Specific and Stable Fluorescence Labeling of Histidine-Tagged Proteins for Dissecting Multi-Protein Complex Formation

Abstract

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.

Essential role of endocytosis for Interleukin-4 receptor mediated JAK/STAT signalling

“…We extended this here to both IL-4R types by tracking endocytosis of N-terminally His-tagged IL-4Rα by pulse-chase labelling with the hexahistidine-specific dye trisNTA-Alexa-Fluor-647 (Gandhi et al, 2014;Lata et al, 2006). At the 0-min time point, no vesicular structures were labelled regardless of ligand presence or IL-4R subtype ( Fig.…”

“…Receptor internalization in the absence of ligand was tracked by a similar loading assay using pulse labelling of cells expressing Nterminally His-tagged receptor subunits with trisNTA-Alexa-Fluor-647 (Gandhi et al, 2014;Lata et al, 2006). Although nonspecific internalization of the trisNTA dye contributes ∼10% to the endosomal signals (Gandhi et al, 2014), uptake of H6-IL-4Rα and H6-IL-2Rγ into cortical endosomes following a chase of 10 min at 37°C was reduced to about 50% of control levels in the presence of EHT-1864 (Fig.…”

“…His tags were detected using trisNTA-Alexa-Fluor-647 (Lata et al, 2006) as described previously (Gandhi et al, 2014).…”

Live In-Cell Visualization of Proteins Using Super Resolution Imaging

“…One method involves fusion of a peptide that recruits a small molecule to the protein of interest (Martin et al 2005;Lata et al 2006). Other methodologies use proteins to recruit the small molecule tag (Marks, Braun, and Nolan 2004;Bonasio et al 2007) which can improve the specificity of binding due to the larger interaction surface although the increased size of this protein can perturb protein/enzyme function.…”

NTAㆍNi2+-Functionalized Quantum Dots for VAMP2 Labeling in Live Cells

“…This decrease in luminous intensity was smaller than the significant fluorophore emission quenching reported previously in systems that used Ni 2+ to bridge modified NTA and His6 components. 20 The paramagnetic Ni 2+ moiety was found to strongly quench the photoluminescence of some organic dyes, limiting their application in biological systems.…”