Mutant Flavin-Based Fluorescent Protein Sensors for Detecting Intracellular Zinc and Copper in Escherichia coli

Flavin-based fluorescent proteins (FbFPs) are a class of fluorescent reporters that undergo oxygen-independent fluorophore incorporation, which is an important advantage over green fluorescent proteins (GFPs) and mFruits. A FbFP derived from Chlamydomonas reinhardtii (CreiLOV) is a promising platform for designing new metal sensors. Some FbFPs are intrinsically quenched by metal ions, but the question of where metals bind and how to tune metal affinity has not been addressed. We used site-directed mutagenesis of CreiLOV to probe a hypothesized copper(II) binding site that led to fluorescence quenching. Most mutations changed the fluorescence quenching level, supporting the proposed site. One key mutation introducing a second cysteine residue in place of asparagine (CreiLOV_N41C) significantly altered metal affinity and selectivity, yielding a zinc sensor. The fluorescence intensity and lifetime of CreiLOV_N41C were reversibly quenched by Zn²⁺ ions with a biologically relevant affinity (apparent dissociation constant, K_d, of 1 nM). Copper quenching of CreiLOV_N41C was retained but with several orders of magnitude higher affinity than CreiLOV (K_d = 0.066 fM for Cu²⁺, 5.4 fM for Cu⁺) and partial reversibility. We also show that CreiLOV_N41C is an excellent intensity- and lifetime-based zinc sensor in aerobic and anaerobic live bacterial cells. Zn²⁺-induced fluorescence quenching is reversible over several cycles in Escherichia coli cell suspensions and can be imaged by fluorescence microscopy. CreiLOV_N41C is a novel oxygen-independent metal sensor that significantly expands the current fluorescent protein-based toolbox of metal sensors and will allow for studies of anaerobic and low oxygen systems previously precluded by the use of oxygen-dependent GFPs.