Proceedings of the National Academy of Sciences

High pressure (HP) can trigger bacterial spore germination, acting either through germinant receptors (GRs) or the SpoVA channel. However, the mechanism by which HP activates these membrane-embedded proteins remains elusive. Here, using Bacillus subtilis, we demonstrate that the GerA germinant receptor (GR) is the primary target of moderate HP (50-300 MPa). Mutagenesis reveals that pore-lining residues within the GerA ion channel are essential for the pressure response, whereas canonical ligand-binding and intramembrane signaling residues are dispensable. We then propose a <underline>s</underline>tretch-<underline>t</underline>o-<underline>o</underline>pen (STO) model, in which HP differentially compresses the more compliant inner membrane (IM) relative to the rigid spore core, generating lateral membrane tension that promotes opening of the GerA channel. In situ membrane tension measurements indicate HP-induced compression of IM phospholipids and elevated membrane tension. This tension-dependent gating is further supported by the pressure-dependent phenotypic rescue of GerA channel mutants. Consistently, HP increases IM permeability to water-soluble and membrane-impermeable agents (propidium iodide and formaldehyde), an effect potentiated by GerA, indicating concomitant opening of GerA by HP. Furthermore, modulating IM fluidity via heat activation or decoating altered membrane physical properties and delayed HP-induced germination, establishing the IM as the critical mechanical transducer. Additionally, computational modeling and calculations support faster compression of the IM than of the core under HP, rationalizing the source of tensile stress. Together, our findings establish a novel mechanism of HP-induced GerA activation via the STO model: HP compresses the IM, generates lateral tension, and promotes opening of the GerA ion channel to trigger bacterial spore germination.

Cellulose is a common component of bacterial biofilms where it interacts with other biopolymers to form a 3-dimensional matrix enclosing the bacteria. Synthesized and secreted by the synthase-dependent biosynthesis pathway common to many bacterial exopolysaccharides, its surface exposure depends on the presence of the periplasmic cellulase BcsZ. During export across the periplasm, E. coli and other Enterobacteriaceae modify cellulose with lipid-derived phosphoethanolamine (pEtN). How BcsZ hydrolyzes pEtN-cellulose in the periplasm is unknown and so is the native distribution pattern of pEtN on cellulose. Here, we used carbohydrate synthesis, X-ray crystallography, native mass spectrometry, and super-resolution MINFLUX nanoscopy to delineate the role of BcsZ during cellulose biosynthesis. Crystal structures of BcsZ bound to chemically synthesized pEtN cello-oligosaccharides identify how the enzyme recognizes pEtN-modified glucosyl units. Comparing mono and double substituted cellohexaoses, we identify varying binding poses that are determined by two pEtN coordination sites within the BcsZ catalytic pocket. Combined, our structural analyses reveal an ideal BcsZ cellohexaose ligand containing two pEtN modified units separated by an unmodified cellotriosyl unit. The enzyme binds and hydrolyzes this compound with substantially increased affinity and efficiency. Further, BcsZ digestion of native pEtN cellulose combined with native mass spectrometry analyses reveals the stochastic distribution of pEtN on biofilm cellulose. Additionally, MINFLUX co-localization of BcsZ with other components of the biosynthetic complex demonstrates the random distribution of BcsZ across the periplasm. Our data suggest BcsZ functions independently of the biosynthetic complex to clear mislocalized pEtN cellulose from the periplasm.

The earliest stages of protein evolution remain a mystery: the nature of the first protein forms, their roles in emergent biological systems, and the forces that shaped them are largely unknown. Here, we combine insights from metabolic modelling, the organization of protein structure space, and protein folding mechanisms to probe the emergence of two ubiquitous cofactor- binding folds: Rossmanns and P-loop NTPases. While both folds are essential for contemporary life, we show that Rossmanns catalyze reactions deeper within the metabolic core and are more central in structure space than P-loop NTPases. Folding mechanism analysis further reveals that, whereas P-loop NTPases may require non-local interactions to fold, Rossmann folding can be nucleated by a structural module at the heart of the fold that contains a nucleotide-binding motif. Because this motif also directly mediates biochemical activity, this result suggests how early proteins may have compactly satisfied both folding and biochemical activity. Our results imply that folding constraints favored early enzymatic forms with compact binding motifs and modest catalytic roles. We conclude that the early emergence of the Rossmann fold reflects the chemical and physical constraints of protein folding, explaining both its profound antiquity and sustained longevity.

Long-range correlated motions couple distant regions of a protein, providing a physical basis for allosteric communication, cooperative conformational change, and the balance between structural stability and sensitivity to perturbations. Yet membrane proteins operate within a strongly anisotropic lipid bilayer, and how this environment reshapes such system-spanning coordination remains unclear. Using an implicit-membrane anisotropic network model, we perform a proteome-wide analysis of more than 3,000 human transmembrane proteins. We find that long-range correlations remain scale free under membrane constraints but become strongly direction dependent. Across protein sizes and topologies, their correlation lengths continue to scale with the corresponding molecular dimensions, while increasing membrane anisotropy extends in-plane correlations and shortens those along the membrane normal. Because spontaneous correlations and perturbation responses arise from the same underlying mechanics, we further resolve residue-level responses into in-plane and normal components. The resulting directional mechanical susceptibility provides new predictions of mutation-sensitive sites in GPCRs beyond those captured by conventional scalar flexibility measures. Together, these results show how environmental symmetry breaking can organize protein mechanics across scales, linking collective dynamics to the functional sensitivity of individual residues and connecting a general physical mechanism to experimentally measurable protein function.

Tau is a neuron-specific microtubule-associated protein that can self-associate into pathological insoluble aggregates or phase separate into condensates whose (patho)physiological role is debated. Recent studies suggest that intracellular surfaces can locally promote biomolecular condensation, even at low molecular concentrations. While microtubules in neurons provide an abundant tau-interaction surface, their role in tau phase separation remains unclear. Through a dialogue between experiments and theory, we demonstrate that tau forms multilayered condensates on microtubules at physiological concentrations via a prewetting-like transition. Concomitant tau-microtubule and tau-tau interactions explain the experimentally observed cooperative binding of the innermost tau layer directly adsorbed to the microtubule. The formation of this layer is dictated by the spacing of tubulin dimers within the microtubule lattice. Additional tau layers, driven by tau-tau interactions and independent of lattice spacing, are finite in thickness and unstable away from the microtubule surface. While the microtubule-adsorbed tau can selectively restrict proteins from the microtubule surface, the multilayered tau condensates can recruit tau interactors, such as RNA or soluble tubulin, highlighting the distinct roles of the condensate layers. Our results suggest that a prewetting-like transition constitutes a general physical mechanism for organizing liquid-like biomolecular layers of defined composition on charged intracellular surfaces.

Photosystem II (PSII) is in eukaryotic phototrophs is generally considered to operate within a more restricted spectral range than photosystem I (PSI), in which long-wavelength chlorophylls are a well-established feature of the peripheral antenna. Whether eukaryotic PSII can acquire comparable far-red-associated properties through lineage-specific antenna diversification has remained unclear. Here we present a 3.09 [A] cryo-electron microscopy structure of the C2S2M2L2 PSII supercomplex from Euglena gracilis, a euglenophyte species harbouring a secondary plastid and unusual light-harvesting system. We show that the euglenophyte-specific antenna protein LhcE9 occupies the position corresponding to canonical Lhcb5, but in a markedly different orientation that creates a distinct interface with the PSII core, particularly with CP43. Combined structural, spectroscopic, mutagenesis and proteomic analyses support LhcE9 as the stably bound PSII antenna subunit most closely associated with the far-red state in the supercomplex. Excitation-energy-transfer calculations further indicate two fast lineage-specific antenna-to-core routes mediated by LhcE9 and PsbX. Together, these findings reveal an unexpected mode of PSII antenna diversification and provide a structural framework for far-red-associated light harvesting in PSII.

Enzymatic reactions in biomolecular condensates are often assumed to be regulated through local enrichment of reactants. However, condensates also reshape molecular transport and reaction kinetics, making it unclear how phase separation controls catalysis in living cells. Here, we develop a quantitative theory of biomolecular catalysis in phase-separated systems and find that liquid condensates can act as tunable catalytic switches, transitioning between regimes of enhanced and suppressed enzymatic activity, exhibiting optimal responses at biologically relevant condensate sizes. We show that condensate-mediated catalysis cannot be understood from reactant enrichment alone, but instead emerges from the coupled interplay of molecular partitioning, diffusive transport, and phase-dependent reaction kinetics. The strongest regulatory effects occur under rapid interphase exchange, where the spatially heterogeneous catalytic network admits a system-level Michaelis-Menten description governed by system-averaged concentrations and reaction kinetics. Our framework predicts that micron-sized condensates can either enhance or suppress enzymatic activity by up to two orders of magnitude, and that optimal catalytic regulation can emerge at condensate sizes comparable to many biomolecular condensates. These results provide experimentally testable predictions for condensate-mediated catalysis and establish quantitative principles for understanding and engineering enzyme-catalysed reactions in biomolecular condensates.

Metabolites are generally viewed as substrates, products, cofactors, or regulators of individual proteins, whereas metabolites recurring across many protein families are often regarded as promiscuous binders. Here, we analyzed 989,058 BioLiP2 protein-ligand binding sites and assigned 929,546 sites to ECOD v295 homologous groups to quantify ligand specificity, cross-fold scatter, structural breadth, and metabolite-mediated connectivity across protein-family space. Many ancient metabolites preferentially occupied cognate structural groups, demonstrating that broad evolutionary reuse can coexist with local structural discrimination. After excluding elemental metals, BioLiP potential-artifact/dual-use ligands, and metabolites containing fewer than six heavy atoms, 32 ancient metabolites occupied a mean of 185.38 ECOD F-groups per metabolite, compared with 6.32 F-groups for 2,540 mapped filtered non-ancient metabolites-- a 29.35-fold enrichment (bootstrap 95% CI, 18.66-43.46). The complete 40-ancient-metabolite network connected all 6,798 associated F-groups into a single giant connected component (GCC). Even after stringent filtering, all 3,135 ancient-metabolite-associated F-groups remained in one GCC. Degree-preserving configuration-model randomizations and maximum-degree capping showed that this connectivity follows from the broad, recurrent distribution of metabolite binding rather than dependence on a few extreme hubs or a specialized higher-order topology. Differences between ancient and filtered non-ancient networks were not explained by metabolite size, whereas generic crystallization additives preferentially occupied smaller pockets. These results indicate that a limited ancient chemical repertoire established a globally connected protein- family architecture that subsequent metabolite diversification expanded while preserving its basic organization. SignificanceMetabolites are conventionally viewed as substrates, products, cofactors, or regulators acting on individual proteins. Global examination of experimentally observed metabolite-protein interactions reveals a broader organizing principle. Ancient metabolites combine local binding discrimination with extraordinary reuse across protein families, such that only 40 metabolites generate an almost completely connected network spanning thousands of ECOD (evolutionary classification of domains) protein families. The much larger non-ancient metabolite repertoire expands the protein-family space covered by this network, while preserving near-global connectivity. Thus, metabolite diversification appears to have elaborated, rather than created, a chemically connected protein architecture established early in evolution, suggesting that overlapping metabolite-binding repertoires could coordinate proteins, pathways, and cellular processes.

Soil microalgae endure harsh terrestrial stressors, such as intense light. Eustigmatophytes are an independent evolutionary branch within stramenopiles and occupy diverse aquatic and terrestrial environments, but the structural organization of their photosynthetic apparatus remains poorly understood. Here, we determined the cryo-electron microscopy structure of a photosystem I-light-harvesting complex I (PSI-LHCI) supercomplex bound with ferredoxin-NADP+ oxidoreductase (FNR) from the terrestrial eustigmatophyte Vischeria stellata at 2.44 [A] resolution. The supercomplex contains a monomeric PSI core associated with only three LHCI subunits, representing the smallest PSI-LHCI reported among structurally characterized red-lineage PSI complexes composed of violaxanthin-Chl a proteins (VCPs). The three VCPIs with distinct structure features and arrangements form a compact belt along the PsaL-PsaI-PsaM side of PSI. The structure also resolves a 43-residue N-terminal segment of FNR (FNR-N) bound to the PSI stromal surface, which is stabilized by both a eustigmatophyte-conserved insertion in PsaL and the N-terminal region of PsaD. In contrast, the catalytic region of FNR was not resolved, suggesting conformational flexibility. Computational simulations indicate potential excitation-energy-transfer pathways connecting the three VCPI subunits to the PSI core and highlight lineage-specific pigments that maintain energetic connectivity within the exceptionally compact antenna. Our analysis further reveals conservation of FNR tethering despite pronounced diversification of antenna size and organization. These findings uncover a modular evolutionary principle in which PSI acceptor-side organization is retained while the light-harvesting antenna is extensively remodeled, providing a framework for understanding the diversification of photosynthetic energy conversion across ecological transitions.

Gap junction channels formed by connexins mediate direct intercellular communication and are essential for electrical signaling and tissue homeostasis. Despite their large, solvent-accessible pores, connexin channels exhibit distinct conductance, selectivity, and rectification properties, but the molecular mechanisms underlying these behaviors remain incompletely understood. Here, we performed [~]67 s of all-atom computational electrophysiology simulations of connexin-46 (Cx46), connexin-50 (Cx50), and heterotypic Cx46/50 gap junction channels based on high-resolution open-state structures, enabling characterization of both ion permeation and long-timescale channel dynamics. Simulations reveal a multi-ion, multi-pathway permeation mechanism governed by isoform-specific energetic barriers and transient ion coordination sites that shape conductance and selectivity. In heterotypic Cx46/50 channels, asymmetric energetic landscapes establish a mechanistic basis for rectification. Unexpectedly, the microsecond-timescale simulations further revealed dynamic interactions between the intracellular loop (ICL) region and N-terminal domain (NT) that transiently constrict the pore and attenuate ionic currents. These findings suggest that the open-state comprises an ensemble of rapidly interconverting conductance microstates rather than a single static conformation, providing structural information of potential mechanistic importance beyond what has been learned from cryo-EM studies. Together, our results provide a mechanistic basis for ion permeation and current modulation in gap junction channels and highlight the importance of long-timescale protein dynamics in shaping intercellular communication.

Autotransporter proteins are major virulence factors in Gram-negative pathogens, yet how they fold during secretion remains incompletely understood. A longstanding puzzle is why pertactin folds and is secreted in vivo within minutes but refolds in vitro over hours to days. We introduce BEAM, a multiscale framework that learns slow collective variables from coarse-grained simulations to guide all-atom enhanced sampling. Applied to a C-terminal segment of the pertactin passenger domain from Bordetella pertussis, BEAM achieved four- to six-fold greater conformational coverage than traditional collective-variable-guided adaptive sampling or unbiased molecular dynamics. The resulting free-energy landscape revealed a compact, non-native intermediate accessible in bulk solution but geometrically incompatible with vectorial translocation across the outer membrane. Kinetic simulations show that access to this intermediate slows folding, whereas excluding it produces rapid, in vivo-like kinetics. Together, these results explain how vectorial secretion accelerates pertactin folding by excluding an off-pathway kinetic trap. More broadly, BEAM provides a multiscale strategy for revealing hidden conformational states at atomic resolution.

Inner mitochondrial membrane proteins must be sufficiently hydrophilic to withstand aqueous exposure during translation and transit to the mitochondria. Meanwhile, their transmembrane segments must be sufficiently hydrophobic to stably embed in the lipid membrane. We hypothesized that sequence-level adaptations evolved to balance these constraints. Here, we integrate structure-informed evolutionary analyses of mitochondrial proteins with atomistic simulations and cell-based experiments to identify aliphatic-to-threonine substitutions (ATS) as a potential solution to these constraints. With high statistical confidence, this transmembrane segment-specific adaptation is recurrently and convergently observed throughout mitochondrial evolution. Conformational analyses show that threonine interacts with both water and the transmembrane helix backbone, thereby lowering hydrophobicity without destabilizing secondary structure. In the extremely hydrophobic ATP6 protein, reverting threonines to aliphatic residues disrupts mitochondrial targeting, while introducing threonines into a poorly targeted variant improves its mitochondrial localization. These findings have implications for mitochondrial genome evolution, the rational design of membrane proteins, and potentially mitochondrial gene therapy.

White-barked birches extend to the northern limit of tree growth, and their bark is known to reduce solar damage during winter and early-spring by limiting solar heating and the incidence of harmful freeze-thaw events. The physical basis for this protection, however, has remained unclear. Here, we show that extracted betulin, the dominant triterpenoid responsible for the bark's whiteness, and Himalayan birch bark, both exhibit passive radiative cooling. Under low solar irradiance, bark and betulin-pellets reach temperatures below that of a shaded reference, and pellets cool more than bark. The cooling arises from high solar reflectance, which suppresses solar heating, and substantial mid-infrared emission, which drives radiative heat loss toward outer space. These findings help explain how bark-whiteness may contribute to protecting birch trees from solar-induced thermal stress.

Information is fundamental to biological survival, but the amount of information and its biological value are not equivalent. Shannon information quantifies uncertainty reduction, whereas Volkenstein's value of information measures how information changes the probability of a biologically relevant outcome. Although originally developed for molecular biology contexts, the latter concept has rarely been applied to environmental sensing and ecological interactions. Here we develop a value-of-information framework for microbial predator-prey interactions based on hydrodynamic sensing, in which prey detect fluid disturbances generated by approaching predators. Using a mechanistic model that incorporates sensory thresholds, memory, false alarms, biological benefits and costs, and predator encounter probability, we characterize mutual information from three hierarchical measures of biological value: encounter-conditional value, ecological value, and lifetime fitness value. The framework reveals how small amounts of sensory information can produce disproportionately large survival benefits during predator encounters, generating encounter-level value amplification in which biological value exceeds Shannon information. However, although global sensitivity analysis shows that such amplification is common, it is not universal and becomes progressively diluted at broader ecological and lifetime scales by encounter rarity, background noise, and sensory costs. Across most parameter combinations, the encounter-conditional value exceeded the ecological value, which in turn exceeded the lifetime fitness value. These results demonstrate that environmental sensing should be evaluated not only by how accurately it represents the external world, but by how strongly it changes biologically relevant outcomes. More broadly, the framework extends Volkenstein's concept of information value to ecological interactions and provides a quantitative framework for predicting when environmental information enhances survival and fitness, thereby providing a platform for explaining the evolution, maintenance, diversification, and loss of sensory systems.

Cellular activity drives epithelial fluidization -- a widespread phenomenon observed during tissue development, remodeling, and repair both in vivo and in vitro. Yet the physical origins and spatial organization of active forces vary widely across biological systems and are often represented by a single generic mechanism in theoretical models. Here, using an active vertex model, we systematically compare four modes of epithelial activity spanning subcellular to tissue scales: apolar motility, polar motility, fluctuating contractility, and mechanochemical regulation. Although all four mechanisms drive the same global transition from a solid-like rectangular tissue to a fluid-like circular morphology, they reach this state through distinct pathways -- differing in the rates and topology of junctional rearrangements, cell elimination, and collective motion and leave distinguishable signatures in tissue architecture, cell dynamics, and mechanical relaxation. Among these observables, spatial velocity correlations directly capture the spatial organization of activity: their correlation length and functional form together resolve all four mechanisms. The robustness of these signatures across activity strengths suggests that spatial velocity correlations offer an experimentally accessible means of identifying the physical origin of epithelial activity from live-cell imaging alone.

Auxin impacts on nearly every aspect of plant growth and development. Its exogenous application therefore results in pleiotropic growth responses. Exploiting this activity for plant propagation requires avoiding or minimizing such off-target effects and is generally achieved as a trade-off between toxicity and organogenetic efficacity. We recently identified the compound HYSPARIN (HYS) with potent, and uniquely selective adventitious root inductive activity. Unlike other root-inducing compounds, HYS preferentially activates auxin responses in the shoot via an unknown mechanism. Here, we show that HYS acts as a shoot-specific proauxin. Rather than acting through auxin homeostasis, we found that HYS is hydrolysed in planta independently of ILR1/ILL amidohydrolases to release the synthetic auxin MCPA. Structure-activity relationship analysis confirmed a strong dependence on its MCPA moiety for activating auxin responses, and identified its promoiety as a determinant of shoot-specificity and activity. Selective application of MCPA also potently induces AR is consistent with a model in which HYS metabolism produces a spatially restricted, AR inductive auxin signal. The activation mechanism of HYS thus provides a conceptual framework for tissue-specific metabolic delivery of auxin and may enable the programmable delivery of other xenobiotics in plants.

Photoprotection against excess energy is essential for the survival of photosynthetic organisms under adverse conditions. In plants, excess energy can be dissipated as heat through non-photochemical quenching (NPQ) of chlorophyll fluorescence, involving the trimeric light-harvesting complex II (LHCII), the major antenna of photosystem II. How NPQ affects antenna proteins remains debated, especially as most studies focus on short-lived components artificially induced in vitro. Here, we characterize the effects of qH, a long-lived NPQ component, on the fluorescence properties of natively quenched LHCII. Single-molecule fluorescence measurements, combined with biochemical and biophysical ensemble approaches, reveal a larger and more quenched subpopulation of LHCII trimers exhibiting fluorescence intermittency in samples with qH compared to those without. This behavior is linked to a small conformational change that stabilizes a quenched state, enhancing photoprotection at the antenna level. These findings provide new insights into sustained NPQ and its role in regulating energy dissipation under natural light conditions.

Chlorophyll (Chl) is essential for oxygenic photosynthesis, binding to membrane proteins for light harvesting and electron transfer. In angiosperms, Chl synthesis is halted in darkness, preventing accumulation of Chl-binding photosynthetic complexes in etioplasts. However, etioplasts assemble a dimeric Cytochrome b6f (Cyt b6f) complex, uniquely binding protochlorophyll (Pchl), the esterified derivative of protochlorophyllide (Pchlide). This indicates an evolutionarily conserved structural or functional role for Pchl distinct from the Chl bound in Cyt b6f in chloroplasts. Here we show that upon light-induced Chl synthesis in-vivo and in-vitro, Chl accumulation in Cyt b6f dimers precedes photosystems I and II. We find that chlorophyllide and Chl bind to the light-harvesting-like protein 3 (LIL3), supporting a role for LIL3 in early Chl allocation that extends its described role in stabilizing geranylgeranyl reductase. We determine a dissociation constant of 246.6 {+/-} 37 nM for Chlide binding to LIL3 in-vitro and show that Cyt b6f monomers and LIL3 co-migrate with Chlide in native PAGE, whereas Cyt b6f dimers and LIL3 co-migrate with Chl. These results indicate that Chlide binding to LIL3 chaperones esterification to Chl and reduction of geranylgeraniol, and that Chl release with Cyt b6f dimerization prioritizes Chl binding to Cyt b6f assembly during de-etiolation.

Respiratory disease seasonality is widely recognized, yet its mechanistic basis remains poorly understood. Winter environmental conditions have dominated explanations for influenza seasonality in temperate regions, while human behavior's role has been overlooked. Integrating high-resolution data on influenza cases and human behavior in built environments, we show US influenza seasonality emerges from behavioral adaptations to the environment rather than environmental effects alone. Southeastern US counties maintain persistent summer circulation shaped by weather-driven indoor crowding and built environment vulnerabilities, seeding epidemics as early as August that then spread nationwide. A transmission model incorporating intercounty connectivity, school calendars, and building-modulated transmissibility reproduces this spatiotemporal invasion pattern. These results suggest behavior, both routine mixing and environmental adaptation, shapes influenza seasonality, complementing explanations centered on aerosol persistence or viral survival. Our framework supports epidemic preparedness: modeling southern reservoirs' connectivity to northern counties, shifting vaccination timing to precede local epidemic onset could reduce influenza burden.

A central open problem in the study of de novo gene origination is that the molecular mechanisms and functions driving the emergence of such evolutionarily young, de novo protein-coding genes remain poorly understood. Metal chelation, a simple, directly selectable activity that both requires no specific interaction partners and is also compatible with intrinsic disorder, is one possible function. This possibility was tested using sequence signatures in 7,849 transcriptionally supported, still-segregating Drosophila melanogaster de novo open reading frames. Interestingly, these new open reading frames (neORFs) are enriched for bis-histidine motifs at the metal-coordination-competent spacings H-x-H and H-x-x-x-H and show no enrichment at the incompatible even spacings relative to repeat-masked intergenic ORFs. Notably, these neORFs were also found to lack the C-x-x-C grammar of canonical metal-binding proteins. I report that this H-x-H bis-histidine signal is generated by translation of (CA) microsatellites into His-Thr-His in Drosophila melanogaster, as evidenced by a CAC-codon bias within H-x-H motifs and a fourfold threonine enrichment at the central position. I propose that recurrent microsatellite expansion supplies Drosophila with a distributed, independently originated class of candidate metal-binding de novo peptides. I then trace one neORF (ZMEG) from conserved ancestral non-coding sequence to a transcribed, melanogaster-lineage open reading frame whose (CA)9-derived His run presents a candidate His32-His36 bis-histidine site. I propose that such de novo proteins may constitute a class of molecules united not by common descent but by their shared origin in evolvable repeat sequence, highlighting the importance of emergence bias in molecular evolution.