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

• Published in: Angewandte Chemie International Edition Vol. 62; no. 26; pp. e202302304 - n/a
• Main Authors: Ong, Whitney S. Y., Ji, Ke, Pathiranage, Vishaka, Maydew, Caden, Baek, Kiheon, Villones, Rhiza Lyne E., Meloni, Gabriele, Walker, Alice R., Dodani, Sheel C.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 26.06.2023
• Edition: International ed. in English
• Subjects: Anions; Anions - chemistry; Aqueous solutions; Binding; Fluorescence; Green fluorescent protein; Green Fluorescent Proteins - genetics; Kinetics; Molecular Dynamics; Mutagenesis; Protein Engineering; Proteins; Sensors; Sulfates; Supramolecular Chemistry; Anions; Molecular Dynamics; Sensors; Supramolecular Chemistry; Protein Engineering
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202302304

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. The serendipitous discovery of an intrinsic fluorescent protein‐based sensor for sulfate called SulfOFF‐1 is reported. This function was unlocked by introducing mutations in a solvent‐exposed region of the fluorescent protein mNeonGreen. An integrated approach encompassing in vitro spectroscopy and in silico modeling provides a comprehensive understanding of the sensor mechanism.