation by varying the relative amounts of the component cell extracts.