Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation.
Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.
Lata
Suman
S
Institut für Biochemie, Johann Wolfgang Goethe-University, Biozentrum N210, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany.
Gavutis
Martynas
M
Tampé
Robert
R
Piehler
Jacob
J
D002614:Chelating Agents / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D016898:Interferon-alpha / Q000737:chemistry*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*; D010777:Photochemistry; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002614:Chelating Agents; D005456:Fluorescent Dyes; D016898:Interferon-alpha; D008565:Membrane Proteins; D009842:Oligopeptides; D017471:Receptors, Interferon; D053633:Receptor, Interferon alpha-beta; D006639:Histidine; D009571:Nitrilotriacetic Acid