Dynamics of NF kappa B and Ikappa Balpha 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 kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB 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 IkappaBalpha 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-kappaB-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(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.
Schmid
J A
JA
Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna A-1235, Austria. johannes.schmid@univie.ac.at
Birbach
A
A
Hofer-Warbinek
R
R
Pengg
M
M
Burner
U
U
Furtmüller
P G
PG
Binder
B R
BR
de Martin
R
R
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D020672:I-kappa B Proteins / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D051996:Transcription Factor RelA
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D008164:Luminescent Proteins; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D049452:Green Fluorescent Proteins; D004247:DNA