Light-driven Anion-Pumping Rhodopsin with Unique Cytoplasmic Anion-release Mechanism.

Microbial rhodopsins are photoreceptive membrane proteins found in microorganisms with an all-trans-retinal chromophore. The function of many microbial rhodopsins is determined by three residues in the third transmembrane helix called motif residues. Here, we report a group of microbial rhodopsins with a novel Thr-Thr-Gly (TTG) motif. The ion-transport assay revealed that they function as light-driven inward anion pumps similar to halorhodopsins previously found in archaea and bacteria. Based on the characteristic glycine residue in their motif and light-driven anion-pumping function, these new rhodopsins are called glycylhalorhodopsins (GHRs). X-ray crystallographic analysis found large cavities on the cytoplasmic side, which are produced by the small side-chain volume of the glycine residue in the motif. The opened structure of GHR on the cytoplasmic side is related to the anion releasing process to the cytoplasm during the photoreaction compared to canonical halorhodopsin from Natronomonas pharaonis (NpHR). GHR also transports SO42- and the extracellular glutamate residue plays an essential role in extracellular SO42- uptake. In summary, we have identified TTG motif-containing microbial rhodopsins that display an anion-releasing mechanism.

Time-resolved detection of light-induced conformational changes of heliorhodopsin.

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 µs and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 µs). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process.

Fracture Analysis of Short-Scale Corroded/Healthy Reinforced Concrete Beams under Bending Using Acoustic Emission.

In this work, the acoustic emission (AE) technique is used to evaluate the fracture process of corroded and healthy reinforced concrete (RC) beams subjected to a monotonic bending test. In fact, many researchers have conducted laboratory experiments considering different conditions to perform rebar corrosion monitoring in RC structures using the AE method. However, previous studies have not investigated the evolution of the bending performance of RC beams at different corrosion degrees, considering the interaction of single rebar with concrete. In this study, healthy and electrically corroded RC beams are evaluated, considering different corrosion levels. The analysis of the moving average of the AE maximum amplitudes was consistent to distinguish four stages of mechanical behavior that the healthy, and corroded specimens with low and medium corrosion levels of 0.9% and 3.2% experienced up to failure. Three damage stages were identified in the case of a high corrosion level of 9.3%. Then, the AE maximum amplitudes were suitable to establish an efficient clustering, which enabled the classification of the fractures into minor, medium, and major classes. Furthermore, the digital analysis method proposed in this study was suitable to visually reveal the influence of the preexisting corrosion-induced damages on the bending failure process of the RC beams.

Kinetic study on the molecular mechanism of light-driven inward proton transport by schizorhodopsins.

Schizorhodopsins (SzRs) are light-driven inward proton pumping membrane proteins. A H+ is released to the cytoplasmic solvent from the chromophore, retinal Schiff base (RSB), after light absorption, and then another H+ is bound to the RSB at the end of photocyclic reaction. However, the mechanistic detail of H+ transfers in SzR is almost unknown. Here we studied the deuterium isotope effect and the temperature dependence of the reaction rate constants of elementary steps in the photocycles of SzRs. The former indicated that deprotonation and reprotonation of RSB is mainly accomplished by H+ hopping between heavy atoms with similar H+ affinity. Furthermore, the temperature dependence of the rate constants revealed that most of H+ transfer events have a high entropy barrier. In contrast, the activation enthalpy and entropy of extremely thermostable SzR (MsSzR) are significantly higher than other types of SzRs (SzR1 and MtSzR) suggesting that its highly thermostable structure is optimized with at the cost of slower reaction rates at ambient temperatures.

Saccharibacteria harness light energy using type-1 rhodopsins that may rely on retinal sourced from microbial hosts.

Microbial rhodopsins are a family of photoreceptive membrane proteins with a wide distribution across the Tree of Life. Within the candidate phyla radiation (CPR), a diverse group of putatively episymbiotic bacteria, the genetic potential to produce rhodopsins appears to be confined to a small clade of organisms from sunlit environments. Here, we characterize the metabolic context and biophysical features of Saccharibacteria Type-1 rhodopsin sequences derived from metagenomic surveys and show that these proteins function as outward proton pumps. This provides one of the only known mechanisms by which CPR can generate a proton gradient for ATP synthesis. These Saccharibacteria do not encode the genetic machinery to produce all-trans-retinal, the chromophore essential for rhodopsin function, but their rhodopsins are able to rapidly uptake this cofactor when provided in experimental assays. We found consistent evidence for the capacity to produce retinal from ß-carotene in microorganisms co-occurring with Saccharibacteria, and this genetic potential was dominated by members of the Actinobacteria, which are known hosts of Saccharibacteria in other habitats. If Actinobacteria serve as hosts for Saccharibacteria in freshwater environments, exchange of retinal for use by rhodopsin may be a feature of their associations.

Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.