BIOPHYSICS OF THE IKBA/NF-KB INTERACTION DYNAMICS

University Of California, San Diego, La Jolla CA

Investigators

Linked publications & trials

Abstract

The overall goal of the project is to provide a biophysical description of the dynamics of the NF-KB/lKBa[unreadable] interaction. Free IKBa has a helical CD spectrum but binds ANS, and has solvent-exposed amides. These[unreadable] results suggest that regions of IKBa may not be compact. When bound to NF-KB, the helical signiture of[unreadable] IKBa does not change, and it still binds substantial ANS, though less than was observed for free IKBa.[unreadable] Although the intracellular half-life of free IKBa is less than 10 min, the intracellular half-life of the complex[unreadable] between IKBa and NF-KB is greater than 48 hr. In AIM 1, we will "take-apart" the NF-KB(p50/p65) to[unreadable] understand, in kinetic and thermodynamic terms, the roles of the NLS, the dimerization domain, and the Nterminal[unreadable] domain of p65 in binding to IKBa. Preliminary data shows that the NLS by itself, binds to IKBa.[unreadable] With Jane Dyson, we will explore the details of the NF-KB/lKBa binding interaction beginning with the NF-KB[unreadable] NLS and then the NLS with the dimerization domain. In AIM 2, we will use amide H/2H exchange to[unreadable] study the complex between IKBa and NF-KB. These experiments will show where differences in solvent[unreadable] accessibility occur in each protein upon binding and will help us formulate hypotheses to explain the large[unreadable] stabilization of IKBa upon binding to NF-KB. These first two aims will provide a solid biophysical[unreadable] characterization of the interaction of wild-type IKBa with NF-KB. In AIM 3, we will generate a panel of[unreadable] mutants based on folding phi values predicted by Wolynes (Project by Wolynes). For each mutant, folding kinetics and[unreadable] in vitro thermodynamic stability will be measured. Phi values for folding will be measured and directly[unreadable] compared to the predicted ones. Binding kinetics and thermodynamics will be determined by SPR and ITC.[unreadable] The binding data will be directly compared to binding predictions from Wolynes. In AIM 4, we will study[unreadable] the kinetics of the interaction between IKBa and the NF-KB/DNA complex. Preliminary SPR experiments[unreadable] show that IKBa promotes dissociation of NF-KB from the DNA. These results will provide parameters for[unreadable] Alex Hoffmann's signaling model.