# Physical Mechanisms of an Unconventional Green Fluorescent Protein Indicator for Chloride

## TL;DR

This paper explores how a unique green fluorescent protein from a jellyfish responds to different anions like chloride, using simulations and experiments to understand its behavior.

## Contribution

The study reveals a new mechanism of anion sensitivity in a green fluorescent protein through combined theoretical and experimental approaches.

## Key findings

- cgreGFP is sensitive to anions including chloride, bromide, iodide, and nitrate.
- Anion binding alters the chromophore equilibrium, causing a turn-off fluorescence response at acidic pH.
- Mutagenesis of the anion entry pathway validates the theoretical predictions.

## Abstract

Fluorescent proteins
bearing an intrinsic tripeptide chromophore
exhibit diverse, tunable photophysical features that are exceptional
for biosensing applications. However, atomic-level details of these
sensing mechanisms are obscured experimentally, particularly as protein
motions, including the chromophore, cannot be divined from the structure
alone. Molecular dynamics (MD) simulations can bridge this gap, providing
a landscape of global and local protein motions with resolution to
key amino acids connected to function and potential for engineering.
In this study, we uncover that the green fluorescent protein from
the jellyfish Clytia gregaria (cgreGFP) is sensitive
to anions, including chloride, bromide, iodide, and nitrate, with
a combination of theoretical and experimental investigations. Constant
pH molecular dynamics (CpHMD) simulations reveal a coordinated entry
of all four anions into an unconventional binding cavity near the
chromophore. Photophysical measurements of the wild-type protein confirm
this behavior and demonstrate that anion binding tunes the chromophore
equilibrium, resulting in a turn-off fluorescence response at acidic
pH with the affinity trend iodide > nitrate > bromide > chloride.
Finally, targeted mutagenesis of the anion entry pathway emphasizes
the guiding force of theory to understand cgreGFP-like indicators
and beyond.