Alpha and beta interferons (IFN-␣Type I interferons (IFNs) form a family of multifunctional cytokines initially described for their direct antiviral effect but now also recognized as major elements of the immune response (19,46). Differential activities of IFN subtypes have been reported (3) and used in the clinic for the treatment of various pathologies, including viral hepatitis (IFN-␣2) and multiple sclerosis (IFN-␤) (32). All type I IFNs are recognized by a single shared receptor composed of two transmembrane proteins, IFNAR1 and IFNAR2. Because of the much faster k on and much slower k off of IFN-␣2 towards IFNAR2 than those measured for IFNAR1 (18,34,40), a two-step assembling mechanism was proposed for the interaction between IFN and the two receptors (Fig. 1B). After binding of IFN-␣2 to IFNAR2 (k a1 ), IFNAR1 transiently associates in a second step to the complex (k a2 ) (18,25). Owing to the short lifetime of the IFN-␣2-IFNAR1 interaction, the complex dissociates (k d2 ) and reassociates (k a2 ) in a fast manner. Thus, depending on the receptor surface concentrations and ligand binding affinities, only part of the bound ligand is involved in the active ternary complex.After formation of the ternary complex, the interferon signal is transduced through the receptor-associated JAK kinases, with the STAT transcription factors as their main targets (3).

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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.

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We describe an experimental approach for studying ligand-receptor interactions in the plane of the membrane. The extracellular domains of the type I interferon receptor subunits ifnar1-EC and ifnar2-EC were tethered in an oriented fashion onto solid-supported, fluid lipid bilayers, thus mimicking membrane anchoring and lateral diffusion of the receptor. Ligand-induced receptor assembling was investigated by simultaneous total internal reflection fluorescence spectroscopy and reflectance interferometry (RIf). Based on a rigorous characterization of the interactions of fluorescence-labeled IFNalpha2 with each of the receptor subunits, the dynamics of the ternary complex formation on the fluid lipid bilayer was addressed in further detail making use of the features of the simultaneous detection. All these measurements supported the formation of a ternary complex in two steps, i.e., association of the ligand to ifnar2-EC and subsequent recruitment of ifnar1-EC on the surface of the membrane. Based on the ability to control and quantify the receptor surface concentrations, equilibrium, and rate constants of the interaction in the plane of the membrane were determined by monitoring ligand dissociation at different receptor surface concentrations. Using mutants of IFNalpha2 binding to ifnar2-EC with different association rate constants, the key role of the association rate constants for the assembling mechanism was demonstrated.