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-ka ppa KB) by using fusion proteins of the p65 subunit with mutants of green f luorescent protein (GFP). GFP-NF-kappa B chimeras were functional both in v itro 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 w ere linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual reg ulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappa B-binding oligonucleotides a lso resulted in FRET. This effect was DNA sequence-specific and exhibited s aturation characteristics. Application of stopped-flow fluorometry to measu re the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fas t increase of acceptor fluorescence with a plateau after about 10 ms. The o bserved initial binding rate showed a temperature-dependent linear correlat ion with the oligonucleotide concentration. The association constant calcul ated according to pre-steady state kinetics was 3 x 10(6) M-1, although equ ilibrium binding studies implied significantly higher values. This observat ion suggests that the binding process involves a rapid association with a r ather high off-rate followed by a conformational change resulting in an inc rease of the association constant.