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NMR studies of the transcriptional inhibitor I kappa B alpha and its interaction with the transcription factor NF kappa B

  • Author(s): Cervantes, Carla F.
  • et al.
Abstract

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. Previous data has indicated that the ankyrin repeat domain of I[kappa]B[alpha], the primary inhibitor of NF-[kappa]B, may be incompletely folded in the absence of NF-[kappa]B. Based on this premise, the initial goal of this work was to investigate the solution structure and dynamics of the ankyrin repeat domain of Ikappa]B[alpha] in its free state by NMR. In the Chapters 2 and 3, solution NMR experiments on free I[kappa]B[alpha](67-206) protein, that allowed us to characterize the structure and dynamics of this "folded" part of the I[kappa]B[alpha] ankyrin repeat domain will be presented. A battery of NMR experiments, including chemical shifts, NOEs, amide proton exchange, backbone relaxation and residual I[kappa]B[alpha] and a peptide representing the nuclear localization sequence of NF- [kappa]B is characterized by NMR. Again, a battery of NMR and binding experiments are presented that show that the NLS polypeptide (residues 293-321 of NF-[kappa]B(p65) folds upon binding to I[kappa]B[alpha] providing a mechanistic explanation of how it is sequestered by inhibitor binding. In the final chapter, the full-length ankyrin repeat domain of I[kappa]B[alpha] ; is characterized. The fifth and sixth ankyrin repeats show very few cross peaks in the NMR indicating that they are weakly folded and in conformational exchange. By taking advantage of NMR data obtained on the bound protein as well as on a stabilized mutant, a good number of resonance assignments could be made. These assignments facilitated the interpretation of backbone dynamics experiments that allowed us to characterize the dynamics of the weakly- folded fifth and sixth repeats of the IκBα ankyrin repeat domain. Since the function of I[kappa]B[alpha] is so intimately related to its folded state, the experiments described herein should provide not only a detailed characterization of the free form of I[kappa]B[alpha], but also important insights into its function in vivo through characterization of its complex with NF-[kappa]B

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