title: A microarray-based approach to evaluate the functional significance of protein-binding motifs.
abstract: Intracellular proteins comprise numerous peptide motifs that interact with protein-binding domains. However, using sequence information alone, the identification of functionally relevant interaction motifs remains a challenge. Here, we present a microarray-based approach for the evaluation of peptides as protein-binding motifs. To this end, peptides corresponding to protein interaction motifs were spotted as a microarray. First, peptides were titrated with a pan-specific binder and the apparent K(d) value of this binder for each peptide was determined. For phosphotyrosine-containing peptides, an anti-phosphotyrosine antibody was employed. Then, in the presence of the pan-specific binder, arrays were competitively titrated with cell lysate and competition constants were determined. Using the Cheng-Prusoff equation, binding constants for the pan-specific binder and inhibition constants for the lysates were converted into affinity constants for the lysate. We experimentally validate this method using a phosphotyrosine-binding SH2 domain as a further reference. Furthermore, strong binders correlated with binding motifs engaging in numerous interactions as predicted by Scansite. This method provides a highly parallel and robust approach to identify peptides corresponding to interaction motifs with strong binding capacity for proteins in the cell lysate.
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Sinzinger
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Yi-Da
authors.initials: YD
authors.lastname: Chung
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
authors.forename: Merel J W
authors.initials: MJ
authors.lastname: Adjobo-Hermans
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands. roland.brock@radboudumc.nl.
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
mesh_terms: D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
chemical_list: D011506:Proteins
keywords: Cheng-Prusoff equation; Dissociation constant; EC50; Peptide microarray; Protein interaction; T cell signalling