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

Oana N Antonescu,Kristoffer E Johansson,Alexander Nesterov-Mueller,Jakob R Winther,Ditte F Heidemann,Roman Popov,Nicole A M Damm,Andreas Rasmussen,Ravi Pratap Barnwal

doi:10.1371/journal.pone.0241461

Oana N Antonescu, Kristoffer E Johansson + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0241461

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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.

Highlights

Tens of new split fluorescent proteins have been developed since the first reassembly of a splitFP was achieved 20 years ago [1]
LOO10-GFP was obtained from a circularly permuted superfolder GFP, with βstrand 10 engineered in the N-terminal, employing minor modifications to an established
Peptide arrays for split GFP screening protocol [12]

Summary

Introduction

Tens of new split fluorescent proteins (splitFPs) have been developed since the first reassembly of a splitFP was achieved 20 years ago [1]. Fluorescent proteins (FPs) have been split in many creative ways, by removing fragments ranging from around half of the FP β-barrel [1] to one [2] or two [3] secondary elements. The splitFP fragments obtained have low or no fluorescence on their own, but can reassemble to form a fully functional FP. These properties have made splitFPs desirable for many bioanalytical applications, from sensing protein-protein interactions to protein detection and localization, and as tools in super-resolution microscopy [4,5].

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