Unknown

Dataset Information

0

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.

SUBMITTER: Sinzinger MD 

PROVIDER: S-EPMC4830892 | biostudies-other | 2016 May

REPOSITORIES: biostudies-other

altmetric image

Publications

A microarray-based approach to evaluate the functional significance of protein-binding motifs.

Sinzinger Michael D MD   Chung Yi-Da YD   Adjobo-Hermans Merel J W MJ   Brock Roland R  

Analytical and bioanalytical chemistry 20160218 12


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  ...[more]

Similar Datasets

| S-EPMC2425478 | biostudies-literature
| S-EPMC3460961 | biostudies-literature
| S-EPMC2754467 | biostudies-literature
| S-EPMC9881048 | biostudies-literature
| S-EPMC2640396 | biostudies-literature
| S-EPMC3486348 | biostudies-literature
| S-EPMC8903255 | biostudies-literature
| S-EPMC4552278 | biostudies-literature
| S-EPMC5920051 | biostudies-literature
| S-EPMC3330829 | biostudies-literature