Hydrogen bond flexibility correlates with Stokes shift in mPlum variants

Area of Science:

• Molecular Biology
• Biophysics
• Spectroscopy

Background:

• Fluorescent proteins are crucial for tracking subcellular processes with high precision.
• Proteins emitting beyond 650 nm allow deeper tissue penetration and longer imaging times.
• The origin of the extended Stokes shift in red fluorescent proteins remains unclear.

Purpose of the Study:

• To investigate the link between chromophore environment flexibility and Stokes shift in the mPlum fluorescent protein.
• To understand the excited state solvation dynamics in mPlum and its mutants.

Main Methods:

• Spectrally resolved transient grating spectroscopy was used to study solvation dynamics.
• Molecular dynamics simulations were employed to analyze the chromophore environment.
• Point mutants of residues E16 and I65 in mPlum were examined.

Main Results:

• Two relaxation constants (picoseconds and tens of picoseconds) were observed, corresponding to direct and water-mediated hydrogen bond survival times.
• The mPlum variant I65V, exhibiting the largest Stokes shift, showed significant decay on both time scales.
• A correlation was found between the bathochromic shift and facile switching between direct and water-mediated hydrogen bonds.

Conclusions:

• Environmental flexibility, particularly the dynamic switching of hydrogen bonds at the chromophore site, is key to the extended Stokes shift in mPlum.
• This dynamic model provides insights into excited state solvation mechanisms.
• The findings offer a framework for engineering novel red fluorescent proteins with improved imaging capabilities.