Dynamics of NF κB and IκBα Studied with Green Fluorescent Protein (GFP) Fusion Proteins
Journal of Biological Chemistry
We investigated the dynamics of nuclear transcription factor B (NF-B) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-B chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IB␣ similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence
... ance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-B-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature-dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 ؋ 10 6 M ؊1 , although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant.