The overall goal of the project is to provide a biophysical description of the dynamics of the NF-KB/lKBainteraction. Free IKBa has a helical CD spectrum but binds ANS, and has solvent-exposed amides. Theseresults suggest that regions of IKBa may not be compact. When bound to NF-KB, the helical signiture ofIKBa 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 complexbetween IKBa and NF-KB is greater than 48 hr.
In AIM 1, we will 'take-apart' the NF-KB(p50/p65) tounderstand, in kinetic and thermodynamic terms, the roles of the NLS, the dimerization domain, and the Nterminaldomain 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-KBNLS and then the NLS with the dimerization domain.
In AIM 2, we will use amide H/2H exchange tostudy the complex between IKBa and NF-KB. These experiments will show where differences in solventaccessibility occur in each protein upon binding and will help us formulate hypotheses to explain the largestabilization of IKBa upon binding to NF-KB. These first two aims will provide a solid biophysicalcharacterization of the interaction of wild-type IKBa with NF-KB.
In AIM 3, we will generate a panel ofmutants based on folding phi values predicted by Wolynes (Project by Wolynes). For each mutant, folding kinetics andin vitro thermodynamic stability will be measured. Phi values for folding will be measured and directlycompared 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 studythe kinetics of the interaction between IKBa and the NF-KB/DNA complex. Preliminary SPR experimentsshow that IKBa promotes dissociation of NF-KB from the DNA. These results will provide parameters forAlex Hoffmann's signaling model.
Showing the most recent 10 out of 108 publications
