Surviving extreme radiation.

Tiny animals known as tardigrades use a combination of DNA repair machinery and a novel protein to mend their genome after intense ionizing radiation.

Water content, transition temperature and fragility influence protection and anhydrobiotic capacity.

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. 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 glycerol, quantifying their enzyme-protective capacity and their material properties in a dry state. 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. Next, we examined the material properties of a dry desiccation tolerant and intolerant life stage from three different organisms. The dried desiccation tolerant life stage of all organisms had an increased glass transition temperature and reduced glass former fragility relative to its dried desiccation intolerant life stage. These results suggest in nature organismal desiccation tolerance relies on a combination of various material 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. Statement of significance: For 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.

Helicity of a tardigrade disordered protein contributes to its protective function during desiccation.

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 the tardigrade Hypsibius exemplaris, 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, or replacement of hydrophobic amino acids with serine or glycine residues results in variants with different degrees of helical structure. Importantly, correlation of protective capacity and helical content in variants generated through different helix perturbing modalities does 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.

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.

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 proteome-wide protein unfolding and aggregation. For cells to survive this stress, at least some of the proteome must disaggregate and resume function upon rehydration. The molecular determinants that underlie the ability of proteins to do this remain largely unknown. Here, we apply quantitative and structural proteomic mass spectrometry to desiccated and rehydrated yeast extracts to show that some proteins possess an innate capacity to survive extreme water loss. Structural analysis correlates the ability of proteins to resist desiccation with their surface chemistry. Remarkably, highly resistant proteins are responsible for the production of the cell's building blocks - amino acids, metabolites, and sugars. Conversely, those proteins that are most desiccation-sensitive are involved in ribosome biogenesis and other energy consuming processes. As a result, the rehydrated proteome is preferentially enriched with metabolite and small molecule producers and depleted of some of the cell's heaviest consumers. We propose this functional bias enables cells to kickstart their metabolism and promote cell survival following desiccation and rehydration.