Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions.
Fiche publication
Date publication
mai 2020
Journal
Analytical biochemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr TRAVE Gilles, Mr EBERLING Pascal , Dr NOMINE Yves, Dr GOGL Gergo
Tous les auteurs :
Bonhoure A, Forster A, Babah KO, Gógl G, Eberling P, Kostmann C, Volkmer R, Mancilla VT, Travé G, Nominé Y
Lien Pubmed
Résumé
Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range, that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the "holdup" principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities.
Mots clés
Conservative replacement, Linear motif, Oncoprotein, Papillomavirus, Peptide array, Protein-protein interaction
Référence
Anal. Biochem.. 2020 May 16;:113772