Dynamics of NF Kappa B and I Kappa B alpha Studied with Green Fluorescent Protein (GFP) Fusion Proteins INVESTIGATION OF GFP-p65 BINDING TO DNA BY FLUORESCENCE RESONANCE ENERGY TRANSFER

We investigated the dynamics of nuclear transcription factor Kappa B (NF- Kappa B) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP- NF- Kappa 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 I Kappa B alpha 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 resonance 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- Kappa 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 x 10 super(6) M super(-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.