A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range

Abstract

We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics.

Playing with the Molecules of Life

“…205–207 Target proteins are efficiently and selectively labeled and background nonspecific proteome labeling is minimal or undetectable, despite a significant number of endogenous genes that terminate in amber codons 208 . The use of Cys and an ncAA, or two ncAAs, with bio-orthogonal reactivity allows the site-specific labeling of proteins with two different moieties via sequential or one-pot bioorthogonal reactions for applications such as Forster resonance energy transfer (FRET) measurements 17, 133, 136 with excellent dynamic range 209 .…”

Luciferases with Tunable Emission Wavelengths

“…To demonstrate the feasibility of this approach, we redshifted ap reviously introduced LUCID for the anti-cancer and anti-inflammatory drug methotrexate (LUCID-MTX). [9] LUCID-MTX is based on acircular permutation of dihydrofolate reductase as receptor protein and trimethoprim as the tethered ligand, which competes with free methotrexate for binding.F or the generation of ar ed-shifted LUCID-MTX, LUCID RS -MTX (Figure 4a), we replaced NanoLuc by H-Luc and labeled H-Luc with CPY.Atrimethoprim-Cy5 derivative was linked to SNAP-tag via aPEG 11 -linker [11] (Figure 4b). In the absence of methotrexate,t he sensor is in its closed state and astrong BRET from H-Luc-CPY to Cy5 occurs,resulting in an emission maximum of 669 nm.…”

Engineering BRET-Sensor Proteins

“…3C and D). In recent years, the ratiometric response of the Snifit sensors has been further optimized by introducing a relatively rigid 30 amino-acid polyproline linker between the CLIP-and the SNAP-tag domains (Brun et al, 2011), and by introducing donor fluorophores directly into the ligand-binding domain by the site-specific incorporation of nonnatural amino acids (Xue, Prifti, & Johnsson, 2016).…”