Fluorescent proteins are well known for their excellent ability to act as biosensors, which helps to investigate the biochemistry of living cells and organisms. mNeonGreen(mNG) is one of the brightest green fluorescent proteins, has been identified as a turn on fluorescent protein sensor for chloride, and confirmed with a resolved x-ray crystal structure with bound chloride. Experimental data has also shown that mNG shows a turn off response for sulfate at physiological pH. In this work, we explore the mechanisms involved in sulfate binding and how mutations on flexible loops located close to the chromophore could help to further engineer this biosensor. We applied constant pH molecular dynamics simulations on the wild type and several mutated systems at pH 7. Through these simulations we were able to observe a random diffusion of sulphate ion into the beta barrel of mNG, where the location and orientation of entry residues acted as the limiting factor for the entry of ions. Specifically, the gating motion shown by W138 had a major effect on the entry of sulfate ions into the beta barrel. Among the mutated systems, the double mutant D137G/W138G (2G) has the the largest entry point opening (~20 Å) when sulfate is unbound. Once sulfate binds, the inner binding pocket rearranges accordingly, locking the sulfate in place. This is in good agreement with the experimental result that 2G is the most efficient mutant kinetically. Upon binding of sulfate, a change in the chromophore conformation was also observed in all the systems, which provides a potential avenue of exploration for determining the mechanism of the turn-off response. In summary, we were able to identify likely binding pathways for sulfate in mNG, key binding residues, and determine atomic-level details for the mutagenesis experiments to improve kinetic efficiency of mNG as a sulfate sensor.

Computational studies of mNeonGreen as a sulfate sensor

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