Ultrafast Backbone Protonation in Channelrhodopsin-1 Captured by Polarization Resolved Fs Vis-pump—IR-Probe Spectroscopy and Computational Methods

Abstract

Channelrhodopsins (ChR) are light-gated ion-channels heavily used in optogenetics. Upon light excitation an ultrafast all-trans to 13-cis isomerization of the retinal chromophore takes place. It is still uncertain by what means this reaction leads to further protein changes and channel conductivity. Channelrhodopsin-1 in Chlamydomonas augustae exhibits a 100 fs photoisomerization and a protonated counterion complex. By polarization resolved ultrafast spectroscopy in the mid-IR we show that the initial reaction of the retinal is accompanied by changes in the protein backbone and ultrafast protonation changes at the counterion complex comprising Asp299 and Glu169. In combination with homology modelling and quantum mechanics/molecular mechanics (QM/MM) geometry optimization we assign the protonation dynamics to ultrafast deprotonation of Glu169, and transient protonation of the Glu169 backbone, followed by a proton transfer from the backbone to the carboxylate group of Asp299 on a timescale of tens of picoseconds. The second proton transfer is not related to retinal dynamics and reflects pure protein changes in the first photoproduct. We assume these protein dynamics to be the first steps in a cascade of protein-wide changes resulting in channel conductivity.

Impact of Ultrafast Electric Field Changes on Photoreceptor Protein Dynamics

“…This proton transfer to D299 depicted in Figure 2c reflects protein dynamics significantly after the isomerization process. 7,30 The established protonation pattern paves the way for the proton transfer from the retinal to Glu169 (Figure 2d) in the transition of the long-living intermediates P1 to P2. 33,34 These observations demonstrate processes steering protein dynamics both prior to and beyond chromophore isomerization.…”

“…Since neither an overlap of the retinal excited-state wave function to the carboxylic group Glu169 or to the protein backbone exists, nor a steric interaction gives rise to the observed changes on this time-scale, the structural changes of the chromophore in the excited state including isomerization cannot be the causal reason for the protonation dynamics. 30 Thus, we propose the ultrafast altered electric field change to be responsible for these ultrafast protonation changes.…”

“…Recently, we were able to demonstrate ultrafast protein protonation changes prior to the conformational change of the chromophore. 30 In CaChR1, the protonated Schiff base of the retinal chromophore interacts with a counterion consisting of protonated Glu169, deprotonated Asp299, and water molecules. The electrostatic interaction between these groups stabilizes the conformation of the counterion around the Schiff base (see Figure 2a).…”

“…The backbone dynamics are assigned to a transient protonation of the E169 carbonyl group. 30 Deprotonation of E169 occurs prior to the isomerization process. With the decay of the electronic excited state, all-trans retinal isomerizes with a time constant of 100 fs and adopts the 13-cis conformation.…”

“…21 In CaChR1, the initial deprotonation of E169 leads to protonation of D299 on a longer time-scale, relevant for further downstream processes. 30 Thus, ultrafast protein changes, faster than isomerization, are significant for protein changes on a longer time-scale. The driving force is presumably the altered Coulomb force inducing transient electrostatic changes propagating in time and space in the protein.…”