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Originally published In Press as doi:10.1074/jbc.M000291200 on March 15, 2000

J. Biol. Chem., Vol. 275, Issue 22, 17035-17042, June 2, 2000
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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*

Johannes A. SchmidDagger §, Andreas BirbachDagger , Renate Hofer-WarbinekDagger , Margarete PenggDagger , Ursula Burner, Paul G. Furtmüller, Bernd R. BinderDagger , and Rainer de MartinDagger

From the Dagger  Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna A-1235, Austria and the  Department of Biochemistry, Institute of Chemistry, University of Agricultural Sciences, Vienna A-1190, Austria

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 Ikappa Balpha 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 × 106 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.

* This study was supported by a grant from the Austrian Science Foundation (SFB5-12).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Vascular Biology and Thrombosis Research, University of Vienna, Brunnerstr. 59, A-1235 Vienna, Austria. Tel.: 43-1-86634-471; Fax: 43-1-86634-623; E-mail: johannes.schmid@univie.ac.at.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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