Solution structure and dynamics of polyubiquitin chains

描述(申请人提供)：这项建议是为了描述多泛素(PolyUb)链的溶液结构、动力学和相互作用，PolyUb链是真核生物中一种普遍的蛋白质分解信号。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，它影响着多种重要的细胞事件。了解Lys48连接的PolyUb链是如何被26S蛋白酶体和其他下游效应分子识别的，是我们理解调控机制的核心。值得注意的是，通过不同的赖氨酸连接的PolyUb链也起到了细胞信号的作用，尽管是调节的而不是蛋白质分解的。尽管有重要证据表明，不同的PolyUb链的特异性识别是基于构象决定因素，但到目前为止，对于这些分子在溶液中的构象性质还没有明确的看法。获得这样的信息是绝对必要的，以便发展对不同链如何能够作为特定信号的分子理解。我们将使用核磁共振方法来确定Lys48连接的四联Ub链的三维构象，以及与蛋白酶体受体的分子模拟物hHR23a和S5a结合的二和四联Ub链的三维构象。这将使用由自旋松弛和剩余偶极耦合测量得出的长程、取向约束的组合来执行，并由顺磁松弛增强和顺磁标记引入的伪接触位移来补充距离信息。这些新的核磁共振方法将与基于NOES的传统方法结合使用，并基于化学位移扰动和溶剂可及性测量绘制结构域间或分子间界面。我们将使用一组具有代表性的UBA结构域来确定各种Ub链受体的链接特异性的结构决定因素。结构域运动在PolyUb链与各种受体相互作用的能力中起着至关重要的作用。我们将在Ub-Ub界面的残基中引入突变，以确定影响界面稳定性和动力学的决定因素。我们将测量不同pH和温度条件下di-Ub和Tetra-Ub中的蛋白质动力学，以确定结构域间运动的贡献，并表征PolyUb链的构象灵活性。

DESCRIPTION (provided by applicant): This proposal is to characterize the solution structure, dynamics, and interactions of polyubiquitin (polyUb) chains, a universal proteolytic signal in eukaryotes. The ubiquitin-proteasome proteolytic pathway is the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how Lys48-linked polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Remarkably, polyUb chains linked via different lysines also act as cellular signals, although regulatory rather than proteolytic. Although significant evidence suggests that the specific recognition of different polyUb chains is based on conformational determinants, as yet there is no clear view of the conformational properties of these molecules in solution. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. We will use NMR approaches to determine the three-dimensional conformation of Lys48-linked tetra-Ub chain, free in solution, and of di- and tetra-Ub bound to molecular mimics of the proteasomal receptors, hHR23A and S5a. This will be performed using a combination of long-range, orientational constraints derived from spin-relaxation and residual dipolar coupling measurements, complemented with distance information from paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels. These novel NMR approaches will be used in combination with the conventional methods based on NOEs and mapping of the interdomain or intermolecular interface based on chemical shift perturbations and solvent accessibility measurements. We will use a representative set of UBA domains in order to identify structural determinants of the linkage specificity of various Ub-chain receptors. Domain motions play essential role in polyUb chain's ability to interact with various receptors. We will introduce mutations in the residues at the Ub-Ub interface in order to identify determining factors contributing to the interface stability and dynamics. We will measure protein dynamics in di-Ub and tetra-Ub under various pH and temperature conditions in order to determine the contribution from interdomain motions and to characterize the conformational flexibility of polyUb chains.