Large Stokes shift (LSS) fluorescent proteins, characterized by significant energy gaps between absorption and emission, are invaluable for biological imaging. While most reported LSS systems rely on photoacidic chromophores undergoing excited-state proton transfer (ESPT), photobasic variants have remained underexplored. Here, we construct an LSS complex by incorporating the photobasic fluorophore FR-1V into an engineered rhodopsin mimic hCRBPII mutant M1. Through ultrafast spectroscopy, we reveal pH-dependent ESPT dynamics: at pH 8, ESPT occurs as a single kinetic process (τ = 1. 8 ps), whereas at pH 11, it proceeds via two distinct processes (τ = 1. 0 ps, τ = 13 ps). To reconcile transient absorption spectroscopy (TAS) and time-correlated single-photon counting (TCSPC) results, we hypothesized pH-dependent heterogeneity in the hydrogen-bonding networks of the ground-state Schiff base. Molecular dynamics simulations further support this model, revealing two distinct conformational states: One with stable water-bridged hydrogen-bond networks that facilitate ESPT, and another lacking such networks where proton transfer is structurally impeded. These findings establish a mechanistic framework for pH-responsive biosensors and advance the understanding of protein-chromophore interactions in photobasic systems.
Meng et al. (Sun,) studied this question.