Conformation of the c-Fos/c-Jun complex in vivo

A combined FRET, FCCS, and MD-modeling study

G. Vámosi, Nina Baudendistel, Claus Wilhelm Von Der Lieth, Nikoletta Szalóki, Gábor Mocsár, Gabriele Müller, Péter Brázda, Waldemar Waldeck, S. Damjanovich, Jörg Langowski, Katalin Tóth

Research output: Contribution to journalArticle

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Abstract

The activator protein-1 transcription factor is a heterodimer containing one of each of the Fos and Jun subfamilies of basic-region leucine-zipper proteins. We have previously shown by fluorescence cross-correlation spectroscopy (FCCS) that the fluorescent fusion proteins Fos-EGFP and Jun-mRFP1, cotransfected in HeLa cells, formed stable complexes in situ. Here we studied the relative position of the C-terminal domains via fluorescence resonance energy transfer (FRET) measured by flow cytometry and confocal microscopy. To get a more detailed insight into the conformation of the C-terminal domains of the complex we constructed C-terminal labeled full-length and truncated forms of Fos. We developed a novel iterative evaluation method to determine accurate FRET efficiencies regardless of relative protein expression levels, using a spectral- or intensitybased approach. The full-length C-terminal-labeled Jun and Fos proteins displayed a FRET-measured average distance of 8 ± 1 nm. Deletion of the last 164 amino acids at the C-terminus of Fos resulted in a distance of 6.1 ± 1 nm between the labels. FCCS shows that Jun-mRFP1 and the truncated Fos-EGFP also interact stably in the nucleus, although they bind to nuclear components with lower affinity. Thus, the C-terminal end of Fos may play a role in the stabilization of the interaction between activator protein-1 and DNA. Molecular dynamics simulations predict a dye-to-dye distance of 6.7 ± 0.1 nm for the dimer between Jun-mRFP1 and the truncated Fos-EGFP, in good agreement with our FRET data. A wide variety of models could be developed for the full-length dimer, with possible dye-to-dye distances varying largely between 6 and 20 nm. However, from our FRET results we can conclude that more than half of the occurring dye-to-dye distances are between 6 and 10 nm.

Original languageEnglish
Pages (from-to)2859-2868
Number of pages10
JournalBiophysical Journal
Volume94
Issue number7
DOIs
Publication statusPublished - Apr 2008

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Fluorescence Resonance Energy Transfer
Spectrum Analysis
Coloring Agents
Fluorescence
Transcription Factor AP-1
Leucine Zippers
Proteins
Molecular Dynamics Simulation
HeLa Cells
Confocal Microscopy
Flow Cytometry
Transcription Factors
Amino Acids
DNA

ASJC Scopus subject areas

  • Biophysics

Cite this

Vámosi, G., Baudendistel, N., Von Der Lieth, C. W., Szalóki, N., Mocsár, G., Müller, G., ... Tóth, K. (2008). Conformation of the c-Fos/c-Jun complex in vivo: A combined FRET, FCCS, and MD-modeling study. Biophysical Journal, 94(7), 2859-2868. https://doi.org/10.1529/biophysj.107.120766

Conformation of the c-Fos/c-Jun complex in vivo : A combined FRET, FCCS, and MD-modeling study. / Vámosi, G.; Baudendistel, Nina; Von Der Lieth, Claus Wilhelm; Szalóki, Nikoletta; Mocsár, Gábor; Müller, Gabriele; Brázda, Péter; Waldeck, Waldemar; Damjanovich, S.; Langowski, Jörg; Tóth, Katalin.

In: Biophysical Journal, Vol. 94, No. 7, 04.2008, p. 2859-2868.

Research output: Contribution to journalArticle

Vámosi, G, Baudendistel, N, Von Der Lieth, CW, Szalóki, N, Mocsár, G, Müller, G, Brázda, P, Waldeck, W, Damjanovich, S, Langowski, J & Tóth, K 2008, 'Conformation of the c-Fos/c-Jun complex in vivo: A combined FRET, FCCS, and MD-modeling study', Biophysical Journal, vol. 94, no. 7, pp. 2859-2868. https://doi.org/10.1529/biophysj.107.120766
Vámosi, G. ; Baudendistel, Nina ; Von Der Lieth, Claus Wilhelm ; Szalóki, Nikoletta ; Mocsár, Gábor ; Müller, Gabriele ; Brázda, Péter ; Waldeck, Waldemar ; Damjanovich, S. ; Langowski, Jörg ; Tóth, Katalin. / Conformation of the c-Fos/c-Jun complex in vivo : A combined FRET, FCCS, and MD-modeling study. In: Biophysical Journal. 2008 ; Vol. 94, No. 7. pp. 2859-2868.
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