Front Cover: Identification of mNeonGreen as a pH‐Dependent, Turn‐On Fluorescent Protein Sensor for Chloride (ChemBioChem 14/2019)

Abstract

Chloride-sensitivef luorescent proteins generated from laboratory evolution have ac haracteristict yrosine residue that interacts with ac hloride ion and p-stacks with the chromophore. However,t he engineered yellow-green fluorescent protein mNeonGreen lacks this interaction but still binds chloride, as seen in ar ecently reported crystal structure. Based on its unique coordination sphere, we were curious if chloride could influencet he opticalp roperties of mNeonGreen. Here, we present the structure-guided identification and spectroscopic characterization of mNeonGreen as at urn-on fluorescent protein sensor for chloride. Our results show that chloride binding lowers the chromophore pK a and shifts the equilibrium away from the weaklyf luorescent phenol form to the highly fluorescent phenolate form, resultingi napH-dependent,t urn-on fluorescencer esponse. Moreover,t hrough mutagenesis, we link this sensing mechanism to an on-coordinating residue in the chloride binding pocket. This discoverys ets the stage to furthere ngineerm NeonGreen as an ew fluorescent proteinbased tool for imaging cellular chloride.[a] J.

What biologists want from their chloride reporters – a conversation between chemists and biologists

“…However, the Cl − dissociation constant is out of the physiological range even at acidic pH, making it impractical for deployment at physiological pH. Similarly, the tetrameric YFP from Brachiostoma lanceolatum (lanYFP) has a Cl − -binding pocket (Tutol et al, 2019b). Monomerization of lanYFP maintains the Cl − -binding pocket to form mNeonGreen, a turn-on sensor for Cl − .…”

A Single Point Mutation Converts a Proton-pumping Rhodopsin into a Turn-on Fluorescent Sensor for Chloride

“…[51] Based on this, we recently reported that chloride binding decreases the pKa of the chromophore and shifts the equilibrium from the non-fluorescent phenol state to the highly fluorescent phenolate state, generating a turn-on fluorescence response. [49] Furthermore, we attributed this unique sensing mechanism to a non-coordinating arginine residue, instead of a tyrosine residue, above the chromophore. Together, these homologues highlight how differences in the amino acid residues, within and outside of the chloride binding pocket and even in the chromophore, can influence sensor properties.…”

“…[42][43][44][45][46][47] Relative to these literature precedents, we have identified low sequence identity homologues that are also sensitive to chloride. [48,49] The chloride binding pocket in the naturally occurring yellow fluorescent protein from the jellyfish Phialidium sp. (phiYFP) is identical to that of avYFP-H148Q.…”

A Metal-Based Receptor for Selective Coordination and Fluorescent Sensing of Chloride

“…For this reason, many sensor systems to detect chloride have been developed [32][33][34], many of which involve binding chloride by H-bonding or charge-charge interactions, while a few exploit the photochemical response of a metallo-receptor able to bind chloride [35][36][37][38].…”