SOLUTION STRUCTURE AND DYNAMICS OF IKBA

功能性蛋白质在体内可能展开或部分折叠的原因之一是相对容易和容易 当它们不与其生物靶标复合时，它们可以被快速降解。初步数据 表明在缺乏 NF-KB 的情况下，IKBa 的锚蛋白重复结构域可能不完全折叠。的 该项目的总体目标是通过比较 IKBa 的结构和动力学来检验这一假设 溶液中不含蛋白质，并与 NF-KB 形成复合物。该项目包括两个主要具体目标，一是 涉及游离 lKBa[67-287] 的 NMR 表征，另一个涉及 IKBa 之间的复合物 和 NF-KB。在特定目标 1a 中，解决方案 NMR 共振分配将免费进行 lKBa[67-287] Komives 项目将对其进行广泛表征。初步核磁共振谱为 质量好，说明这个任务是可行的。具体目标 1b 将使用共振分配 利用一组 NMR 实验，包括化学位移、NOE、残余偶极耦合和顺磁弛豫 通过旋转标签。特别是，酰胺质子交换率将通过各种 NMR 来测量 实验，以提供特定地点的信息以供 Wolynes 的项目使用。多肽链动力学， 主链和侧链都将使用 NMR 弛豫测量进行评估。比较 Komives 项目中制备的稳定性更高的突变体的溶液 NMR 行为，并进行体内评估 Hoffmann 项目中的功能并在 Ghosh 项目中的蛋白酶体降解测定中进行测试将 这一具体目标的重要组成部分。在特定目标 2a 中，IKBa 和肽之间的复合物 代表 NF-KB 核定位序列的序列将通过 NMR 进行表征。这个序列有 预计可与 IKBa 结合（Wolynes 项目），并已显示可与 lKBa[67-287] 以 mu M 结合 亲和力（Komives 项目）。具体目标 2b 将检查 lKBa[67-287] 和 NF-KB[p50(245-350)p65(191-321)] 之间的复合体。对于 NMR 研究来说，这是一个极具挑战性的课题，但应该给出 关于在游离蛋白中观察到的 IKBa 灵活性保留程度的重要信息 在综合体中。由于 IKBa 的功能与其折叠状态密切相关，因此实验 本文描述的不仅应提供 IKBa 游离形式的详细表征，而且还应提供 通过对其与 NF-KB 复合物的表征，对其体内功能有了重要的了解。

One of the reasons why functional proteins might be unfolded or partly folded in vivo is the relative ease and rapidity by which they can be degraded when not in complex with their biological target. Preliminary data indicate that the ankyrin repeat domain of IKBa may be incompletely folded in the absence of NF-KB. The overall goal of this Project is to test this hypothesis by comparing the structure and dynamics of the IKBa protein free in solution and in complex with NF-KB. The project consists of two major specific aims, one concerned with NMR characterization of free lKBa[67-287] and the other with the complex between IKBa and NF-KB. In Specific Aim 1a, solution NMR resonance assignments will be made for free lKBa[67-287] protein, which is to be extensively characterized in Project by Komives. Preliminary NMR spectra are of good quality, indicating that this task will be feasible. Specific Aim 1b will use the resonance assignments obtained in Aim 1a to characterize the solution structure and dynamics of lKBa[67-287], utilizing a battery of NMR experiments, including chemical shifts, NOEs, residual dipolar couplings and paramagnetic relaxation by spin labels. In particular, amide proton exchange rates will be measured by a variety of NMR experiments, to provide site-specific information for use in Project by Wolynes. Polypeptide chain dynamics, both backbone and side chain, will be evaluated using NMR relaxation measurements. Comparison of the solution NMR behavior of the higher-stability mutants prepared in Project by Komives, evaluated for in vivo function in Project by Hoffmann and tested in the proteasome degradation assay in Project by Ghosh will be an important part of this Specific Aim. In Specific Aim 2a, the complex between IKBa and a peptide representing the nuclear localization sequence of NF-KB will be characterized by NMR. This sequence has been predicted to bind to IKBa (Project by Wolynes) and has been shown to bind to lKBa[67-287] with mu M affinity (Project by Komives). Specific Aim 2b will examine the complex between lKBa[67-287] and NF-KB[p50(245-350)p65(191-321)]. This is an extremely challenging subject for NMR study, but should give important information on the extent to which the flexibility of IKBa observed in the free protein is preserved in the complex. Since the function of IKBa is so intimately related to its folded state, the experiments described herein should provide not only a detailed characterization of the free form of IKBa, but also important insights into its function in vivo through characterization of its complex with NF-KB.