SOLUTION STRUCTURE AND DYNAMICS OF IKBA

University Of California, San Diego, La Jolla CA

Investigators

Linked publications & trials

Abstract

One of the reasons why functional proteins might be unfolded or partly folded in vivo is the relative ease and[unreadable] rapidity by which they can be degraded when not in complex with their biological target. Preliminary data[unreadable] indicate that the ankyrin repeat domain of IKBa may be incompletely folded in the absence of NF-KB. The[unreadable] overall goal of this Project is to test this hypothesis by comparing the structure and dynamics of the IKBa[unreadable] protein free in solution and in complex with NF-KB. The project consists of two major specific aims, one[unreadable] concerned with NMR characterization of free lKBa[67-287] and the other with the complex between IKBa[unreadable] and NF-KB. In Specific Aim 1a, solution NMR resonance assignments will be made for free lKBa[67-287][unreadable] protein, which is to be extensively characterized in Project by Komives. Preliminary NMR spectra are of[unreadable] good quality, indicating that this task will be feasible. Specific Aim 1b will use the resonance assignments[unreadable] obtained in Aim 1a to characterize the solution structure and dynamics of lKBa[67-287], utilizing a battery of[unreadable] NMR experiments, including chemical shifts, NOEs, residual dipolar couplings and paramagnetic relaxation[unreadable] by spin labels. In particular, amide proton exchange rates will be measured by a variety of NMR[unreadable] experiments, to provide site-specific information for use in Project by Wolynes. Polypeptide chain dynamics,[unreadable] both backbone and side chain, will be evaluated using NMR relaxation measurements. Comparison of the[unreadable] solution NMR behavior of the higher-stability mutants prepared in Project by Komives, evaluated for in vivo[unreadable] function in Project by Hoffmann and tested in the proteasome degradation assay in Project by Ghosh will be[unreadable] an important part of this Specific Aim. In Specific Aim 2a, the complex between IKBa and a peptide[unreadable] representing the nuclear localization sequence of NF-KB will be characterized by NMR. This sequence has[unreadable] been predicted to bind to IKBa (Project by Wolynes) and has been shown to bind to lKBa[67-287] with mu M[unreadable] 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[unreadable] important information on the extent to which the flexibility of IKBa observed in the free protein is preserved[unreadable] in the complex. Since the function of IKBa is so intimately related to its folded state, the experiments[unreadable] described herein should provide not only a detailed characterization of the free form of IKBa, but also[unreadable] important insights into its function in vivo through characterization of its complex with NF-KB.