Reaction Dynamics in the Chrimson Channelrhodopsin: Observation of Product-State Evolution and Slow Diffusive Protein Motions

Chrimson is a red-light absorbing channelrhodopsin useful for deep-tissue optogenetics applications. Here, we present the Chrimson reaction dynamics from femtoseconds to seconds, analyzed with target analysis methods to disentangle spectrally and temporally overlapping excited-and product-state dynamics. We found multiple phases ranging from approximate to 100 fs to approximate to 20 ps in the excited-state decay, where spectral features overlapping with stimulated emission components were assigned to early dynamics of K-like species on a 10 ps time scale. Selective excitation at the maximum or the blue edge of the absorption spectrum resulted in spectrally distinct but kinetically similar excited-state and product-state species, which gradually became indistinguishable on the mu s to 100 mu s time scales. Hence, by removing specific protein conformations within an inhomoge-neously broadened ensemble, we resolved slow protein backbone and amino acid side-chain motions in the dark that underlie inhomogeneous broadening, demonstrating that the latter represents a dynamic interconversion between protein substates.

Automated summary

In this study, the reaction dynamics of Chrimson channelrhodopsin were analyzed from femtoseconds to seconds. Multiple phases in the decay of excited state were identified, and early dynamics of K-like species on a 10 ps time scale were observed. Selective excitation at different wavelengths resulted in distinct spectral features, which gradually became indistinguishable over time. Protein motions underlying inhomogeneous broadening were resolved, demonstrating a dynamic interconversion between protein substates.