Biology subjects

Boothby, T. C.

Publications and source records attributed to Boothby, T. C..

13 recordsLinked to original sources

bioRxiv2026

A molecular grammar for environmental sensitivity in intrinsically disordered protein regions

Cells must continuously sense and respond to changes in their physicochemical environment, yet the molecular mechanisms underlying this process remain incompletely understood. Intrinsically disordered protein regions (IDRs) constitute a substantial fraction of the eukaryotic proteome and participate in nearly every major cellular process. IDRs exist as dynamic conformational ensembles whose properties are strongly influenced by both amino acid sequence and the physicochemical environment. Although this intrinsic sensitivity has suggested that IDRs may contribute to environmental sensing of the cellular environment, the sequence-encoded principles that enable such sensing remain poorly understood. Here we identify a molecular grammar that captures key sequence features underlying variation in environmental ensemble sensitivity across diverse IDRs. Using 188 IDRs derived from organisms across all kingdoms of life, we systematically quantify changes in ensemble properties in response to hyperosmotic stress in living cells. We show that ensemble sensitivity is significantly associated with specific sequence features and predicted ensemble dimensions, with homogeneous patterning of oppositely charged residues emerging as a strong predictor of high sensitivity in charged IDRs. This sequence organization is necessary for high sensitivity in naturally-occurring IDRs and sufficient to confer high responsiveness in de novo designed low-complexity sequences. We further demonstrate that the presence of additional structured domains within IDR-containing proteins can substantially modulate ensemble sensitivity relative to isolated IDRs, revealing an additional layer of modulation at the multi-domain protein level. Applying the molecular grammar to the with-no-lysine (WNK) kinase crowding-sensing system, we find that regions within the disordered C-terminal domain - the domain necessary for crowding-induced phase separation and cell volume recovery - are predicted as hypersensitive, a property that is conserved across distant WNK homologues despite sequence divergence, and that is shared by heterologous IDRs capable of functionally replacing it. Together, our results establish environmental ensemble sensitivity as a sequence-encoded, context-dependent, and quantitatively tunable property of IDRs, provide a framework for understanding how physicochemical changes can be transduced in cells without dedicated receptor architectures as exemplified by the WNK crowding-sensing system, and offer design principles for engineering synthetic protein-based environmental sensors.

biophysics↗

bioRxiv2026

Drying parameters and aging modulate protective properties of vitrified trehalose

Storage of biological materials is essential for medical, research, and biotechnological applications. While cold-chain preservation is effective, it is costly, infrastructure-dependent, and vulnerable to disruption. Room-temperature dry storage, inspired by desiccation-tolerant organisms, provides an alternative by stabilizing biomolecules in vitrified ("glass-like") matrices that limit molecular motion which if not reduced can lead to breakdown and loss of integrity. Trehalose is widely used as a vitrifying agent, but its protective capacity depends on glassy properties shaped by drying methods, environmental conditions, storage duration, and the type of preserved molecule. Systematic studies linking these factors to short and long-term stability remain limited. Here, we examine how drying conditions and storage duration influence the stability of DNA, RNA, and enzymes in vitrified trehalose systems. DNA remained stable under all conditions, independent of trehalose or drying parameters, reflecting its intrinsic resistance to desiccation-induced damage. RNA showed moderate sensitivity to drying without trehalose but was stabilized in its presence, although RNA integrity did not consistently correlate with measured vitrified properties. In contrast, enzymes were highly sensitive to drying without trehalose and were strongly protected under conditions that promoted favorable vitrified properties. Enzyme protection after 30 min correlated with high glass transition temperature. However, during prolonged drying, increased glass transition temperature was inversely correlated with enzyme protection and was a better indicator of detrimental physical aging of the vitrified system. These findings present the first insight into how drying methods, environmental conditions, and storage duration shape vitrified properties and stability. They guide optimization of room-temperature preservation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/700019v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@796fc0org.highwire.dtl.DTLVardef@97ca65org.highwire.dtl.DTLVardef@168d532org.highwire.dtl.DTLVardef@184ee58_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINucleic acids are generally stable under various drying conditions/vitrified systems C_LIO_LIProtein preservation benefits from specific mediators and vitrified properties C_LIO_LIDrying method and parameters tune vitrified properties and protection C_LIO_LIVitrified systems age over time, changing properties and protectiveness C_LIO_LIInitially high Tg is protective, increasing Tg and fragility over time indicate aging C_LI

biochemistry↗

bioRxiv2025

Functional diversity of Arabidopsis late embryogenesis abundant proteins in response to changes in the physicochemical environment

Some desiccation-tolerant organisms accumulate intrinsically disordered proteins (IDPs), such as Late Embryogenesis Abundant (LEA) proteins, which help protect other proteins from inactivation and/or aggregation during desiccation. Like other IDPs, LEA proteins adopt ensembles of extended conformations that shift in response to environmental changes. Desiccation causes dramatic changes in the cellular environment, but it is unclear how the structural malleability of LEAs is related to their protective function. In this work, we measured the in vitro protective function and structural sensitivity to changes in the environment of four Arabidopsis thaliana LEA proteins from different families. We found that all LEAs showed different protection efficiencies of the labile enzyme lactate dehydrogenase under desiccation in vitro. In line with this, we identified distinct ensemble structural changes when these LEA proteins were exposed to different physicochemical environments. Specifically, AtEM1, AtLEA7, and AtLEA4-5 showed compaction when the solution was crowded with polymers, whereas AtLEA6-2.2 showed larger structural changes when salt concentrations were increased. Furthermore, the ensembles of AtEM1, AtLEA7, and AtLEA4-5 gained helicity under desiccated conditions, while that of AtLEA6-2.2 remained largely disordered. Our results highlight how ensemble properties of LEA proteins contribute to their distinct functional activities in vitro. This work advances our understanding of the molecular features that contribute to functional diversity in desiccation-related IDPs.

biophysics↗

bioRxiv2025

FLOE1 maintains cellular viscosity in rehydrating Arabidopsis embryos

Most plant embryos can survive for years in a dry state of less than 10% water(1). Rehydration during seed germination is critical but risky-too little environmental water can dehydrate and kill the developing embryo. Plants avoid this by germinating only when sufficient water is present. However, the mechanisms by which sufficient water is sensed and how it triggers germination remain unknown. FLOE1 suppresses seed germination under water limitation by undergoing reversible phase changes in response to water potential(2). To understand how FLOE1 affects germination, we compared water behavior in wild type and floe1-1 null mutants. While both had similar amounts of water when dry, floe1-1 seeds hydrated less than the wild type after imbibition. Additionally, bound water was less restricted in floe1-1 embryos, suggesting FLOE1 promotes macromolecular water binding. By developing plant-compatible versions of Genetically Encoded Multimeric nanoparticles (GEMs)(3), we show that FLOE1 increases cytoplasmic viscosity during embryo rehydration. FLOE1 in yeast was sufficient to increase cytoplasmic viscosity. FLOE1 also increased cytoplasmic viscosity during hyperosmotic stress in yeast, and this ability was ablated by FLOE1 domain deletions. Our study identifies FLOE1 as a regulator of water content, dynamics, and cytoplasmic viscosity, linking molecular water control to cellular physical properties.

plant biology↗

bioRxiv2024

LEA_4 motifs function alone and in conjunction with synergistic cosolutes to protect a labile enzyme during desiccation

Organisms from all kingdoms of life depend on Late Embryogenesis Abundant (LEA) proteins to survive desiccation. LEA proteins are divided into broad families distinguished by the presence of family-specific motif sequences. The LEA_4 family, characterized by eleven-residue motifs, plays a crucial role in the desiccation tolerance of numerous species. However, the role of these motifs in the function of LEA_4 proteins is unclear, with some studies finding that they recapitulate the function of full-length LEA_4 proteins in vivo, and other studies finding the opposite result. In this study, we characterize the ability of LEA_4 motifs to protect a desiccation-sensitive enzyme, citrate synthase, from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of citrate synthase during desiccation, but also that they can do so more robustly via synergistically interactions with cosolutes. Our analysis further suggests that cosolutes induce synergy with LEA_4 motifs in a manner that correlates with transfer free energy (TFE). This research advances our understanding of LEA_4 proteins by demonstrating that during desiccation their motifs can protect specific clients to varying degrees and that their protective capacity is modulated by their chemical environment. Our findings extend beyond the realm of desiccation tolerance, offering insights into the interplay between IDPs and cosolutes. By investigating the function of LEA_4 motifs, we highlight broader strategies for understanding protein stability and function.

biochemistry↗

bioRxiv2024

Protein surface chemistry encodes an adaptive resistance to desiccation

Cellular desiccation - the loss of nearly all water from the cell - is a recurring stress in an increasing number of ecosystems that can drive protein unfolding and aggregation. For cells to survive, at least some of the proteome must resume function upon rehydration. Which proteins tolerate desiccation, and the molecular determinants that underlie this tolerance, are largely unknown. Here, we apply quantitative and structural proteomic mass spectrometry to show that certain proteins possess an innate capacity to tolerate rehydration following extreme water loss. Structural analysis points to protein surface chemistry as a key determinant for desiccation tolerance, which we test by showing that rational surface mutants can convert a desiccation sensitive protein into a tolerant one. Desiccation tolerance also has strong overlap with cellular function, with highly tolerant proteins responsible for production of small molecule building blocks, and intolerant proteins involved in energy-consuming processes such as ribosome biogenesis. As a result, the rehydrated proteome is preferentially enriched with metabolite and small molecule producers and depleted of some of the cells heaviest consumers. We propose this functional bias enables cells to kickstart their metabolism and promote cell survival following desiccation and rehydration. TeaserProteins can resist extreme dryness by tuning the amino acids on their surfaces.

biophysics↗

bioRxiv2024

Disordered proteins interact with the chemical environment to tune their protective function during drying

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

biochemistry↗

bioRxiv2023

Helicity of a tardigrade disordered protein promotes desiccation tolerance

In order to survive extreme drying (anhydrobiosis), many organisms, spanning every kingdom of life, accumulate intrinsically disordered proteins (IDPs). For decades, the ability of anhydrobiosis-related IDPs to form transient amphipathic helices has been suggested to be important for promoting desiccation tolerance. However, evidence empirically supporting the necessity and/or sufficiency of helicity in mediating anhydrobiosis is lacking. Here we demonstrate that the linker region of CAHS D, a desiccation-related IDP from tardigrades that contains significant helical structure, is the protective portion of this protein. Perturbing the sequence composition and grammar of the linker region of CAHS D, through the insertion of helix-breaking prolines, modulating the identity of charged residues, sequence scrambling, or replacement of hydrophobic amino acids with serine or glycine residues results in variants with different degrees of helical structure. Importantly, the resulting helicity of these variants generated through similar helix breaking modalities correlates strongly with their ability to promote desiccation tolerance, providing direct evidence that helical structure is necessary for robust protection conferred by this desiccation-related IDP. However, correlation of protective capacity and helical content in variants generated through different helix perturbing modalities do not show as strong a trend, suggesting that while helicity is important it is not the only property that makes a protein protective during desiccation. These results provide direct evidence for the decades old theory that helicity of desiccation-related IDPs is linked to their anhydrobiotic capacity.

biochemistry↗

bioRxiv2023

Labile assembly of a tardigrade protein induces biostasis

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins that form gels. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of gelation for CAHS D similar to that of intermediate filaments. We show that gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fiber-like condensates during osmotic stress. Condensation of CAHS D improves survival of osmotically shocked cells through at least two mechanisms: reduction of cell volume change and reduction of metabolic activity. Importantly, condensation of CAHS D is reversible and metabolic rates return to control levels after CAHS condensates are resolved. This work provides insights into how tardigrades induce biostasis through the self-assembly of CAHS gels.

physiology↗

bioRxiv2023

Distinct material properties of sugar-glasses correlate with anhydrobiosis in vitro and in vivo

Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or anhydrobiosis (Greek for "life without water"). Increased intracellular viscosity, leading to the formation of a vitrified state is necessary, but not sufficient, for survival while dry. What properties of a vitrified system make it desiccation-tolerant or -sensitive are unknown. We have analyzed 18 different in vitro vitrified systems, composed of one of three protective disaccharides (trehalose, sucrose, or maltose) and varying amounts of glycerol, quantifying their enzyme-protective capacity and their material properties in a dry state. We find that protection conferred by mixtures containing maltose correlates strongly with increased water content, increased glass-transition temperature, and reduced glass former fragility, while the protection of glasses formed with sucrose correlates with increased glass transition temperature and the protection conferred by trehalose glasses correlates with reduced glass former fragility. Thus, in vitro different vitrified sugars confer protection through distinct material properties. Extending on this, we have examined the material properties of a dry desiccation tolerant and intolerant life stage from three different organisms. In all cases, the dried desiccation tolerant life stage of an organism had an increased glass transition temperature relative to its dried desiccation intolerant life stage, and this trend is also seen in all three organisms when considering reduced glass former fragility. These results suggest that while drying of different protective sugars in vitro results in vitrified systems with distinct material properties that correlate with their enzyme-protective capacity, in nature organismal desiccation tolerance relies on a combination of these properties. This study advances our understanding of how protective and non-protective glasses differ in terms of material properties that promote anhydrobiosis. This knowledge presents avenues to develop novel stabilization technologies for pharmaceuticals that currently rely on the cold-chain. 1.1 Statement of significanceFor the past three decades the anhydrobiosis field has lived with a paradox, while vitrification is necessary for survival in the dry state, it is not sufficient. Understanding what property(s) distinguishes a desiccation tolerant from an intolerant vitrified system and how anhydrobiotic organisms survive drying is one of the enduring mysteries of organismal physiology. Here we show in vitro the enzyme-protective capacity of different vitrifying sugars can be correlated with distinct material properties. However, in vivo, diverse desiccation tolerant organisms appear to combine these material properties to promote their survival in a dry state. 3.1 HighlightsO_LIThe enzyme-protective capacities of different glass forming sugars correlate with distinct material properties. C_LIO_LIMaterial properties of dried anhydrobiotic organisms differ dramatically when examined in desiccation tolerant and intolerant life stages. C_LIO_LIOrganismal desiccation tolerance is concomitant with changes in glassy properties including increased glass transition temperature and reduced glass former fragility. C_LI

biophysics↗

bioRxiv2022

Natural and engineered mediators of desiccation tolerance stabilize Human Blood Clotting Factor VIII in a dry state

Biologics, pharmaceuticals containing or derived from living organisms, such as vaccines, antibodies, stem cells, blood, and blood products are a cornerstone of modern medicine. However, nearly all biologics have a major deficiency: they are inherently unstable, requiring storage under constant cold conditions. The so-called cold-chain, while effective, represents a serious economic and logistical hurdle for deploying biologics in remote, underdeveloped, or austere settings where access to cold-chain infrastructure ranging from refrigerators and freezers to stable electricity is limited. To address this issue, we explore the possibility of using anhydrobiosis, the ability of organisms such as tardigrades to enter a reversible state of suspended animation brought on by extreme drying, as a jumping off point in the development of dry storage technology that would allow biologics to be kept in a desiccated state under ambient or even elevated temperatures. Here we examine the ability of different protein and sugar-based mediators of anhydrobiosis derived from tardigrades and other anhydrobiotic organisms to stabilize Human Blood Clotting Factor VIII under repeated dehydration/rehydration cycles, thermal stress, and long-term dry storage conditions. We find that while both protein and sugarbased protectants can stabilize the biologic pharmaceutical Human Blood Clotting Factor FVIII under all these conditions, protein-based mediators offer more accessible avenues for engineering and thus tuning of protective function. Using classic protein engineering approaches, we fine tune the biophysical properties of a protein-based mediator of anhydrobiosis derived from a tardigrade, CAHS D. Modulating the ability of CAHS D to form hydrogels made the protein better or worse at providing protection to Human Blood Clotting Factor VIII under different conditions. This study demonstrates the effectiveness of tardigrade CAHS proteins and other mediators of desiccation tolerance at preserving the function of a biologic without the need for the cold-chain. In addition, our study demonstrates that engineering approaches can tune natural products to serve specific protective functions, such as coping with desiccation cycling versus thermal stress. Ultimately, these findings provide a proof of principle that our reliance on the cold-chain to stabilize life-saving pharmaceuticals can be broken using natural and engineered mediators of desiccation tolerance.

pharmacology and toxicology↗

bioRxiv2021

Molecular Swiss Army Knives: Tardigrade CAHS Proteins Mediate Desiccation Tolerance Through Multiple Mechanisms

Tardigrades, also known as water bears, make up a phylum of small but extremely hardy animals, renowned for their ability to survive extreme stresses, including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins, such as gelatin, our data support a mechanism in which gel formation of CAHS D is driven by intermolecular {beta}-{beta} interactions. We find that gel formation corresponds with strong coordination of water and slowing of water diffusion. The degree of water coordination correlates with the ability of CAHS D to protect lactate dehydrogenase from unfolding when dried. This implies that the mechanism for unfolding protection can be attributed to a combination of hydration and slowed molecular motion. Conversely, rapid diffusion leading to efficient molecular shielding appears to be the predominant mechanism preventing protein aggregation. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as molecular Swiss Army Knives capable of providing protection through several different mechanisms simultaneously.

biophysics↗

bioRxiv2017

LITE microscopy: a technology for high numerical aperture, low photobleaching fluorescence imaging

Fluorescence microscopy is a powerful approach for studying sub-cellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel LSFM method: Lateral Interference Tilted Excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically-limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

cell biology↗