# Multiphoton Absorption Spectra of Channelrhodopsin‑2 via Multiscale Simulation Methods

## TL;DR

This paper introduces a detailed computational method to study the light absorption properties of a key optogenetics protein, Channelrhodopsin-2, using advanced simulation techniques.

## Contribution

The paper presents a novel multiscale simulation methodology to compute one-, two-, and three-photon absorption spectra of ChR2.

## Key findings

- The multiscale method accurately computes one-photon absorption spectra matching experimental data.
- Theoretical two- and three-photon absorption spectra of ChR2 are reported for the first time.
- Spectral differences arise from structural variations captured by classical and quantum simulations.

## Abstract

Channelrhodopsin-2 (ChR2) is a 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 (RSB) 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 molecular
mechanics (MM) molecular dynamics (MD), quantum mechanics/molecular
mechanics (QM/MM)-MD, and fragment-based polarizable embedding (PE)
to derive environment-specific PE potentials from the explicit protein–lipid-solvent
environment. The final step in the methodology is to use these potentials
to compute accurate spectra via PE-time-dependent density functional
theory (PE-TD-DFT). 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. We compare the multiphoton absorption
(MPA) spectra where the two RSB moieties are sampled using classical
MD and QM/MM-MD, respectively. The resulting spectral differences
are attributed to variations in key structural parameters that we
analyze and document.