Proton Release Reactions in the Inward H+ Pump NsXeR.

Directional ion transport across biological membranes plays a central role in many cellular processes. Elucidating the molecular determinants for vectorial ion transport is key to understanding the functional mechanism of membrane-bound ion pumps. The extensive investigation of the light-driven proton pump bacteriorhodopsin from Halobacterium salinarum(HsBR) enabled a detailed description of outward proton transport. Although the structure of inward-directed proton pumping rhodopsins is very similar to HsBR, little is known about their protonation pathway, and hence, the molecular reasons for the vectoriality of proton translocation remain unclear. Here, we employ a combined experimental and theoretical approach to tracking protonation steps in the light-driven inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR). Time-resolved infrared spectroscopy reveals the transient deprotonation of D220 concomitantly with deprotonation of the retinal Schiff base. Our molecular dynamics simulations support a proton release pathway from the retinal Schiff base via a hydrogen-bonded water wire leading to D220 that could provide a putative gating point for the proton release and with allosteric interactions to the retinal Schiff base. Our findings support the key role of D220 in mediating proton release to the cytoplasmic side and provide evidence that this residue is not the primary proton acceptor of the proton transiently released by the retinal Schiff base.

Monitoring the Progression of Cell-Free Expression of Microbial Rhodopsins by Surface Enhanced IR Spectroscopy: Resolving a Branch Point for Successful/Unsuccessful Folding.

The translocon-unassisted folding process of transmembrane domains of the microbial rhodopsins sensory rhodopsin I (HsSRI) and II (HsSRII), channelrhodopsin II (CrChR2), and bacteriorhodopsin (HsBR) during cell-free expression has been investigated by Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS). Up to now, only a limited number of rhodopsins have been expressed and folded into the functional holoprotein in cell free expression systems, while other microbial rhodopsins fail to properly bind the chromophore all-trans retinal as indicated by the missing visible absorption. SEIRAS experiments suggest that all investigated rhodopsins lead to the production of polypeptides, which are co-translationally inserted into a solid-supported lipid bilayer during the first hour after the in-vitro expression is initiated. Secondary structure analysis of the IR spectra revealed that the polypeptides form a comparable amount of α-helical structure during the initial phase of insertion into the lipid bilayer. As the process progressed (>1 h), only HsBR exhibited a further increase and association of α-helices to form a compact tertiary structure, while the helical contents of the other rhodopsins stagnated. This result suggests that the molecular reason for the unsuccessful cell-free expression of the two sensory rhodopsins and of CrChR2 is not due to the translation process, but rather to the folding process during the post-translational period. Taking our previous observation into account that HsBR fails to form a tertiary structure in the absence of its retinal, we infer that the chromophore retinal is an integral component of the compaction of the polypeptide into its tertiary structure and the formation of a fully functional protein.

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

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.

The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis.

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.

Biofabricating a Silk Scaffold as a Functional Microbial Trap.

Silk fibroin produced from silkworms has been intensively utilized as a scaffold material for a variety of biotechnological applications owing to its remarkable mechanical strength, extensibility, biocompatibility, and ease of biofunctionalization. In this research, we engineered silk as a novel trap platform capable of capturing microorganisms. Specifically, we first fabricated the silk material into a silk sponge by lyophilization, yielding a 3D scaffold with porous microstructures. The sponge stability in water was significantly improved by ethanol treatment with elevated ß-sheet content and crystallinity of silk. Next, we biofunctionalized the silk sponge with a poly-specific microbial targeting molecule, ApoH (apolipoprotein H), to enable a novel silk-based microbial trap. The recombinant ApoH engineered with an additional penta-tyrosine was assembled onto the silk sponge through the horseradish peroxidase (HRP) mediated dityrosine cross-linking. Last, the ApoH-decorated silk sponge was demonstrated to be functional in capturing our model microorganism targets, E. coli and norovirus-like particles. We envision that this biofabricated silk platform, capable of trapping a variety of microbial entities, could serve as a versatile scaffold for rapid isolation and enrichment of microbial samples toward future diagnostics and therapeutics. This strategy, in turn, can expedite advancing future biodevices with functionality and sustainability.

The Blue-Green Sensory Rhodopsin SRM from Haloarcula marismortui Attenuates Both Phototactic Responses Mediated by Sensory Rhodopsin I and II in Halobacterium salinarum.

Haloarchaea utilize various microbial rhodopsins to harvest light energy or to mediate phototaxis in search of optimal environmental niches. To date, only the red light-sensing sensory rhodopsin I (SRI) and the blue light-sensing sensory rhodopsin II (SRII) have been shown to mediate positive and negative phototaxis, respectively. In this work, we demonstrated that a blue-green light-sensing (504 nm) sensory rhodopsin from Haloarcula marismortui, SRM, attenuated both positive and negative phototaxis through its sensing region. The H. marismortui genome encodes three sensory rhodopsins: SRI, SRII and SRM. Using spectroscopic assays, we first demonstrated the interaction between SRM and its cognate transducer, HtrM. We then transformed an SRM-HtrM fusion protein into Halobacterium salinarum, which contains only SRI and SRII, and observed that SRM-HtrM fusion protein decreased both positive and negative phototaxis of H. salinarum. Together, our results suggested a novel phototaxis signalling system in H. marismortui comprised of three sensory rhodopsins in which the phototactic response of SRI and SRII were attenuated by SRM.