Year: 2026
Type: preprint
Abstract: Understanding the protonation equilibria of key residues in fluorescent proteins, particularly those buried and involved in hydrogen bonding with the chromophore, is crucial for modeling their photodynamical behavior. Using mCherry as a model system, we investigate the protonation effects of two ionizable residues, GLU 215 and LYS 70, located within 4 Å of the chromophore, on the protein's photodynamics. Although GLU and LYS should be fully deprotonated and protonated respectively at pH=7.5 and 300K in water, constant pH molecular dynamics simulations reveal that the fractions of protonated GLU 215 and LYS 70 are both near 50% in mCherry at pH=7.5 and 300K. These co-existing protonation microstates modulate chromophore geometry and hydrogen bonding patterns on the ground state, affecting the accessibility of different non-radiative decay pathways on the first singlet excited state (S1). Upon deprotonation of GLU 215 and loss of its hydrogen bond with the chromophore, the protein environment both sterically restricts chromophore twisting and removes the electrostatic driving force for this motion on S1. These findings provide valuable insights for designing brighter red fluorescent proteins by selective stabilization of residue protonation states that block non-radiative decay pathways.
Source: ChemRxiv
Authors: Mingning Zhu, Elisa Pieri, Todd J. Martínez
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7221933773