Environmental Dipolar Relaxation during Excited-State Proton Transfer in Green Fluorescent Protein

Area of Science:

• Biophysics
• Photochemistry
• Protein Engineering

Background:

• Green Fluorescent Protein (GFP) variants are essential tools for biological imaging.
• Excited-state proton transfer (ESPT) is a key process in GFP chromophore function.
• The role of environmental dipolar relaxation during GFP's ESPT is not well understood.

Purpose of the Study:

• To provide the first experimental evidence for dipolar relaxation during ESPT in GFP.
• To investigate the influence of protein matrix relaxation on the ESPT reaction pathway.
• To explore the implications for designing novel fluorescent proteins.

Main Methods:

• Utilized fluorescence spectroscopy to study GFP variants.
• Excited the neutral phenolic ground-state absorption band along its red edge.
• Analyzed changes in the center of spectral mass (CSM) of emission spectra.

Main Results:

• Observed minimal change in CSM across excitation wavelengths, unlike other biological chromophores.
• Demonstrated that dipolar relaxation occurs on the subnanosecond timescale, concurrent with ESPT.
• Provided evidence that this relaxation is a key determinant of the ESPT reaction pathway.

Conclusions:

• Environmental dipolar relaxation is integral to the adiabatic nature of ESPT in GFP.
• The protein matrix relaxation kinetics match ESPT timescales, explaining its adiabaticity.
• Findings can guide the rational design of fluorescent proteins with optimized photophysical properties.