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.

Keywords: Anions; Molecular Dynamics; Protein Engineering; Sensors; Supramolecular Chemistry.

Figure 1.

(A) X-ray crystal structure of mNeonGreen (mNG) with the chromophore (CRO) (PDB ID: 5LTP). The residues in the anion binding pocket and β-bulge are highlighted. Each residue is labeled with the single letter amino acid code and position number. (B) The chromophore equilibrium of mNG can be tuned between the non-fluorescent phenol and fluorescent phenolate states by anions at pH 4.5.

Figure 2.

SulfOFF-1 (mNG-D137G-W138G) is a turn-off fluorescent sensor for sulfate. (A) Absorption and (B) emission spectra of SulfOFF-1 in the presence of 0 (black), 0.025, 0.05, 0.075, 0.125, 0.25, 0.5, 0.75, 1, 2, 5, and 10 mM (red) sulfate. The inset shows the normalized emission response at 520 nm as a function of increasing sulfate concentrations. (C) The fluorescence response (F_f / F_i) at 520 nm of SulfOFF-1 (apo, F_i) in the presence of 10 mM (F_f) sodium chloride (Cl⁻), bromide (Br⁻), iodide (I⁻), nitrate (NO₃⁻), phosphate (H₂PO₄⁻/HPO₄²⁻), sulfate (SO₄²⁻), and gluconate (Gluc). Data was collected in triplicate from two protein preparations and is reported as the average with standard error of the mean. All experiments were carried out at room temperature (23–25 °C) with 4 μM protein in 50 mM MOPS and 1 mM NaCl buffer at pH 7 (λ_ex = 485 nm, λ_em = 500–650 nm).

Figure 3.

Surface potential map of mNG generated by molecular dynamics (MD) simulations where a high density of positive charge is in blue and negative charge is in red. Sulfate binding hotspots are associated with the solvent-exposed residues R103, R140 and R172 in the region of positive charge density. The key R residues near the β-bulge and the surface-bound sulfate ions prior to entry are shown.

Figure 4.

MD simulations reveal a pathway for sulfate entry and binding in mNG at the β-bulge gate. Representative snapshots are shown for the apo and sulfate-bound phases with possible hydrogen bonding and electrostatic interactions within 4 Å (dashed lines).

Figure 5.

MD simulations indicate that SulfOFF-1 has the widest opening to facilitate the binding of sulfate. (A) Representative snapshot of SulfOFF-1 to highlight the opening that is defined as the distance (dashed line) between the Cα atoms of residues R140 and E198. (B) Analysis of the distances for mNG (purple) and SulfOFF-1 (red) as a function of time for a representative trajectory.

Figure 6.

MD simulations show how sulfate binding induces a change in the chromophore orientation leading to fluorescence quenching in (A) mNG and (B) SulfOFF-1. A snapshot from each sulfate-bound trajectory is shown in the top panel. Possible hydrogen bonding or electrostatic interactions within 4 Å of sulfate are shown with dashed lines. Comparison of the chromophores in the apo (white) and sulfate-bound (yellow) trajectories for each protein is shown in the bottom panel. All snapshots are taken from the last frame of a representative trajectory.