Biology subjects

Baker, B. J.

Publications and source records attributed to Baker, B. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Asgard archaeal origin of microtubules

Eukaryotic cells change their shapes, actively segregate their DNA and contain membrane networks, facilitated by a complex cytoskeleton containing actin filaments, microtubules made from tubulin, and other components. These filaments have ancient evolutionary origins since actin- and tubulin-like proteins form prokaryotic cytoskeletons in archaea and bacteria. Bona fide eukaryotic F-actin can be traced back to crenarchaea and Asgard archaea, which are the closest known relatives of eukaryotes. A possible Asgard archaeal origin of microtubules was suggested recently with the discovery of a lokiarchaeon containing AtubAB mini microtubules that share architectural features with their eukaryotic counterparts. Using phylogenetic analyses of metagenomic data, here we report the broad occurrence of tubulins in Asgard archaea. Biochemical and structural analyses showed that one of our newly discovered heimdallarchaeial AtubAB tubulin pairs forms four-protofilament mini-microtubules that show dynamic instability and are inhibited by the tubulin drug maytansine. Our work raises the possibility that microtubule architecture and dynamics evolved in Asgard archaea prior to eukaryogenesis.

cell biology↗

bioRxiv2026

Turning the Fluorescent Protein Barrel into a Programmable Electrostatic Device Reveals Differential Bleaching States of the Chromophore

Genetically encoded voltage indicators (GEVIs) based on fluorescent proteins (FPs) report membrane potential changes through voltage-driven conformational rearrangements of a voltage-sensing domain that perturb the electrostatic and hydrogen-bonding environment of a fused FP chromophore. While the chromophore is largely protected from the external environment by the FP {beta}-can, previous results suggest that this structure can also act as a programmable electrostatic grid that influences chromophore flexibility and fluorescence transition states. Here, we show that varying the external polar offset of {beta}-sheet residues flanking a chromophore-proximal position systematically reshapes fluorescence transitions, altering response polarity and kinetics. Importantly, the influence of these external residues depends strongly on the internal sidechain chemistry, revealing reciprocal coupling between {beta}-sheet electrostatics and chromophore-proximal residues. We further show that the functional effects of glutamine and asparagine substitutions at this position are strongly context-dependent and can be tuned by appropriate external polar offsets, consistent with electrostatic control of internal sidechain orientation and expanding the molecular switch repertoire. Disrupting steady-state fluorescence further revealed that the secondary component of the voltage-dependent fluorescence transition is preferentially light-sensitive and can be selectively diminished by repeated stimulation, indicating that it arises from a photophysically distinct process separable from the primary response. In addition, altering the chromophore protonation equilibrium (e.g., T65S) substantially changes response kinetics, indicating that chromophore state influences how {beta}-can electrostatics couple to fluorescence transitions. Together, these results define general programming rules by which the electrostatic grid of the FP {beta}-barrel can be tuned to control chromophore optical properties both at rest and during perturbation of the steady state.

biophysics↗

bioRxiv2025

An Asgard archaeon with internal membrane compartments

The emergence of eukaryotes from a merger between an archaeon and a bacterial cell [~]two billion years ago involved a profound change in cellular organisation. While the order in which different features of the eukaryotic cell arose remains a matter of controversy1-3, close archaeal relatives of eukaryotes have recently been identified that possess homologues of eukaryotic trafficking machinery4,5 and a complex cell architecture6,7. The members of this phylum, the Asgard archaea (syn. Prometheoarchaeota) described so far, however, lack internal membrane-bound compartments, and therefore have shed little light on origins of the hallmark eukaryotic endomembrane system. Here we report the cell biological analysis of a member of the Heimdallarchaeia, Candidatus Y. umbracryptum in enriched mixed microbial communities. Possessing a small genome encoding few homologues of eukaryotic membrane remodelling machinery8, Ca. Y. umbracryptum cells in late-stage cultures resemble previously described Asgard archaea with extensive cellular protrusions. Surprisingly, during early stages of culture growth Ca. Y. umbracryptum cells have fewer protrusions but possess numerous intracellular vesicles, most of which have a luminal surface that morphologically resembles the outer coat of the plasma membrane. These data alter our view of eukaryogenesis by identifying a close archaeal relative of eukaryotes with an endomembrane system, whose appearance changes with cell state.

cell biology↗

bioRxiv2025

Archaeal lineages related to eukaryotes encode functional diterpenoid cyclases

The first eukaryotic cell originated through the union of an archaeon and a bacterium. Phylogenetic evidence suggests that the archaeal partner was a member of phylum Asgardarchaeota, likely within the Heimdallarchaeia clade. Although little is known about the molecular basis of eukaryogenesis, insight can be gained from studying the unique biochemistry of modern Asgardarchaeota. Here, we investigated the potential for Asgards to produce cyclic terpenoids. Phylogenomics coupled with structural prediction revealed that organisms from the clades Hodarchaeales and Kariarchaeaceae encode for diterpenoid cyclases. We tested the functionality of these enzymes in vitro, showing that they cyclize geranylgeranyl pyrophosphate to form bicyclic halimadienyl pyrophosphate. Halimadienyl nucleosides have previously been shown to mediate intracellular persistence of Mycobacterium tuberculosis in host endosomes, and may function similarly in Asgardarchaeota. The characterization of Asgard diterpenoid cyclases provides insight into the biochemistry of an elusive group of microbes that are pivotal in the evolution of complex cellular life.

microbiology↗

bioRxiv2024

Methane production from hydrocarbons by consortia dominated by ANME archaea

Understanding microbial production and consumption of methane, a potent greenhouse gas in the atmosphere, is critical for bridging knowledge gaps in global carbon cycling. In anoxic environments, methane is known to be produced through hydrogenotrophic, acetoclastic or methylotrophic mechanisms. Methane production from hydrocarbons may also be important, especially in hydrocarbon-rich environments, like the Gulf of California, but the mechanism of this hydrocarbonoclastic methanogenesis remains unclear. The activity of consortia of anaerobic methane oxidizing (ANME) archaea and bacteria limits the release of methane to the atmosphere by consuming methane in anoxic environments globally. Here we used isotopic-labeling to track the conversion of hydrocarbons (hexadecane and naphthalene) to methane in enrichments from hydrothermally impacted, hydrocarbon-rich sediments from the Gulf of California. Methane was produced directly from hexadecane and naphthalene, in both the presence and absence of sulfate. We reconstructed metagenomic assembled-genomes (MAGs) from these experiments which revealed a mixture of bacteria dominated by Desulfobacteriota and Bacteroidota, and archaea dominated by Aeinigmarchaeota, Thermoplasmatota, and ANME group 2c. The ANME-2c were the only MAGs that encoded methyl coenzyme M reductases (McrA) and complete Wood-Ljungdahl pathways (WLP). This suggests that ANME-2c archaea may be involved in the production of methane along the seafloor, and that our understanding of the roles of these globally important microbes is not yet fully appreciated.

microbiology↗

bioRxiv2024

Structure-based inference of eukaryotic complexity in Asgard archaea

Asgard archaea played a key role in the origin of the eukaryotic cell. While previous studies found that Asgard genomes encode diverse eukaryotic signature proteins (ESPs), representing homologs of proteins that play important roles in the complex organization of eukaryotic cells, the cellular characteristics and complexity of the Asgard archaeal ancestor of eukaryotes remain unclear. Here, we used de novo protein structure modeling and sensitive sequence similarity detection algorithms within an expanded Asgard archaeal genomic dataset to build a structural catalogue of the Asgard archaeal pangenome and identify 908 new isomorphic ESPs (iESPs), representing clusters of protein structures most similar to eukaryotic proteins and that likely underwent extensive sequence divergence. While most previously identified ESPs were involved in cellular processes and signaling, iESPs are enriched in information storage and processing functions, with several being potentially implicated in facilitating cellular complexity. By expanding the complement of eukaryotic proteins in Asgard archaea, this study indicates that the archaeal ancestor of eukaryotes was more complex than previously assumed.

evolutionary biology↗

bioRxiv2024

Oxygen metabolism in descendants of the archaeal-eukaryotic ancestor

Asgard archaea were pivotal in the origin of complex cellular life. Hodarchaeales (Asgardarchaeota class Heimdallarchaeia) were recently shown to be the closest relatives of eukaryotes. However, limited sampling of these archaea constrains our understanding of their ecology and evolution1-3, including their anticipated role in eukaryogenesis. Here, we nearly double the number of Asgardarchaeota metagenome-assembled genomes (MAGs) to 869, including 136 new Heimdallarchaeia (49 Hodarchaeales) and several novel lineages. Examining global distribution revealed Hodarcheales are primarily found in coastal marine sediments. Detailed analysis of their metabolic capabilities revealed guilds of Heimdallarchaeia are distinct from other Asgardarchaeota. These archaea encode hallmarks of aerobic eukaryotes, including electron transport chain complexes (III and IV), biosynthesis of heme, and response to reactive oxygen species (ROS). The predicted structural architecture of Heimdallarchaeia membrane-bound hydrogenases includes additional Complex-I-like subunits potentially increasing the proton motive force and ATP synthesis. Heimdallarchaeia genomes encode CoxD, which regulates the electron transport chain (ETC) in eukaryotes. Thus, key hallmarks for aerobic respiration may have been present in the Asgard-eukaryotic ancestor. Moreover, we found that Heimdallarchaeia is present in a variety of oxic marine environments. This expanded diversity reveals these Archaea likely conferred energetic advantages during early stages of eukaryogenesis, fueling cellular complexity.

microbiology↗

bioRxiv2024

Culture-dependent identification of rare marine sediment bacteria from the Gulf of Mexico and Antarctica

Laboratory-viable cultivars of previously uncultured bacteria further taxonomic understanding. Despite many years of modern microbiological investigations, the vast majority of bacterial taxonomy remains uncharacterized. While many attempts have been made to decrease this knowledge gap, culture-based approaches parse away at the unknown and are critical for improvement of both culturing techniques and computational prediction efficacy. To this end of providing culture-based approaches, we present a multi-faceted approach to recovering marine environmental bacteria. We employ combinations of nutritional availability, inoculation techniques, and incubation parameters in our recovery of marine sediment-associated bacteria from the Gulf of Mexico and Antarctica. The recovered biodiversity spans several taxa, with 16S-ITS-23S rRNA gene-based identification of multiple isolates belonging to rarer genera increasingly undergoing phylogenetic rearrangements. Our modifications to traditional culturing techniques have not only recovered rarer taxa, but also resulted in the recovery of biotechnologically promising bacteria. Together, we propose our stepwise combinations of recovery parameters as a viable approach to decreasing the bacterial knowledge gap.

microbiology↗

bioRxiv2024

Genetic elements and defense systems drive diversification and evolution in Asgard archaea

Asgard Archaea are of great interest as the progenitors of Eukaryotes, but little is known about the mobile genetic elements (MGEs) that may shape their ongoing evolution. Here, we describe MGEs that replicate in Atabeyarchaeia, wetland Asgard archaea phylum represented by two complete genomes. We used soil depth-resolved population metagenomic datasets to track 18 MGEs for which genome structures were defined and precise chromosome integration sites could be identified for confident host linkage. Additionally, we identified a complete 20.67 kilobase pair (kbp) circular plasmid (the first reported for Asgard archaea) and two groups of viruses linked to Atabeyarchaeia, via CRISPR spacer targeting. Closely related 40 kbp viruses possess a hypervariable genomic region encoding combinations of specific genes for small cysteine-rich proteins structurally similar to restriction-homing endonucleases. One 10.9 kbp circularizable plasmid-like MGE integrates genomically into an Atabeyarchaeia chromosome and has a 2.5 kbp circularizable element integrated within it. The 10.9 kbp MGE encodes a highly expressed methylase with a sequence specificity matching an active methylation motif identified by PacBio sequencing. Restriction-modification of Atabeyarchaeia differs from that of another coexisting Asgard archaea Freyarchaeia which has few identified MGEs but possesses diverse defense mechanisms, including DISARM and Hachiman not found in Atabeyarchaeia. Overall, defense systems and methylation mechanisms of Asgard archaea likely modulate their interactions with MGEs, and integration/excision and copy number variation of MGEs in turn enable host genetic versatility.

microbiology↗

bioRxiv2024

Bacterial ecology and evolution converge on seasonal and decadal scales

Ecology and evolution are often viewed as distinct processes, which interact on contemporary time scales in microbiomes. To observe these processes in a natural system, we collected a two-decade, 471-sample freshwater lake time series, creating the longest metagenome dataset to date. Among 2,855 species-representative genomes, diverse species and strains followed cyclical seasonal patterns, and one in five species experienced decadal shifts in strain composition. The most globally abundant freshwater bacterium had constant species-level abundance, but environmental extremes appeared to trigger a shift in strain composition and positive selection of amino acid and nucleic acid metabolism genes. These genes identify organic nitrogen compounds as potential drivers of freshwater responses to global change. Seasonal and long-term strain dynamics could be regarded as ecological processes or equivalently as evolutionary change. Rather than as distinct processes that interact, we propose a conceptualization where ecology and evolution converge along a continuum to better describe change in diverse microbial communities.

evolutionary biology↗

bioRxiv2023

S1 hydrophobic residues alter the voltage sensing phosphatase enzymatic function and voltage sensing

The voltage sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage sensing proteins, the VSDs do not interact with one another and the S1-S3 helices are considered mainly as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a voltage-regulated enzyme and Hvs are channels that only have VSDs. To investigate the S1 contribution to VSP function, we individually mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134 and L137). We also combined these mutations to generate quadruple mutation designated S1-Q. Most of these mutations shifted the voltage dependence of activity to higher voltages though interestingly, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions were consistently shifted to lower voltages and indicated a second voltage dependent motion. Co-immunoprecipitation demonstrated that none of the mutations broke the VSP dimer indicating that the S1 impact could stem from intrasubunit and/or intersubunit interactions. Lastly, when the same alanine mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzymes conformational response to membrane potential transients and influencing the function of the VSD.

biophysics↗

bioRxiv2023

Imaging Electrical Activity of Retinal Ganglion Cells with Fluores-cent Voltage and Calcium Indicator Proteins in Retinal Degenera-tive rd1 Blind Mice

In order to understand the retinal network, it is essential to identify functional connectivity among retinal neurons. For this purpose, imaging neuronal activity through fluorescent indicator proteins has been a promising approach offering simultaneous measurements of neuronal activities from different regions of the circuit. In this study, we used genetically encoded voltage and calcium indicators, Bongwoori-R3 and GCaMP6f, to visualize membrane voltage and calcium dynamics in the form of the spatial map within retinal ganglion cells from retina tissues of the photoreceptor degenerated rd1 mice. Retinal voltage imaging confirmed current-evoked responses from somatic spiking and intercellular conduction, while calcium imaging showed current evoked changes in calcium concentrations of presynaptic neurons. These results indicate that the combination of fluorescent protein sensors and high-speed imaging methods permits imaging electrical activity with cellular precision and millisecond resolution. Hence, we expect our method will provide a potent experimental platform for the study of retinal signaling pathways as well as the development of retinal stimulation strategies in visual prosthesis.

neuroscience↗

bioRxiv2023

Asgard archaea modulate potential methanogenesis substrates in wetland soil

The roles of Asgard archaea in eukaryogenesis and marine biogeochemical cycles are well studied, yet their contributions in soil ecosystems are unknown. Of particular interest are Asgard archaeal contributions to methane cycling in wetland soils. To investigate this, we reconstructed two complete genomes for soil-associated Atabeyarchaeia, a new Asgard lineage, and the first complete genome of Freyarchaeia, and defined their metabolism in situ. Metatranscriptomics highlights high expression of [NiFe]-hydrogenases, pyruvate oxidation and carbon fixation via the Wood-Ljungdahl pathway genes. Also highly expressed are genes encoding enzymes for amino acid metabolism, anaerobic aldehyde oxidation, hydrogen peroxide detoxification and glycerol and carbohydrate breakdown to acetate and formate. Overall, soil-associated Asgard archaea are predicted to be non-methanogenic acetogens, likely impacting reservoirs of substrates for methane production in terrestrial ecosystems. One-Sentence SummaryComplete genomes of Asgard archaea, coupled with metatranscriptomic data, indicate roles in production and consumption of carbon compounds that are known to serve as substrates for methane production in wetlands.

microbiology↗

bioRxiv2023

Phylogenomics and ancestral reconstruction of Korarchaeota reveals genomic adaptation to habitat switching

Our knowledge of archaeal diversity and evolution has expanded rapidly in the past decade. However, hardly any genomes of the phylum Korarchaeota have been obtained due to the difficulty in accessing their natural habitats and - possibly - their limited abundance. As a result, many aspects of Korarchaeota biology, physiology and evolution remain enigmatic. Here, we expand this phylum with five high-quality metagenome-assembled genomes. This improved taxon sampling combined with sophisticated phylogenomic analyses robustly places Korarchaeota at the base of TACK and Asgard clades, revisiting the phylums long-assumed position. Furthermore, we observe a clear split between terrestrial and marine thermal clades. Gene tree-aware ancestral reconstructions suggest that the last Korarchaeota common ancestor was a thermophilic autotroph. In contrast, Korarchaeaceae, the lineage where environmental transitions occurred, shifted towards a heterotrophic lifestyle. Terrestrial Korarchaeota gained many cas and CARF genes indicating they may need to manage viral infections. Together, our study provides new insights into these early diverging Archaea and suggests that gradual gene gain and loss shaped their adaptation to different thermal environments. ImportanceKorarchaeota are an ancient group of archaea, but their biology, physiology and evolution have remained obscure. Analysis of five novel Korarchaeota MAGs, and publicly available reference data provides robust phylogenomic evidence that Korarchaeota are placed at the base of Asgard archaea and TACK, revisiting the phylums long-assumed position. Gene content reconstruction suggests a versatile thermophilic and autotrophic last Korarchaeota common ancestor. Environmental distribution surveying of public databases places all Korarchaeota in thermophilic environments and indicates that their habitat is limited to hydrothermal vents and hot springs. Our modeling indicates at least two transitions linked to habitat switching between these environments in the evolutionary history of Korarchaeota. Both are linked to a significant alteration of the inferred ancestral gene content, including a shift towards a heterotrophic and potential scavenging lifestyle. Furthermore, hot spring Korarchaeota acquired various genes participating in resistance to viruses, suggesting they may need to manage frequent viral threats.

microbiology↗

bioRxiv2023

Asgard archaea defense systems and their roles in the origin of immunity in eukaryotes

Immune systems are integral to survival against viral infection. Recently, dozens of new antiviral systems have been characterized in bacteria1. Some of these systems are present in eukaryotes and appear to have originated in prokaryotes. However, little is known about these defense mechanisms in archaea. Here, we identified 2,610 complete defense systems in archaea related to eukaryotes, the Asgardarchaeota2. These comprise 89 unique systems, including argonaute, NLR, mokosh, viperin, lassamu, and CBASS. Asgard viperin (asVip) and argonaute (asAgo) proteins are present at high frequencies compared to bacteria and have structural homology to eukaryotes. Phylogenetic analyses revealed asVips are ancestral eukaryotic proteins. Heterologous expression of asVips in bacteria, including the lineage closest to eukaryotes, Hodarchaeales, showed anti-phage activity. Eukaryotic- and bacterial-argonaute proteins appear to have originated in Asgardarchaeota and preserve ancient structural characteristics. asAgos have argonaute-PIWI domains which are key components of the RNA interference (RNAi) in eukaryotes. Characterization of hundreds of defense systems in the Asgardarchaeota revealed these archaea played important roles in the innovation of viral protection in eukaryotes. Given their relationship to eukaryotes, these defense systems may have applications in biomedicine and biotechnology.

microbiology↗

bioRxiv2023

Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes

In the ongoing debates about eukaryogenesis, the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors, members of the Asgard archaea play a key role as the closest archaeal relatives of eukaryotes. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved. Here, we analyze distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea, as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree/species tree reconciliation approaches, we show that, in analogy to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared to other archaea. Finally, we infer that the last common ancestor of Asgard archaea likely was a thermophilic chemolithotroph, and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and the platform for the emergence of cellular complexity in eukaryotic cells.

evolutionary biology↗

bioRxiv2022

Autotrophic biofilms sustained by deeply-sourced groundwater host diverse CPR bacteria implicated in sulfur and hydrogen metabolism

BackgroundCandidate Phyla Radiation (CPR) bacteria are commonly detected yet enigmatic members of diverse microbial communities. Their host associations, metabolic capabilities, and potential roles in biogeochemical cycles remain under-explored. We studied chemoautotrophically-based biofilms that host diverse CPR bacteria and grow in sulfide-rich springs using bulk geochemical analysis, genome-resolved metagenomics and scanning transmission x-ray microscopy (STXM) at room temperature and 87{degrees} K. ResultsCPR-affiliated Gracilibacteria, Absconditabacteria, Saccharibacteria, Peregrinibacteria, Berkelbacteria, Microgenomates, and Parcubacteria are members of two biofilm communities dominated by chemolithotrophic sulfur-oxidizing bacteria including Thiothrix or Beggiatoa. STXM imaging revealed ultra-small cells along the surfaces of filamentous bacteria that we interpret are CPR bacterial episymbionts. STXM and NEXAFS spectroscopy at carbon K and sulfur L2,3 edges show protein-encapsulated elemental sulfur spherical granules associated with filamentous bacteria, indicating that they are sulfur-oxidizers, likely Thiothrix. Berkelbacteria and Moranbacteria in the same biofilm sample are predicted to have a novel electron bifurcating group 3b [NiFe]-hydrogenase, putatively a sulfhydrogenase, potentially linked to sulfur metabolism via redox cofactors. This complex could potentially underpin a symbiosis involving Berkelbacteria and/or Moranbacteria and filamentous sulfur-oxidizing bacteria such as Thiothrix that is based on cryptic sulfur cycling. One Doudnabacteria genome encodes adjacent sulfur dioxygenase and rhodanese genes that may convert thiosulfate to sulfite. We find similar conserved genomic architecture associated with CPR bacteria from other sulfur-rich subsurface ecosystems. ConclusionsOur combined metagenomic, geochemical, spectromicroscopic and structural bioinformatics analyses link some CPR bacteria to sulfur-oxidizing Proteobacteria, likely Thiothrix, and indicate roles for CPR bacteria in sulfur and hydrogen cycling.

microbiology↗

bioRxiv2022

Precise temporal control of GEVI conformations enables the visualization of charge migration in a fluorescent protein resulting in an improved optical response

Rapid and reproducible optical transitions of a fluorescent protein (FP) can be achieved with a Genetically Encoded Voltage Indicator (GEVI) via manipulation of the membrane potential. These transitions revealed novel effects of internal mutations near the chromophore that would not be detected under steady state conditions. Mutating an internal threonine (T203) affected the speed of the voltage-dependent fluorescence transition suggesting a conformational change inside the protein. These optical transitions also demonstrated interplay between internal and externally oriented sidechains of the {beta}-can structure. Replacing the steric hindrance of a phenylalanine near the chromophore with threonine (F165T) did not alter the resting fluorescence but resulted in a more complex fluorescent transition providing evidence for a flexible chromophore undergoing conformational changes. F165T orientation was influenced by the flanking external amino acids at positions 164 and 166 with 164F/165T/166T exacerbating the complexity of the voltage-dependent transition while 164T/165T/166F reduced the flexibility of the chromophore resembling the transition pattern of the original F165 version. Alphafold predictions reveal a threonine switch with different orientations of the F165T internal side chain depending on the direction of the offset in polarity at external positions 164 and 166. The crystal structures of the pH-sensitive FP, Super Ecliptic pHluorin and two derivatives solved in varying pH conditions also indicate interactions between the external protein surface and the internal environment providing another example of a threonine switch near the chromophore at T203. This ability to orient internal sidechains has led to the development of a novel GEVI that gets brighter upon depolarization of the plasma membrane, works at low light levels, is less susceptible to physiological pH, and provides in vivo signals. These observations affecting fluorescent transitions should also prove valuable to the development of any FP-based biosensor.

neuroscience↗