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 super-resolution microscopy. We have here 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 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify 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 [21]. Second, none of the 15 most highly ranking substitutions are highly deleterious (all variants are fully functional).…”

Selective Peptide Binders to the Perfluorinated Sulfonic Acid Ionomer Nafion

“…[ 8 ] We propose a method to screen for peptide binders to an ionomer according to the lock‐and‐key principle. [ 9 ] Such binding, well known for antibodies, is accompanied by a stable ligand orientation to the trapping molecule. This kind of binding is especially useful because the ionomer does not need to be chemically modified.…”