Multiphoton Absorption Spectra of Channelrhodopsin-2 via Multiscale Simulation Methods

Channelrhodopsin 2 (ChR2) is a transmembrane protein, light-gated ion channel widely used in optogenetics, a technique that enables precise control of neuronal activity by genetically engineering light-sensitive proteins into cell membranes. This protein exists in dimeric form, with each monomer containing a retinal Schiff base moiety covalently bonded that undergoes trans-cis isomerization upon light absorption. However, the limited penetration depth of visible light in biological tissues motivates the use of multiphoton-absorption techniques, which enhance tissue penetration, improve focality, and reduce phototoxicity, thereby offering a promising alternative for optogenetic applications. In this paper, we present a fully atomistic multiscale methodology for computing the one-, two-, and three-photon absorption spectra of ChR2, where the protein, lipid bilayer, and solvent are explicitly considered throughout the workflow. This methodology integrates classical MD, QM/MM MD sampling, and fragment-based polarizable embedding (PE) to derive adequate embedding potentials, and PE-TD-DFT for accurate spectroscopic calculations. Validation against experimental one-photon absorption spectra demonstrates excellent agreement. For the first time, we report the theoretical two- and three-photon absorption in ChR2, albeit without direct experimental comparison. Furthermore, spectral differences resulting from different sampling treatments of the two RSB moieties are attributed to variations in key structural parameters that have been analyzed and documented.