SOLUTION STRUCTURE AND DYNAMICS OF IKBA

IKBA 的解决方案结构和动态

• 批准号: 7096437
• 负责人: HELEN JANE DYSON
• 金额: $ 18.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7096437
• 关键词: I kappa B beta; ankyrins; biological signal transduction; electron spin resonance spectroscopy; nuclear factor kappa beta; nuclear magnetic resonance spectroscopy; protein protein interaction; protein structure; protein structure function; solutions; structural biology

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.

功能蛋白可能被展开或在体内部分折叠的原因之一是相对容易和 当它们与其生物学靶标不复杂时，它们可以降低它们的速度。初步数据 表明在不存在NF-KB的情况下，IKBA的Ankyrin重复域可能不完全折叠。这 该项目的总体目标是通过比较IKBA的结构和动态来检验这一假设 溶液中无蛋白质，并与NF-KB复合。该项目由两个主要目标组成，一个 与自由LKBA的NMR表征有关[67-287]，而另一个与IKBA之间的复合物有关 和NF-KB。在特定的AIM 1A中，将对LKBA进行NMR共振分配[67-287] 蛋白质，应在项目中广泛表征的蛋白质。初步NMR光谱是 质量良好，表明此任务是可行的。特定目标1B将使用共振分配 在AIM 1A中获得以表征LKBA的溶液结构和动力学[67-287] NMR实验，包括化学位移，NOE，残留偶极耦合和顺磁性弛豫 通过自旋标签。特别是，酰胺质子汇率将通过多种NMR来衡量 实验，以提供特定于网站的信息，以供Wolynes在项目中使用。多肽链动力学， 主链和侧链都将使用NMR松弛测量进行评估。比较 溶液在项目中制备的高稳定性突变体的NMR行为，评估了体内 霍夫曼（Hoffmann 这个特定目标的重要组成部分。在特定的目标2a中，IKBA和肽之间的复合物 代表NF-KB的核定位序列将以NMR为特征。这个序列具有 预计将与IKBA结合（由Wolynes的项目），并已证明与Mu M结合了LKBA [67-287] 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）]之间的复合物。对于NMR研究，这是一个极具挑战性的主题，但应该给予 有关在自由蛋白中观察到的IKBA灵活性的程度的重要信息 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自由形式的详细表征，还应提供 通过表征其与NF-KB的复合物来对其在体内功能的重要见解。