The Voltage Dependent Sidedness of the Reprotonation of the Retinal Schiff Base Determines the Unique Inward Pumping of Xenorhodopsin

Abstract

The new class of microbial rhodopsins,c alled xenorhodopsins (XeRs), [1] extends the versatility of this family by inward H + pumps. [2][3][4] These pumps are an alternative optogenetic tool to the light-gated ion channels( e.g. ChR1,2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions.In this work we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR). [1] The photodynamic behavior of NsXeR was investigated on the ps to st ime scale elucidating the formation of the Ja nd Ka nd ap reviously unknown long-lived intermediate.T he pH dependent kinetics reveal that alkalization of the surrounding medium accelerates the photocycle and the pump turnover.Inpatch-clamp experiments the blue-light illumination of NsXeR in the Ms tate shows ap otential-dependent vectoriality of the photocurrent transients,s uggesting av ariable accessibility of reprotonation of the retinal Schiff base.Insights on the kinetically independent switching mechanism could furthermore be obtained by mutational studies on the putative intracellular H + acceptor D220.

Introduction of Session 1, “Photochemistry of retinal proteins”

“…The results provided a detailed description of the molecular switch that regulates the accessibility of the proton-binding to the retinal Schiff base during the pumping process. The blue-light illumination of NsXeR in the M state showed a potential-dependent vectoriality of the photocurrent, supporting the idea of a variable accessibility for the reprotonation of the retinal Schiff base [4].…”

Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy

“…( Ns XeR) in lipidic nanodiscs ( Supplementary Figure S2 ). Continuous illumination of Ns XeR generates a photostationary state with major contribution from the longest-lived intermediate state ( Supplementary Figure S3 ) ( Weissbecker et al, 2021 ). The corresponding light minus dark FTIR difference spectrum in the ν(C=O) region ( Figure 4 , black line) shows a sigmoidal band with a negative peak at 1,742 cm −1 and a positive peak at 1,725 cm −1 , that are absent in the difference spectrum of the detergent-solubilized sample ( Figure 4 , inset).…”

Proton Release Reactions in the Inward H+ Pump NsXeR

“…9 An additional long-lived intermediate with absorption characteristics very similar to the dark state, termed NsXeR*, was discovered in detergent-solubilized NsXeR. 17 This long-lived intermediate is also present in nanodisc-reconstituted NsXeR, albeit with a lower relative amplitude as compared to that of detergent-solubilized NsXeR (Figure 2A and Figure S6). The kinetics of the other intermediates are also slightly altered by the membrane environment, an effect well known for microbial rhodopsins.…”

“…17 Based on structural data and time-resolved UV/vis experiments, the H48/D220 pair was suggested to act as a proton acceptor of the RSB proton and D76 as a proton donor to the RSB, but the exact protonation steps are largely unclear. 9,17 To gain a deeper understanding of the molecular determinants of vectorial proton transport, we investigate here the proton pathway in NsXeR by time-resolved molecular spectroscopy in the UV/vis and IR range. We also employed molecular dynamics simulations to elucidate the putative proton pathways used for proton release.…”