RESUMO
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.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Motivos de Aminoácidos , Cromatografia de Afinidade/métodos , Ligantes , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
On a past volume of this monograph we have reviewed general aspects of the varied technologies available to generate peptide arrays. Hallmarks in the development of the technology and a main sketch of preparative steps and applications in binding assays were used to walk the reader through details of peptide arrays. In this occasion, we resume from that work and bring in some considerations on quantitative evaluation of measurements as well as on selected reports applying the technology.
