UC San Diego

The NF[kappa]B signaling system is important in gene regulation following cellular stress and is linked with numerous cancers and inflammatory diseases. The inhibitor of NF[kappa]B, I[kappa]B[alpha], holds NF[kappa]B in the cytoplasm until cellular signaling initiates the degradation of I[kappa]B[alpha]. Free NF[kappa]B enters the nucleus and up-regulates many genes, including the one for. Freshly synthesized I[kappa]B[alpha] actively dissociates NF[kappa]B from the DNA, returning to the resting state. The foldedness of I[kappa]B[alpha] plays several important roles in NF[kappa]B signaling. I[kappa]B[alpha] is composed of six ankyrin repeats (ARs), two of which, AR5-6, are weakly folded in free I[kappa]B[alpha] but fully fold upon NF[kappa]B binding. The weakly folded nature of AR5-6 allows quick degradation of free I[kappa]B[alpha], keeping the cellular concentration of the free inhibitor low and ensuring a fast response when NF[kappa]B is activated. The foldedness of AR5-6 is also important in the stripping of NF[kappa]B from DNA by I[kappa]B[alpha]. Here, I examined the foldedness of I[kappa]B[alpha] by measuring the folding and binding kinetics. In Chapter 2, I examined the folding kinetics of free I[kappa]B[alpha], measuring the kinetics of the well-folded AR1-4 fragment, then comparing the folding kinetics of the full ARD to AR1-4. Using a phi- value analysis, I found that the outer helices of AR3-4 fold first, followed by the outer helices of AR1-2 then finally inner helices. The addition of AR5-6 did not change the folding kinetics, showing that AR5-6 do not contribute to the folding of free IκBα. The bioinformatically-derived ankyrin repeat consensus was also shown to be stabilizing, and will be a useful tool providing potentially infinite stabilization of I[kappa]B[alpha]. In Chapter 3, I examined the effect of a hydroxyl modification on the biophysical properties of I[kappa]B[alpha]. We accurately measured the folding, flexibility, and binding of WT and hydroxylated I[kappa]B[alpha] and saw that these properties were unaffected by hydroxylation. In Chapter 4, I studied the coupled folding and binding kinetics of I[kappa]B[alpha] using a tryptophan probe in AR6. Stabilizing mutations had no influence on the binding kinetics, but introduction of mutations in AR6 that pre-fold AR5-6 resulted in a decreased binding rate, suggesting a fly-casting binding mechanism