Although the outward-directed proton transport across biological membranes is well studied and its importance for bioenergetics is clearly understood, inward-directed light-driven proton pumping by microbial rhodopsins has remained a mystery both physiologically and mechanistically. A new family of Antarctic rhodopsins, which is a subgroup within a novel class of schizorhodopsins reported recently, includes a member, denoted as AntR, which proved amenable to extensive characterization with experiments and computation. Phylogenetic analyses identify AntR as distinct from the well-studied microbial rhodopsins that function as outward-directed ion pumps, and bioinformatics sequence analyses reveal amino acid substitutions at conserved sites essential for outward proton pumping. Modeling and numerical simulations of AntR, combined with advanced analyses using the graph theory and centrality measures from social sciences, identify the dynamic three-dimensional network of hydrogen-bonded water molecules and amino acid residues that function as communication hubs in AntR. This network undergoes major rearrangement upon retinal isomerization, showing important changes in the connectivity of the active center, retinal Schiff base, to the opposing sides of the membrane, as required for proton transport. Numerical simulations and experimental studies of the photochemical cycle of AntR by spectroscopy and sitedirected mutagenesis allowed us to identify pathways that could conduct protons in the direction opposite to that commonly known for outward-directed pumps.

It is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids’ collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. 13C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer.

Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations.

Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.

In biological photoreceptors, the energy stored in early transient species is a key feature to drive the photocycle or a chain of reactions. Time-resolved photoacoustics (PA) can explore the energy landscape of transient species formed within few ns after photoexcitation, as well as volumetric changes (ΔV) of these intermediates with respect to the parental state. In this work, PA identified these important parameters for several channelrhodopsins, namely CaChR1 from Chlamydomonas augustae and CrChR2 from Chlamydomonas reinhardtii and various variants. PA has access to the sub-ns formation of the early photoproduct P1 and to its relaxation, provided that this latter process occurs within a few μs. We found that ΔVP1 for CaChR1 is ca. 12 mL/mol, while it is much smaller for CrChR2 (4.7 mL/mol) and for H. salinarum bacteriorhodopsin (HsBR, ΔVK = 2.8 mL/mol). PA experiments on variants strongly indicate that part of this large ΔVP1 value for CaChR1 is caused by the protonation dynamics of the Schiff base counterion complex involving E169 and D299. PA data further show that the energy level of P1 is higher in CrChR2 (ca. 96 kJ/mol) than in CaChr1 (ca. 46 kJ/mol), comparable to the energy level of the K state of HsBR (60 kJ/mol). Instrumental to gain these molecular values from the raw PA data was the estimation of the quantum yield (Φ) for P1 formation via transient spectroscopy; for both channelrhodopsins, ΦP2 was evaluated as ca. 0.4. Graphical Abstract