Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

Abstract

Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site-specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N-terminally thiolated peptide hydrazides by hydrazone ligation, b) on-surface native chemical ligation with self-purified peptide thioesters, c) radical-induced desulfurization, and d) a surface-based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline-rich ligands.