Two key events are associated with IKK activation through the canonical NF-kB pathway. Phosphorylation of two serine residues within the activation loop (AL) of IKK2/b has long be known to be a requirement for IKK activation and serves as the signature for IKK in its active state. What is much less well understood is the necessary role of non- covalent interaction between linear polyubiquitin chains and the NEMO subunit in promoting AL phosphorylation of IKK2/b. In order to better understand the solution behavior of the enigmatic NEMO protein and gain insight into its influence over multisubunit IKK complexes, I undertook biophysical characterization of purified a series of recombinant human NEMO proteins and disease-associated point mutant proteins as well as various NEMO:IKK2/b complexes. Through size exclusion chromatography-multiangle light scattering and analytical ultracentrifugation, NEMO is predominantly a dimer in solution. However, by virtue of its modular coiled coil segments NEMO exhibits complicated solution dynamics that causes it to behave as a significantly elongated molecule. Analyses of NEMO in complex with IKK2 indicate that NEMO preserves this structurally dynamic character within the multisubuit complex and exacerbates the previously observed propensity of IKK2 towards homo-oligomerization. These observations provide critical information on the structural plasticity of NEMO which helps clarify its role in diseases and IKK regulation through oligomerization-dependent phosphorylation of catalytic IKK2 subunit dimers. As further effort to understand how NEMO induces phosphorylation of the IKK2AL serines, I proposed that upon binding to the polyubiquitin chains synthesized during signaling, NEMO might mediate additional contacts with IKK2, which could induce the conformational changes in IKK2 necessary for AL phosphorylation. Using a series of in vitro and cell-based experiments, a short 6-residues segment located near the C-terminus of NEMO weakly contacts IKK2. A peptide derived from NEMO encompassing these contact residues blocks IKK2 activation in vitro and in cells. Finally, I could show that this novel inhibitory NEMO-based peptide protects mice from LPS-induced lethality. Furthermore, the native sequence NEMO peptide disrupts systemic cytokine induction by LPS while a mutated version of the peptide fails to do. Thus, this NEMO peptide could be developed for therapeutic purposes.