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