This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.107.120766v1 94/7/2859    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Vámosi, G. Articles by Tóth, K. Search for Related Content PubMed PubMed Citation Articles by Vámosi, G. Articles by Tóth, K.

Conformation of the c-Fos/c-Jun Complex In Vivo: A Combined FRET, FCCS, and MD-Modeling Study

György Vámosi ^*, Nina Baudendistel , Claus-Wilhelm von der Lieth , Nikoletta Szalóki ^*, Gábor Mocsár ^*, Gabriele Müller , Péter Brázda , Waldemar Waldeck , Sándor Damjanovich ^*, Jörg Langowski  and Katalin Tóth 

^* Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; Biophysics of Macromolecules Division, Spectroscopic Department, Molecular Modeling, German Cancer Research Center, Heidelberg, Germany; and Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary

Correspondence: Address reprint requests to Katalin Tóth, Tel.: 49-6221-42-3390; E-mail: kt{at}dkfz.de.

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 intensity-based 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.