SOLUTION STRUCTURE AND DYNAMICS OF IKBA

功能性蛋白质在体内可能是未折叠或部分折叠的原因之一是相对容易和 当不与其生物靶标复合时，它们可以降解的速度。初步数据 表明IKBa的锚蛋白重复结构域在NF-κ B不存在时可能不完全折叠。的 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 溶液中无蛋白质，与NF-κ B复合。该项目包括两个主要具体目标，一是 关于游离IKBa的NMR表征[67-287]，另一个涉及IKBa与 和NF-κ B。在具体目标1a中，将对游离1 KBa进行溶液NMR共振归属[67-287] 蛋白质，其将在Komives的Project中被广泛表征。初步NMR谱图为 质量好，表明这项任务是可行的。具体目标1b将使用共振分配 在目标1a中获得，以表征lKBa的溶液结构和动力学[67-287]，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 spin标签。特别地，酰胺质子交换率将通过各种NMR测量。 实验，以提供特定地点的信息，供Wolynes项目使用。多肽链动力学， 主链和侧链都将使用NMR弛豫测量进行评价。比较 Komives项目中制备的高稳定性突变体的溶液NMR行为，体内评价 Hoffmann项目中的功能，并在Ghosh项目中的蛋白酶体降解试验中进行测试， 这是这一具体目标的重要组成部分。在特异性目标2a中，IKBa和肽之间的复合物 代表NF-κ B的核定位序列的序列将通过NMR表征。该序列具有 预测与IKBa结合（Wolynes的项目），并已显示与IKBa结合[67-287]，μ M affinity（Komives的项目）。特异性目标2b将检查lKBa[67-287]和NF-κ B [p50（245-350）p65（191-321）]之间的复合物。这对NMR研究来说是一个极具挑战性的课题，但应该给予 关于在游离蛋白中观察到的IKBa的柔性被保留的程度的重要信息 在复杂的。由于IKBa的功能与其折叠状态密切相关， 本文所述的方法不仅应提供游离形式IKBa的详细表征，而且 通过表征其与NF-κ B的复合物对其体内功能的重要见解。

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.