Mutation E300 recommended by protein language models gives ChrimsonR amplified photocurrent response

A central challenge in rhodopsin engineering is identifying mutations that reliably improve desired functional properties, a task made difficult by the enormous mutation space and limited throughput of electrophysiological screening. Improving rhodopsin properties such as photocurrent amplitude and light sensitivity have the potential to broaden the use of rhodopsins to low-light and deep-tissue applications. With this goal, we applied zero-shot protein language models (ESM-1b/1v) to recommend ChrimsonR mutations and experimentally validated all 17 of these variants using whole-cell patch clamp electrophysiology (n=6 cells per mutation).

Despite many mutations reducing function, protein language models identified both known functional residues and unconventional substitutions that produced large functional gains and synergized with K176R to accelerate channel closing. Two mutations, E300G and E300P, increased sustained photocurrents from 66 pA (control) to 305 pA and 255 pA at 635 nm, reduced \(\hbox _\) at 575 nm from 0.19 mW to 0.07 mW, and altered kinetics (\(\tau _}\) increased from 0.06 s up to 0.40 s relative to ChrimsonR). Our results suggest that protein language models, even without task-specific training, can be used alongside electrophysiological measurements as a strategy for screening rhodopsins for enhanced photocurrent.

ChatGPT was used in the editing of this work. CRF acknowledges the NIH BRAIN Initiative Grant (NEI and NIMH 1-U01-MH106027-01), NIH R01NS102727, NIH Single Cell Grant 1 R01 EY023173, NIH R01DA029639 and NIH RF1AG079269, support from Georgia Tech through the Institute for Bioengineering and Biosciences, Invention Studio, and the George W. Woodruff School of Mechanical Engineering.

S.S. acknowledges the Schmidt Science Fellows for their generous support through the postdoctoral fellowship. ESB acknowledges Lisa Yang, HHMI, NIH 1R01MH123977, NIH R01MH122971, and NIH R01DA029639. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA, USA Wallace H.

Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA Benjamin Magondu, Athena Chien & Craig R. Forest McGovern Institute of Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA Yan Tang Collective, Massachusetts Institute of Technology, Cambridge, MA, USA Koch Institute, Massachusetts Institute of Technology, Cambridge, MA, USA Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA ESB is an inventor on several patents related to optogenetics. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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