NMR Studies or I-kappa B alpha and its interaction with NF-kappa B

转录因子核因子κ B （NF-kB）通过其抑制剂IkBa调控，对正常细胞生长至关重要，调控不当与许多疾病有关。该复合物的结构已通过x射线晶体学得以阐明（Huxford et al. 1998; Jacobs and Harrison 1998），两种蛋白质之间的界面由IkBalpha所有六个β发夹环之间的相互作用组成。游离IkBa结晶的尝试没有成功，酰胺氢交换实验显示，几乎所有的第一、第五和第六锚蛋白重复序列在几分钟内交换，这表明它们在某种程度上是非结构化的（Cray Hughes et al. 2004）。第5和第6个β -发夹在与NF-kB结合后显示出溶剂接近性的显著降低，这不能简单地从界面保护来解释。起初，我们将这些结果解释为IkBa在结合时折叠，但圆二色性实验显示，当IkBalpha与NF-kB结合时，二级结构没有增加（Croy Hughes et al. 2004）。综合这些结果表明，自由的IkBalpha在某种程度上是动态的，可能是熔融球状的，并且它在结合时折叠，但折叠更多的是状态集合的减少，而不是新的二级结构的形成。探测这种事件的最好方法是骨干动力学实验。目标1：自由IkBa的共振分配。采用均匀的13C、15N双标记蛋白和氘化样品以及选择性标记。主要基于0.2 mM野生型IkBa的TROSY-HNCA和hsqc - noasy - hsqc光谱，分配工作取得了进展，我们现在有几个稳定的突变体将同时分配。目的2：阐明溶液中IkBa的动力学。将使用标准R1、R2和异核NOE实验以及rcCPMG实验。目的3：阐明IkBa稳定突变体的骨干动力学。恢复稳定的锚蛋白重复序列的突变比野生型增加了5千卡/摩尔的稳定性。这些实验的结果将显示动力学如何与锚蛋白重复序列蛋白的稳定性相关。目标4。比较游离IkBa和nf - kb结合IkBa的骨架动力学。该项目将有助于了解NF-kB信号通路的调控，NF-kB信号通路对人类健康至关重要，在癌症、关节炎、哮喘、糖尿病、艾滋病和病毒感染等疾病中被错误调控。

DESCRIPTION (provided by applicant): Project Summary The regulation of the transcription factor Nuclear Factor Kappa B (NF-kB) by its inhibitor, IkBa, is critical for normal cell growth and misregulation is associated with many diseases. The structure of the complex has been elucidated by x-ray crystallography (Huxford et al. 1998; Jacobs and Harrison 1998) and the interface between the two proteins is comprised of interactions between all six beta hairpin loops of IkBalpha. Attempts to crystallize free IkBa were unsuccessful and amide hydrogen exchange experiments revealed that nearly all of the first, fifth and sixth ankyrin repeats exchanged within a few minutes suggesting they were somehow unstructured (Cray Hughes et al. 2004). The fifth and sixth beta-hairpins show a marked decrease in solvent accessibility upon binding to NF-kB that can not be explained simply from protection at the interface. At first, we interpreted these results as indicating that IkBa folds upon binding, but circular dichroism experiments showed no increase in secondary structure when IkBalpha bound to NF-kB (Croy Hughes et al. 2004). Taken together the results suggest that free IkBalpha is somehow dynamic and perhaps molten globular and that it folds upon binding but the folding is more a decrease in the ensemble of states rather than formation of new secondary structure. The best way to probe such an event is by backbone dynamics experiments. The Specific Aims are: AIM 1: Resonance Assignments of Free IkBa. Uniformly 13C, 15N double-labeled protein and deuterated samples as well as selective labeling will be used. Progress on the assignments has been made based primarily on the TROSY-HNCA and HSQC-NOESY-HSQC spectra of 0.2 mM wild type IkBa, and we now have several stabilized mutants that will be assigned simultaneously. AIM 2: Elucidation of the Dynamics of IkBa in Solution. Standard R1, R2 and heteronuclear NOE experiments as well as rcCPMG experiments will be used. AIM 3: Elucidation of the backbone dynamics of Stabilized Mutants of IkBa. Mutations that restore the consensus sequence for stable ankyrin repeat proteins increase stability by 5 kcal/mol over wild type. The results from these experiments will show how dynamics relates to stability in ankyrin repeat proteins. AIM 4. Compare the backbone dynamics of free and NF-kB-bound IkBa. Relevance to human health This project will help understand the regulation of the NF-kB signaling pathway that is critical for human health and is misregulated in diseases such as cancer, arthritis, asthma, diabetes, AIDs and viral infections.