SOLUTION STRUCTURE AND DYNAMICS OF IKBA

功能蛋白可能在体内展开或部分折叠的原因之一是相对容易和 当它们不与其生物靶标复合时，它们可以被降解的速度。初步数据 提示在缺乏核因子-KB的情况下，IKBA的Ankyrin重复结构域可能不完全折叠。这个 本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 在溶液中不含蛋白质，与核因子-KB形成复合体。该项目包括两个主要的具体目标，一个 关于游离的1KBa[67-287]和另一个与IKBA之间的络合物的核磁共振表征 和核因子-kB。在特定的目标1a中，将对游离的lKBa[67-287]进行溶液核磁共振指认。 蛋白质，它将在科米维斯的项目中得到广泛的表征。初步的核磁共振波谱为 质量好，说明这项任务是可行的。特定目标1b将使用共鸣作业 在目标1a中获得，以表征lKBa[67-287]的溶液结构和动力学，利用 核磁共振实验，包括化学位移、NOE、残余偶极耦合和顺磁松弛 通过旋转标签。特别是，酰胺的质子交换率将通过各种核磁共振来测量 实验，提供特定地点的信息，供Wolynes在项目中使用。多肽链动力学， 主链和侧链，将使用核磁共振松弛测量进行评估。两个版本的比较 Komives计划中制备的高稳定性突变体的溶液核磁共振行为，体内评估 Hoffmann的项目中的功能和Ghosh的项目中的蛋白酶体降解测试将是 这是这一具体目标的重要组成部分。在特定的靶2a中，IKBA与多肽之间的络合物 代表核因子-KB的核定位序列将通过核磁共振进行表征。该序列具有 被预测与IKBA(Wolynes的项目)结合，并已被证明与LKBA[67-287]结合，具有Mu M 亲和力(Komives的项目)。具体目标2b将研究lkba[67-287]和NF-kB[p50(245-350)p65(191-321)]之间的复合体。对于核磁共振研究来说，这是一个极具挑战性的课题，但应该给出 关于在游离蛋白质中观察到的IKBA弹性被保留的程度的重要信息 在建筑群里。由于IKBA的功能与其折叠状态密切相关，因此实验 这里描述的不仅应该提供对IKBA的自由形式的详细表征，而且还 通过表征其与核因子-KB的复合体，对其在体内的功能有重要的见解。

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.