# Structure, Function and Dynamics of mCoral, a pH-Responsive Engineered Variant of the mCherry Fluorescent Protein with Improved Hydrogen Peroxide Tolerance

## TL;DR

Scientists engineered a new fluorescent protein, mCoral, with improved resistance to hydrogen peroxide and pH responsiveness.

## Contribution

A mutation in mCherry creates mCoral, a pH-responsive fluorescent protein with enhanced hydrogen peroxide tolerance and structural insights.

## Key findings

- mCoral has similar brightness to mCherry but is more resistant to hydrogen peroxide.
- The M66C mutation introduces a water molecule that affects chromophore planarity and spectral properties.
- mCoral's narrower water exit point may explain its increased resistance to hydrogen peroxide.

## Abstract

The red fluorescent protein mCherry is one of the most widely used fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant, termed mCoral (due to its orange fluorescence hue), has similar brightness to mCherry but improved resistance to hydrogen peroxide. The variant is also responsive to pH with low and high pKa forms that have distinct spectral properties, which DFT analysis suggests is due to protonation state changes in the newly introduced thiol group, as well as the phenol group. The structure of mCoral reveals that the M66C mutation creates a space within the β-barrel structure that is filled by a water molecule, which makes new polar interactions, including the backbone carbonyl group of F65. Molecular dynamics simulations suggest that this additional water molecule, together with local solvation around the chromophore, could play a role in promoting planarity of the full conjugated system comprising the chromophore. The mCoral chromophore makes slightly more H-bonds with water than mCherry. The main water exit point for mCherry is also narrower in mCoral, providing a potential explanation for increased resistance to hydrogen peroxide. Overall, a small structural change to mCherry has resulted in a new fluorescent protein with potentially useful characteristics and an insight into the role of dynamics and water in defining the structure–function relationship in red fluorescent proteins.