Biology subjects

Konno, M.

Publications and source records attributed to Konno, M..

15 recordsLinked to original sources

bioRxiv2026

AI-enabled rhodopsin design for blue-light enhanced bacterial growth

We developed an AI-guided design pipeline that generated and validated non-natural microbial rhodopsins with spectral properties not yet known in nature. The pipeline comprised a three-stage in silico design, a genetic algorithm (GA) for sequence generation, a stacked LASSO and XGBoost machine-learning (ML) regressor for spectral prediction and fitness ranking, and a Markov-based sequence plausibility filter to enforce proton pumping like characteristics. Four candidate rhodopsins (APR1, APR2, APR6, and APR7) targeting blue light absorption were designed and AlphaFold3 structural modelling predicted retinal binding pocket architecture consistent with outward proton-pumping function. Experimental characterisation confirmed that all four variants absorbed light at [~]410 nm and significantly promoted the growth of Cupriavidus necator under blue light illumination. This study demonstrates that AI-enabled design can engineer proteins with no natural precedent, generating light-harvesting rhodopsins with novel spectral properties while preserving biological function, marking a significant advance in programmable synthetic biology.

Synthetic Biology↗

bioRxiv2025

Cryo-EM Structures of Methanogenic Schizorhodopsins Reveal Divergent Strategies for Proton Transport and Thermal Adaptation

Schizorhodopsins (SzRs) are light-driven inward proton pumps found in Asgard archaea, contrasting with most microbial rhodopsins that export protons. Some SzRs, including Methanoculleus taiwanensis SzR (MtSzR) and Methanoculleus sp. SzR (MsSzR), exhibit remarkable heat tolerance, suggesting structural adaptations to extreme environments. Here, we report cryo-electron microscopy structures of MtSzR and MsSzR at 2.4 and 2.7 [A] resolutions, respectively, revealing distinct mechanisms for proton transport and thermostability. Both proteins share a cytoplasm-facing proton acceptor, yet MtSzR employs a hydrophobic gating mechanism, whereas MsSzR lacks this barrier and stabilizes its acceptor through a salt-bridge network. Thermostability also diverges: MtSzR relies on proline-rich loops and electrostatic interactions, while MsSzR achieves stability through reinforced trimerization and extensive interhelical aromatic packing. Structural and mutational analyses highlight the adaptability of SzRs and provide a framework for engineering robust photoreceptors for optogenetics and synthetic biology.

biophysics↗

bioRxiv2025

The bacterial schizorhodopsins: novel light-driven inward proton pumps from Antarctic Minisyncoccota (Patescibacteria) and cyanobacteria, with implications for the proton-pumping mechanism

Microbial rhodopsins represent a diverse superfamily of light-sensitive seven-transmembrane proteins with expanding phylogenetic diversity driven by advances in metagenomics. Among these, schizorhodopsins constitute a divergent family originally identified as inward proton pumps from Promethearchaeota (Asgard archaea). Here, we report that in addition to archaeal schizorhodopsins, many members of the family originate from bacteria and detail a comprehensive biophysical characterization of novel schizorhodopsins from Antarctic Minisyncoccota (Patescibacteria) and cyanobacteria, designated as paSzR and psSzR, respectively. Both proteins function as light-driven inward proton pumps, as confirmed through pH measurements in Escherichia coli cells. Laser-flash photolysis experiments identified multiple photointermediates (K, L, and M) characteristic of microbial rhodopsin photocycles, though with slower turnover rates compared to archaeal schizorhodopsins. Site-directed mutagenesis of conserved residues in the third and sixth transmembrane helices demonstrates differential structural requirements between paSzR and psSzR. Our phylogenetic reconstruction reveals that most bacterial schizorhodopsins cluster in a single lineage distinct from archaeal variants. These findings significantly expand our understanding of microbial rhodopsin diversity and provide crucial insights into alternative molecular mechanisms for light-driven proton translocation, with implications for microbial ecology in extreme environments. Statement of significanceSchizorhodopsins represent an unusual family of light-driven proteins that pump protons inwardly--opposite to the classical microbial rhodopsin pumps. Originally discovered in archaea, we revealed their unprecedented prevalence in bacteria. We characterized two novel bacterial schizorhodopsins from the Antarctic: paSzR from Minisyncoccota and psSzR from Cyanobacteriota, demonstrating their inward proton-pumping activity. Our phylogenetic reconstruction showed that most bacterial schizorhodopsins form a distinct evolutionary lineage and two exchange events between Minisyncoccota and cyanobacteria. Through targeted mutagenesis, we identified critical residues that govern spectral tuning and transport efficiency, revealing mechanistic differences between members of the family. These findings expand the known diversity of light-energy conversion mechanisms across microbial life and provide insights into alternative proton translocation strategies, with implications for understanding microbial adaptation to extreme environments.

biophysics↗

bioRxiv2025

Functional characterization of red-shifted rhodopsin channels from giant viruses explored by a machine-learning model for long-wavelength optogenetics

Channelrhodopsins (ChRs) are light-gated ion channels. These proteins are widely used in optogenetics to optically manipulate neural activity. However, manipulation using short-wavelength light to activate ChRs causes cell toxicity and is hampered by low tissue penetration. To overcome these difficulties, although several red-shifted ChR variants have been identified, further red-shift is required for more efficient and noninvasive neural control. While molecular screening of ChRs requires high-cost experiments, recent machine-learning-based protein functionality prediction enables more efficient selection of target proteins for characterization. Here, we constructed an elastic-net machine-learning model trained on 1,163 experimental data to predict the maximum absorption wavelength ({lambda}max) of uncharacterized ChRs. The model suggested several red-shifted candidates, and we identified a viral rhodopsin channel, ChR024, with the second-longest{lambda} max as a cation-conducting ChR ({lambda}max = [~]578 nm) after Chrimson ({lambda}max = [~]580 nm). This result demonstrates the high impact of ML on reducing the screening costs of functional proteins.

biophysics↗

bioRxiv2025

Light-harvesting by antenna-containing xanthorhodopsin from an Antarctic cyanobacterium

AbstractMicrobial rhodopsins are light-sensitive proteins vital to various phototrophic and sensory processes in microorganisms. Xanthorhodopsins, with their dual chromophore system involving retinal and carotenoids, have been predominantly studied in the extreme halophilic bacterium Salinibacter ruber and in the primitive thylakoid-less cyanobacterium Gloeobacter violaceus, where they facilitate light- driven outward proton pumping and enhanced light-harvesting. However, their distribution, binding specificity, and ecological significance in cyanobacteria remain poorly understood. Here we report widespread xanthorhodopsin genes in cyanobacterial genomes and characterize a protein from an Antarctic filamentous cyanobacterium that uniquely binds hydroxylated lutein. Through bioinformatics, spectroscopic, functional and structural assays analyses, we determine the properties and ecological role of this cyanobacterial xanthorhodopsin. Our findings highlight xanthorhodopsins role in modulating light-harvesting efficiency in cyanobacteria, particularly in extreme environments. The lutein binding and associated structural changes likely provide selective advantages for adapting to polar light conditions, contributing to cyanobacterial survival in harsh habitats.

microbiology↗

bioRxiv2024

Light-harvesting by antenna-containing rhodopsins in pelagic Asgard archaea

Aquatic bacterial rhodopsin proton pumps have been recently reported to utilize hydroxylated carotenoids1,2. Here, by combining a marine chromophore extract with purified archaeal rhodopsins identified in marine metagenomes, we report on light energy transfer from diverse hydroxylated carotenoids (lutein, diatoxanthin, and fucoxanthin) to heimdallarchaeial rhodopsins (HeimdallRs)3,4 from uncultured marine planktonic members of the "Ca. Kariarchaeaceae" ("Ca. Asgardarchaeota")5. These light-harvesting antennas absorb in the blue-light range and transfer energy to the green-light absorbing retinal chromophore within HeimdallRs. Furthermore, antenna enhancement of proton pumping by HeimdallRs is also observed under white-light illumination along with a carotenoid-binding-induced structural change in the protein. Our results indicate that the use of light-harvesting antennas in microbial rhodopsins is observed not only in bacteria but also in marine archaea.

microbiology↗

bioRxiv2022

Structural basis for ion selectivity in potassium-selective channelrhodopsins

The KCR channelrhodopsins are recently-discovered light-gated ion channels with high K+ selectivity, a property that has attracted broad attention among biologists- due to intense interest in creating novel inhibitory tools for optogenetics leveraging this K+ selectivity, and due to the mystery of how this selectivity is achieved in the first place. Indeed, the molecular and structural mechanism for K+ selectivity in KCRs has remained especially puzzling since these 7-transmembrane retinal-binding proteins completely lack structural similarity with known K+ channels, which generally coordinate K+ in a precisely symmetric conduction pathway formed by a tight interface among multiple small monomeric channel subunits (presumably not an accessible mechanism for the large KCR rhodopsin proteins). Here we present the cryo-electron microscopy structures of two KCRs from Hyphochytrium catenoides with distinct spectral properties for light absorption and channel actuation, HcKCR1, and HcKCR2, at resolutions of 2.6 and 2.5 [A], respectively. Structural comparison revealed first an unusually-shaped retinal binding pocket which induces rotation of the retinal in HcKCR2, explaining the large spectral difference between HcKCR1 and 2. Next, our combined structural, electrophysiological, computational, and spectroscopic analyses revealed a new solution to the challenging problem of K+-selective transport. KCRs indeed do not exhibit the canonical tetrameric K+ selectivity filter that specifically coordinates dehydrated K+; instead, single KCR monomers form a size exclusion filter using aromatic residues at the extracellular side of the pore which inhibits passage of bulky hydrated ions. This unique feature allows KCRs to function as K+ channels under relevant physiological conditions, providing not only a novel mechanism for achieving high K+ permeability ratios in biological ion channels, but also a framework for designing the next generation of inhibitory optogenetic tools. In BriefThe first structures of K+-selective channelrhodopsins (HcKCR1 and 2) are determined, revealing a K+ selectivity mechanism distinctly different from canonical K+ channels. HighlightsO_LIThe cryo-EM structures of K+-selective channelrhodopsins, HcKCR1 and 2, in nanodisc C_LIO_LIConditions under which naturally-occurring microbial rhodopsins have a 6-s-cis retinal C_LIO_LIIdentification of key residues for high K+ permeability ratios C_LIO_LIThe unique K+ selectivity mechanism of KCRs C_LI

biophysics↗

bioRxiv2022

Energy transfer in ubiquitous rhodopsin pumps with xanthophyll antennas

Energy transfer from light-harvesting ketocarotenoids to light-driven proton pumps xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to rhodopsin proton pumps from the abundant marine and freshwater photoheterotrophs have thus far failed3-5. Here, using functional metagenomics combined with chromophore extraction from the environment, we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet/blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas have a significant impact on rhodopsin phototrophy in the worlds lakes, seas and oceans.

microbiology↗

bioRxiv2022

Proton-transporting heliorhodopsins from marine giant viruses

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-gated proton channel. V2HeR3 absorbs blue-green lights, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a proton accepting group of the Schiff base, whose mutations converted the protein into an outward proton pump. Three environmental viral HeRs from the same group, as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion-transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tools, like type-1 microbial rhodopsins.

biophysics↗

bioRxiv2022

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. Intriguingly, 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 {beta}-carotene in organisms co-occurring with Saccharibacteria, and this genetic potential was dominated by 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.

microbiology↗

bioRxiv2021

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

ChRmine1, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin1, 2, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics1, 3-5. ChRmine and its homologs function as light-gated ion channels, but by primary sequence more closely resemble ion pump rhodopsins; the molecular mechanisms for passive channel conduction in this family of proteins, as well as the unusual properties of ChRmine itself, have remained mysterious. Here we present the cryo-electron microscopy structure of ChRmine at 2.0 [A] resolution. The structure reveals striking architectural features never seen before in channelrhodopsins including trimeric assembly, a short transmembrane-helix 3 unwound in the middle of the membrane, a prominently-twisting extracellular-loop 1, remarkably-large intracellular cavities and extracellular vestibule, and an unprecedented hydrophilic pore that extends through the center of the trimer, separate from the three individual monomer pores. Electrophysiological, spectroscopic, and computational analyses provide insight into conduction and gating of light-gated channels with these distinct design features, and point the way toward structure-guided creation of novel channelrhodopsins for optogenetic applications in biology.

biophysics↗

bioRxiv2020

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs reportedly work as light-driven inward H+ pumps, as xenorhodopsin. Here we report the crystal structure of SzR AM_5_00977 at 2.1 [A] resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 in SzR are shorter than those in the other microbial rhodopsins. Thus, E81 is located near the cytosol, playing a critical role in the inward H+ release. We suggested the H+ is not metastably trapped in E81 and released through the water-mediated transport network from the retinal Schiff base to the cytosol. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

biochemistry↗

bioRxiv2020

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design

Microbial rhodopsins are photoreceptive membrane proteins utilized as molecular tools in optogenetics. In this paper, a machine learning (ML)-based model was constructed to approximate the relationship between amino acid sequences and absorption wavelengths using ~800 rhodopsins with known absorption wavelengths. This ML-based model was specifically designed for screening rhodopsins that are red-shifted from representative rhodopsins in the same subfamily. Among 5,558 candidate rhodopsins suggested by a protein BLAST search of several protein databases, 40 were selected by the ML-based model. The wavelengths of these 40 selected candidates were experimentally investigated, and 32 (80%) showed red-shift gains. In addition, four showed red-shift gains > 20 nm, and two were found to have desirable ion-transporting properties, indicating that they were potentially useful in optogenetics. These findings suggest that an ML-based model can reduce the cost for exploring new functional proteins.

biochemistry↗

bioRxiv2018

Hereditary Pancreatitis Model by Blastocyst Complementation in Mouse

Summary StatementThe present study is the first report for reproducing human disease by utilizing blastocyst complementation method and would lead to the development of novel therapy for human disease.\n\nThe application of pluripotent stem cell is expected to contribute to the elucidation of the unknown mechanism of human diseases. However, in vitro induction of cells in several organs, such as the pancreas and liver, remains difficult; therefore, reproduction of those diseases in a model has not been feasible. To reproduce human hereditary pancreatitis (HP), which is most frequently caused by the mutations in the PRSS1 gene, we performed the blastocyst complementation (BC) method. In the BC method, mouse embryonic stem (ES) cells harboring CRISPR/CAS9-mediated mutations in the Prss1 were injected into the blastocysts of deficient Pdx1 gene mice, which is a critical transcription factor in the pancreas. The results showed that the blastocysts injected into the Prss1-mutant ES cells induced trypsin activation. This implied that the mouse phenotype mimics that of human HP and that the BC method was useful for the reproduction and study of pancreatic disorders. The present study opens the possibility of investigating uncharacterized human diseases by utilizing the BC method.

developmental biology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗