Structural Studies of Large Dynamic Complexes

本MIRA提案中描述的研究的主要总体目标是阐明结构和 大分子及其复合物的动力学特性，通常包含良好的- 结构化和结构化较少的动态或展开区域，使用固态方法的组合， 晶体学或cryo-EM和溶液方法如NMR、SAXS和荧光。两大体系 已经在我们实验室研究了好几年了。转录因子NFκB家族的成员被认为是 在细胞质中通过其结合的抑制剂（Iκ B）处于非活性状态，直到细胞接收到外部信号。 NFκB被IκBα的磷酸化和泛素化激活，然后被蛋白酶体靶向， 降解，释放NFκB转位到细胞核内。在经典的负反馈机制中， NFκB上调IκBα的转录以及信号特异性应激反应基因：新合成 IκBα在动力学上增强NFκB与DNA的解离，我们称之为分子剥离。一 在汽提过程中形成了瞬时三元复合物中间体并且在令人兴奋的新观察中， 最近的研究表明，细胞质中静息的NFκB-IκBα复合物的稳定性通过 与特定的长非编码RNA（lncRNA）相互作用，形成稳定的三元复合物 类似于在分子剥离过程中在细胞核中形成的瞬时NFκB-IκBα-DNA复合物。我们 提出使用各种生物物理技术对NFκB-IκBα-RNA复合物进行结构表征， 包括核磁共振，低温电子显微镜，与加布里埃尔兰德博士合作，和小角度X射线 散射，与约翰泰纳博士合作，并将探讨结构和动态之间的差异， NFκB、IκBα和DNA的二元和三元复合物，以及NFκB-IκBα-RNA三元复合物， 特别是甲基标记的蛋白质。虽然我们对伴侣和共同伴侣有很多了解， 然而，Hsp 90与其客户之间相互作用的结构基础仍然未知。的 一个根本的问题是，我们仍然不了解客户蛋白质的物理状态时，它被绑定到 Hsp 90分子伴侣，我们对Hsp 90分子上客户蛋白的位置只有一个粗略的了解 contact.我们提出了一种创新的方法制备客户蛋白-热休克蛋白90复合物，通过重组 真核细胞的伴侣级联，但在无细胞表达系统的情况下， 细菌细胞提取物。我们将制备雌激素受体配体结合结构域和Hsp 90的复合物， 采用文献中使用的方法来证明这种相互作用的存在， 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 含有根据文献报道用于形成 与HSP 90形成稳定的复合物。这种无细胞系统的优点是，它可以被调整到优化， 复合物的形成通过改变组分细胞提取物的相对量。

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.