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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 are 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. 

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 demonstrate 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. 

Abstract 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 not only ambient but 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 sugar-based protectants can stabilize the biologic pharmaceutical Human Blood Clotting Factor VIII 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 make 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. 

Abstract 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 11‐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 (CS), from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of CS 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. 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. 

Abstract Tardigrades are microscopic animals renowned for their ability to survive extreme desiccation. Unlike many desiccation-tolerant organisms that accumulate high levels of the disaccharide trehalose to protect themselves during drying, tardigrades accumulate little or undetectable levels. Using comparative metabolomics, we find that despite being enriched at low levels, trehalose is a key biomarker distinguishing hydration states of tardigrades. In vitro, naturally occurring stoichiometries of trehalose and CAHS proteins, intrinsically disordered proteins with known protective capabilities, were found to produce synergistic protective effects during desiccation. In vivo, this synergistic interaction is required for robust CAHS-mediated protection. This demonstrates that trehalose acts not only as a protectant, but also as a synergistic cosolute. Beyond desiccation tolerance, our study provides insights into how the solution environment tunes intrinsically disordered proteins’ functions, many of which are vital in biological contexts such as development and disease that are concomitant with large changes in intracellular chemistry. 

Abstract Protein‐based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation. We find that CAHS proteins protect the test enzymes lactate dehydrogenase and lipoprotein lipase against desiccation‐, freezing‐, and lyophilization‐induced deactivation. Our data also show that a variety of globular and disordered protein controls, with no known link to desiccation tolerance, protect our test enzymes. Protection of lactate dehydrogenase correlates, albeit imperfectly, with the charge density of the protein additive, suggesting an approach to tune protection by modifying charge. Our results support the potential use of CAHS proteins as stabilizing excipients in formulations and suggest that other proteins may have similar potential.