Proton transfer kinetics modulated by single-residue substitution in photobasic FR-1V/hCRABPII.

Fluorescent proteins with large Stokes shifts have become indispensable in biological imaging due to their improved signal-to-noise ratio and reduced self-absorption. While traditional photoacid systems have optimization limitations, photobasic fluorescent proteins provide a promising alternative novel excited-state proton transfer (ESPT) mechanisms. We study how single amino acid variations affect the photophysical properties of FR-1V/hCRABPII microenvironment regulation. We used femtosecond time-resolved transient absorption spectroscopy (fs-TA) to compare two mutants: M3/FR-1V (K40E) and M15/FR-1V (K40H). M3/FR-1V demonstrated multi-component dynamics, including rapid ESPT and subsequent conformational relaxation, resulting in efficient fluorescence channels. In contrast, M15/FR-1V exhibits rapid excited-state decay, slower ESPT, and enhanced nonradiative deactivation. Global fitting analysis identified two competitive transfer channels: a favorable conformation that promotes protonated Schiff base formation and radiative transition, and an unfavorable conformation that inhibits proton transfer and non-radiative relaxation. This study offers new molecularlevel insights into the amino acid regulation of ESPT networks in photobasic fluorescent proteins, laying the groundwork for the rational design of next-generation fluorescent imaging tools.