Article

Two-hybrid fluorescence cross-correlation spectroscopy detects protein-protein interactions in vivo

Division Biophysics of Macromolecules (B040), German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
ChemPhysChem (Impact Factor: 3.36). 06/2005; 6(5):984-90. DOI: 10.1002/cphc.200400639
Source: PubMed

ABSTRACT Fluorescence cross-correlation spectroscopy (FCCS) uses the correlated motion of two distinct fluorophores to detect their interaction. Whereas FCCS has been used with chemically or genetically labeled interaction partners in vitro, FCCS has never been demonstrated in vivo between two autofluorescent proteins. At least one reaction partner was always chemically labeled. Fos and Jun, two components of the AP-1 transcription factor, are known to exert their function as a dimer and can therefore serve as a reference for dimer formation. Expressing fusion proteins between Fos and the enhanced green fluorescent protein (EGFP), as well as Jun and the monomeric red fluorescent protein 1 (mRFP1) in HeLa cells, we show here, for the first time, in vivo FCCS detection of protein-protein interactions. The mobility of the dimerized species is slow, indicating that DNA-binding might stabilize dimerization. The technique has rich potential applications for the rapid screening of protein-protein interactions in vivo, which are able to clarify events during the whole life of cells.

Download full-text

Full-text

Available from: Jörg Langowski, Aug 24, 2015
0 Followers
 · 
129 Views
  • Source
    • "The FRT landing site contained in this plasmid was not used for this study. To lower the expression levels, we introduced the early SV40 downstream of the CMV promoter by amplifying the SV40 ''HindIII-fragment'' from pSV-mRFP1-EGFP [44] and subcloning into pcDNA5/FRT using the NheI/HindIII containing primers CTA GCTAGC TGT ACG ACGCGT AGC TTG AGA AAT GGC ATT and CCC AAGCTT AGC TTT TTG CAA AAG CCT AG. The vector system containing both, CMV and a downstream SV40 promoter was called pc2. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transmembrane domains (TMD) connect the inner with the outer world of a living cell. Single TMD containing (bitopic) receptors are of particular interest, because their oligomerization seems to be a common activation mechanism in cell signaling. We analyzed the composition of TMDs in bitopic proteins within the proteomes of 12 model organisms. The average number of strongly polar and charged residues decreases during evolution, while the occurrence of a dimerization motif, GxxxG, remains unchanged. This may reflect the avoidance of unspecific binding within a growing receptor interaction network. In addition, we propose a new experimental approach for studying helix-helix interactions in giant plasma membrane vesicles using scanning fluorescence cross-correlation spectroscopy. Measuring eGFP/mRFP tagged versions of cytokine receptors confirms the homotypic interactions of the erythropoietin receptor in contrast to the Interleukin-4 receptor chains. As a proof of principle, by swapping the TMDs, the interaction potential of erythropoietin receptor was partially transferred to Interleukin-4 receptor α and vice versa. Non-interacting receptors can therefore serve as host molecules for TMDs whose oligomerization capability must be assessed. Computational analysis of the free energy gain resulting from TMD dimer formation strongly corroborates the experimental findings, potentially allowing in silico pre-screening of interacting pairs.
    Proteomics 12/2010; 10(23):4196-208. DOI:10.1002/pmic.201000208 · 3.97 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Unsaturated lipids in cell membranes, including phospholipids and cholesterol, are well-known targets of oxidative modification, which can be induced by a variety of stresses, including ultraviolet A (UVA)- and visible light-induced photodynamic stress. Photodynamic lipid peroxidation has been associated with pathological conditions such as skin phototoxicity and carcinogenesis, as well as therapeutic treatments such as antitumor photodynamic therapy (PDT). Lipid hydroperoxides (LOOHs), including cholesterol hydroperoxides (ChOOHs), are important non-radical intermediates of the peroxidative process which can (i) serve as in situ reporters of type I vs. type II chemistry, (ii) undergo one-electron or two-electron redutive turnover which determines whether peroxidative injury is respectively intensified or suppressed, and (iii) mediate signaling cascades which either fortify antioxidant defenses of cells or evoke apoptotic death if oxidative pressure is too great. The purpose of this chapter is to review current understanding of photodynamic (UVA or visible light-induced) lipid peroxidation with a special focus on these aspects relating to LOOHs. Future goals in this area, many of which depend on continued development of state-of-the-art analytical techniques, will also be discussed.
  • 01/1970: pages 1-38;
Show more