An adaptable, monobody-based biosensor scaffold with FRET output

Protein-based fluorescent biosensors are powerful tools for analyte recognition in vitro and in cells. Numerous proteinaceous binding scaffolds have been developed that recognize ligands with affinity and specificity comparable to those of conventional antibodies, but are smaller, readily overexpressed, and more amenable to engineering. Like antibodies, these binding domains are useful as recognition modules in protein switches and biosensors, but they are not capable of reporting on the binding event by themselves. Here, we engineer a small binding scaffold—a consensus-designed fibronectin 3 monobody—such that it undergoes a conformational change upon ligand binding. This change is detected by Förster resonance energy transfer using chemical dyes or cyan and yellow fluorescent proteins as donor/acceptor groups. By grafting substrate recognition residues from different monobodies onto this scaffold, we create fluorescent biosensors for c-Abl Src homology 2 (SH2) domain, WD40-repeat protein 5 (WDR5), small ubiquitin-like modifier-1 (SUMO), and h-Ras. The biosensors bind their cognate ligands reversibly, with affinities consistent with those of the parent monobodies, and with half times of seconds to minutes. This design serves as generalizable platform for creating a genetically-encoded, ratiometric biosensors by swapping binding residues from known monobodies, with minimal modification.