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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 Intrinsically disordered protein regions (IDRs) are ubiquitous across all kingdoms of life and play a variety of essential cellular roles. IDRs exist in a collection of structurally distinct conformers known as an ensemble. An IDR’s amino acid sequence determines its ensemble, which in turn can play an important role in dictating molecular function. Yet a clear link connecting IDR sequence, its ensemble properties, and its molecular function in living cells has not been directly established. Here, we set out to test this sequence-ensemble-function paradigm using a novel computational method (GOOSE) that enables the rational design of libraries of IDRs by systematically varying specific sequence properties. Using ensemble FRET, we measured the ensemble dimensions of a library of rationally designed IDRs in human-derived cell lines, revealing how IDR sequence influences ensemble dimensionsin situ.Furthermore, we show that the interplay between sequence and ensemble can tune an IDR’s ability to sense changes in cell volume - ade novomolecular function for these synthetic sequences. Our results establish biophysical rules for intracellular sequence-ensemble relationships, enable a new route for understanding how IDR sequences map to function in live cells, and set the ground for the design of synthetic IDRs withde novofunction. 

Proteins must be hydrated to function. Desiccation, a common event in an increasing number of ecosystems, can drive proteome-wide unfolding and aggregation. For cells to survive, proteins must disaggregate and retain their function upon rehydration. The molecular determinants that underlie protein desiccation resistance remain unknown. Here, we use mass spectrometry to show that some proteins possess an innate ability to survive dehydration and subsequent rehydration. Structural analysis correlates the ability of proteins to resist desiccation with their surface area 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 desiccation-sensitive are responsible for ribosome biogenesis. As a result, the rehydrated proteome is preferentially enriched with metabolite and small molecule producers and depleted of ribosomes - the cell's heaviest consumers. We propose this functional bias allows cells to kickstart their metabolism and promote cell survival upon rehydration. 

The molecular machinery that enables life has evolved in water, yet many of the organisms around us are able to survive even extreme desiccation. Especially remarkable are single-cell and sedentary organisms that rely on specialized biomolecular machinery to survive in environments that are routinely subjected to a near-complete lack of water. In this review, we zoom in on the molecular level of what is happening in the cellular environment under water stress. We cover the various mechanisms by which biochemical components of the cell can dysfunction in dehydrated cells and detail the different strategies that organisms have evolved to eliminate or cope with these desiccation-induced perturbations. We specifically focus on two survival strategies: (1) the use of disordered proteins to protect the cellular environment before, during, and in the recovery from desiccation, and (2) the use of biomolecular condensates as a self-assembly mechanism that can sequester or protect specific cellular machinery in times of water stress. We provide a summary of experimental work describing the critical contributions of disordered proteins and biomolecular condensates to the cellular response to water loss and highlight their role in desiccation tolerance. Desiccation biology is an exciting area of cell biology, still far from being completely explored. Understanding it on the molecular level is bound to give us critical new insights in how life adapted/can adapt to the loss of water, spanning from the early colonization of land to how we can deal with climate change in our future. 

Abstract Intrinsically disordered proteins and protein regions (IDPs) are prevalent in all proteomes and are essential to cellular function. Unlike folded proteins, IDPs exist in an ensemble of dissimilar conformations. Despite this structural plasticity, intramolecular interactions create sequence-specific structural biases that determine an IDP ensemble’s three-dimensional shape. Such structural biases can be key to IDP function and are often measured in vitro, but whether those biases are preserved inside the cell is unclear. Here we show that structural biases in IDP ensembles found in vitro are recapitulated inside human-derived cells. We further reveal that structural biases can change in a sequence-dependent manner due to changes in the intracellular milieu, subcellular localization, and intramolecular interactions with tethered well-folded domains. We propose that the structural sensitivity of IDP ensembles can be leveraged for biological function, can be the underlying cause of IDP-driven pathology or can be used to design disorder-based biosensors and actuators. 

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.