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

Abstract

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the β‐bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn‐off sensor SulfOFF‐1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the β‐barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin

“…Thus, the S147D mutation effectively makes EGFP and mClover3 into pH-sensitive GFPs similar to SEP and Lime (Table 2), confirming its ability to modulate the pH sensitivity of GFP variants. It remains to be determined if a similar mutation at the aligned position of FPs from organisms other than Aequorea (e.g., W138 of mNeonGreen) 36 could have a similar influence on pK a .…”