Administrative Supplement to R01 GM065334 for purchase of AKTA Pure purification system

摘要 这项工作将表征多泛素(PolyUb)链的溶液结构、动力学和相互作用， 它们在调节一系列细胞过程中起着信号分子的作用，范围从 细胞周期的进展，到转录激活，抗原处理和囊泡运输 蛋白质。底物与不同连接类型的PolyUb链的结合使目标蛋白 细胞中截然不同的命运。特别是，Lys48连接的PolyUb在泛素- 蛋白酶体蛋白分解途径，是短寿命蛋白周转的主要调节机制， 影响各种重要的细胞事件。了解26S如何识别PolyUb链 蛋白酶体和其他下游效应分子是我们理解其发病机制的核心。 监管。尽管关于细胞过程的信息越来越丰富，但 多泛素化及其与多聚Ub信号结合蛋白的鉴定 在下游事件中，泛素介导的信号多样性的分子基础仍不清楚。这个 不同的PolyUb链对不同结果的信号传递能力的潜在机制仍有待于 在了解PolyUb信号的特异性的起源之前就被阐明了。获取此类信息是 为了发展对不同链如何能够起作用的分子理解是绝对必要的 特定的信号。拟议工作的目标是表征结构和识别特性 各种类型的PolyUb链，以便在分子水平上了解泛素的结构基础 能够充当多功能而又特定的细胞信号。这些研究将集中在所谓的非规范 泛素链：通过Lys48或Lys63以外的赖氨酸连接(例如，Lys6、Lys27)和含有 异质连接(如Lys11和Lys48)，分枝和不分枝。我们还将确定 正则的，Lys48-连接的四Ub链在溶液中的构象，以获得对 它们被细胞受体识别的机制。最后，这些研究将描述相互作用的特征 并确定与最近设计的与PolyUb结合的环肽的PolyUb络合物的结构 具有很高的亲和力和特异性，以便于进一步开发和优化这一全新的 泛素介导的信号通路的调节剂和潜在疗法。我们将使用现代的 核磁共振结合小角X射线和中子散射(SAXS，SANS)确定 多聚Ub链在溶液和TO中的三维结构和构象系综 表征它们的结合偏好和与受体的复合体。

Abstract This work will characterize the solution structure, dynamics, and interactions of polyubiquitin (polyUb) chains, which function as signaling molecules in the regulation of a host of cellular processes, ranging from progression through the cell cycle, to transcriptional activation, antigen processing and vesicular trafficking of proteins. Conjugation of substrates to polyUb chains of different linkage types commits the target protein to distinct fates in the cell. In particular, Lys48-linked polyUb acts as a universal signal in the ubiquitin- proteasome proteolytic pathway, the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Despite an increasing wealth of information on the cellular processes regulated by polyubiquitination and the identification of numerous ubiquitin-binding proteins that tie the polyUb signal to downstream events, the molecular basis of diversity in ubiquitin-mediated signaling remains unclear. The mechanisms underlying the ability of different polyUb chains to signal for distinct outcomes remain to be elucidated before the origin of specificity in polyUb signaling is understood. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. The objective of the proposed work is to characterize the structure and recognition properties of various types of polyUb chains, in order to understand at a molecular level the structural basis for ubiquitin's ability to serve as a versatile yet specific cellular signal. These studies will focus on the so-called non-canonical ubiquitin chains: linked via lysines other than Lys48 or Lys63 (e.g., Lys6, Lys27) and chains containing heterogeneous linkages (e.g., Lys11 & Lys48), branched and unbranched. We will also determine the conformations of the canonical, Lys48-linked tetraUb chains in solution in order to gain insights into the mechanisms of their recognition by cellular receptors. Finally, these studies will characterize the interactions and determine the structures of polyUb complexes with the recently designed cyclic peptides that bind polyUb with high affinity and specificity in order to facilitate further development and optimization of this entirely novel class of modulators and potential therapeutics for ubiquitin-mediated signaling pathways. We will use modern NMR approaches in combination with small-angle X-ray and neutron scattering (SAXS, SANS) to determine the three-dimensional structures and conformational ensembles of polyUb chains in solution and to characterize their binding preferences and complexes with receptors.