When the environment matters most: polarizable-embedding QM/MM for multi-photon absorption in red fluorescent...

Area of Science:

• Computational chemistry
• Biophysics
• Spectroscopy

Background:

• Multi-photon absorption in fluorescent proteins (FPs) is sensitive to their environment.
• Previous computational studies often neglect this environmental influence by isolating the chromophore.
• This limitation hinders a full understanding of protein-specific non-linear optical responses.

Purpose of the Study:

• To investigate two- and three-photon absorption (2PA and 3PA) in seven red fluorescent proteins using a polarizable embedding (PE) quantum mechanical/molecular mechanical (QM/MM) approach.
• To assess the accuracy of the PE-QM/MM method by comparing computed results with experimental data.
• To elucidate the role of the protein environment in modulating the non-linear optical properties of FPs.

Main Methods:

• Employed a polarizable embedding (PE) QM/MM approach to model seven red fluorescent proteins: DsRed, mCherry, mPlum, mStrawberry, TagRFP, mRFP1, and mKate.
• Calculated two-photon absorption (2PA) and three-photon absorption (3PA) cross sections for these proteins.
• Analyzed the correlation between computed non-linear absorption trends and changes in permanent dipole moments (|Δμ|).

Main Results:

• Computed 2PA cross sections (σ2PA) showed strong agreement with experimental values, accurately reproducing magnitudes and relative ordering across the protein series.
• The PE-QM/MM method correctly predicted the highest σ2PA for DsRed, TagRFP, and mKate, consistent with experimental rankings.
• Calculated 3PA cross sections (σ3PA) were reported, with trends mirroring 2PA and showing consistency with available experimental action cross sections.

Conclusions:

• Polarizable embedding (PE) is essential for accurately reproducing multi-photon absorption magnitudes in fluorescent proteins.
• The PE-QM/MM method provides a reliable description of environment-driven charge redistribution affecting non-linear optical properties.
• This work presents the first comprehensive computational study of both 2PA and 3PA across multiple red fluorescent proteins.