Structure, function and dynamics of mCoral, a pH-responsive engineered variant of the mCherry fluorescent protein with improved hydrogen peroxide tolerance

Zitti, Athena, Aksakal, Ozan, Vitsupakorn, Danoo, Rizkallah, Pierre J. ORCID: https://orcid.org/0000-0002-9290-0369, Mikolajek, Halina, Platts, James A. ORCID: https://orcid.org/0000-0002-1008-6595, Menzies, Georgina E. ORCID: https://orcid.org/0000-0002-6600-6507 and Jones, D. Dafydd ORCID: https://orcid.org/0000-0001-7709-3995 2025. Structure, function and dynamics of mCoral, a pH-responsive engineered variant of the mCherry fluorescent protein with improved hydrogen peroxide tolerance. International Journal of Molecular Sciences 27 (1) , 154. 10.3390/ijms27010154

PDF - Published Version Download (5MB)

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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Schools > Chemistry Schools > Medicine Schools > Biosciences
Additional Information:	License information from Publisher: LICENSE 1: URL: https://creativecommons.org/licenses/by/4.0/, Start Date: 2025-12-23
Publisher:	MDPI
ISSN:	1661-6596
Date of First Compliant Deposit:	5 January 2026
Date of Acceptance:	20 December 2025
Last Modified:	05 Jan 2026 12:32
URI:	https://orca.cardiff.ac.uk/id/eprint/183491

Actions (repository staff only)

Edit Item