Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

Abstract

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in superresolution microscopy. In this work, we have established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 complementing peptide variants using high-density peptide microarrays and examined them for binding and functional complementation of the LOO-FP fragment into Green Fluorescent Protein. We have studied the effect of peptide length and the effect of different linkers to the solid support. The peptide microarray platform allowed us to map the effect of all possible amino acid substitutions on each position of the complementing peptides as well as in the context of some single and double amino acid substitutions. As all peptides in different formats were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing determination of robustness and precision of the method.Importantly, we showed that the microarray fluorescence correlated with the affinity in solution between the LOO-FP and peptides. A double substitution yielded a peptide with 9-fold higher affinity than the starting peptide.

Global analysis of multi-mutants to improve protein function

“…4). The latter combination, however, appears to be close to optimal in other high-throughput experiments [24]. Second, none of the 15 most highly ranking substitutions are highly deleterious (all variants are fully functional).…”