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.

Chloride‐sensitive fluorescent proteins generated from laboratory evolution have a characteristic tyrosine residue that interacts with a chloride ion and π‐stacks with the chromophore. However, the engineered yellow‐green fluorescent protein mNeonGreen lacks this interaction but still binds chloride, as seen in a recently reported crystal structure. Based on its unique coordination sphere, we were curious if chloride could influence the optical properties of mNeonGreen. Here, we present the structure‐guided identification and spectroscopic characterization of mNeonGreen as a turn‐on fluorescent protein sensor for chloride. Our results show that chloride binding lowers the chromophore pKa and shifts the equilibrium away from the weakly fluorescent phenol form to the highly fluorescent phenolate form, resulting in a pH‐dependent, turn‐on fluorescence response. Moreover, through mutagenesis, we link this sensing mechanism to a non‐coordinating residue in the chloride binding pocket. This discovery sets the stage to further engineer mNeonGreen as a new fluorescent protein‐based tool for imaging cellular chloride.

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over...

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