The ChlorON Series: Turn-on Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells

Abstract

Chloride is a ubiquitous ion essential for regulating biological processes. Long thought to be just a counterion in biological systems, chloride is emerging as a vital anion that plays a major role in a range of diseases. The ability to visualize intracellular chloride has been enabled by fluorescent protein-based sensors. Although the yellow fluorescent protein from Aequorea victoria, and variants thereof, quench in the presence of chloride and other anions, they have provided valuable insights in biological studies and have established the benchmark for applying protein-based sensors for chloride. However, a single domain fluorescent protein sensor that generates a turn-on response to provide a spatial and temporal map of endogenous chloride has yet to be reported. Here, we highlight how mutagenesis of non-coordinating residues in the binding pocket of mNeonGreen has unlocked this new sensing capability. Our protein engineering efforts coupled with in vitro spectroscopy have led us to discover the ChlorON series as turn-on fluorescent sensors for chloride that operate at physiological pH. Fluorescence microscopy experiments with the ChlorON sensors reveal that cells have baseline levels of chloride, which could not have been directly observed with turn-off sensors. These advancements set the stage for visualizing endogenous chloride dynamics in real-time and uncovering new roles for chloride in biology.

Rational Design of the β‐Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing**

“…[36] The anion binding pocket consists of H62, R88, S153, T173, and Y175 and is near the tyrosine-based chromophore (Figure 1A). [27] The binding of halides and oxyanions shifts the chromophore equilibrium from the phenol to the phenolate state, generating a turn-on fluorescence response (Figure 1B). [23] Interestingly, the opposite is observed with sulfate.…”

“…[23] To explore and develop the sensing potential of this platform, we are applying a structure-guided approach to identify positions for rational protein engineering . [27] Along these lines, the gate post (A136/C139) and β-bulge (D137/W138) region is an attractive starting point because it is spatially near the chromophore and can readily be mutated to tune the photophysical features without significant perturbation to protein folding or chromophore maturation (Figure S1). [37] Closer inspection of this region in mNG reveals that the side chains of A136 and C139 are buried inward, whereas the main chains form hydrogen bonds with the β10 strand (avGFP notation) to stabilize the structure (Figure S1).…”

“…[19][20][21] To decode these supramolecular principles, we are actively investigating, engineering, and applying protein-based hosts for anions. [22][23][24][25][26][27][28] Along these lines, we have a growing program focused on anion sensors derived from the green fluorescent protein (GFP) family.…”

Rational Design of the β‐Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing**

“…[19][20][21] To decode these supramolecular principles, we are actively investigating, engineering, and applying protein-based hosts for anions. [22][23][24][25][26][27][28] Along these lines, we have a growing program focused on anion sensors derived from the green fluorescent protein (GFP) family.…”

“…[36] The anion binding pocket consists of H62, R88, S153, T173, and Y175 and is near the tyrosine-based chromophore (Figure 1A). [27] The binding of halides and oxyanions shifts the chromophore equilibrium from the phenol to the phenolate state, generating a turn-on fluorescence response (Figure 1B). [23] Interestingly, the opposite is observed with sulfate.…”