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 1 a, 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 1 b 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 2 a, 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 2 b 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.
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