SUMMARY Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

Obesity is associated with an impaired immune response, an increased susceptibility to bacterial infection, and a chronic increase in proinflammatory cytokines such as IL-6 and TNFalpha. However, few studies have examined the effect of obesity on the immune response to viral infections. Because infection with influenza is a leading cause of morbidity and mortality worldwide, we investigated the effect of obesity on early immune responses to influenza virus exposure. Diet-induced obese and lean control C57BL/6 mice were infected with influenza A/PR8/34, and lung pathology and immune responses were examined at d 0 (uninfected), 3, and 6, postinfection. Following infection, diet-induced obese mice had a significantly higher mortality rate than the lean controls and elevated lung pathology. Antiviral and proinflammatory cytokine mRNA production in the lungs of the infected mice was markedly different between obese and lean mice. IFNalpha and beta were only minimally expressed in the infected lungs of obese mice and there was a notable delay in expression of the proinflammatory cytokines IL-6 and TNFalpha. Additionally, obese mice had a substantial reduction in NK cell cytotoxicity. These data indicate that obesity inhibits the ability of the immune system to appropriately respond to influenza infection and suggests that obesity may lead to increased morbidity and mortality from viral infections.

An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about protein stability and dynamics in cells.

Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome.T he inside of cells is packed with macromolecules whose concentrations reach 300-400 g/L (1). Compared with the ideal (dilute) environments conventionally used to study proteins, crowding inside cells can significantly alter the biophysical landscape of proteins, including their equilibrium thermodynamic stability (2-6). Experimental and computational efforts establish that crowding effects arise from a combination of short-range (steric) repulsions and longer-range (often called soft) interactions between macromolecules (7-13). Despite a growing number of incell protein studies (2-6), there is no quantitative information about the energetics of quinary interactions.Amide proton exchange experiments have been used for more than 50 y to measure equilibrium protein stability, defined as the Gibbs free energy required to open the protein and expose individual backbone amide protons to solvent, ΔG°′ op (14). For the B1 domain of protein G (GB1), ΔG°′ op equals −RTln(k obs /k uns ), where R is the gas constant, T is the absolute temperature, k obs is the observed rate of exchange, and k uns is the rate in an unstructured peptide (6). We know that the cytoplasm does not affect k uns (15). Most importantly, we know that for exchange under these conditions ΔG°′ op approximates the free energy required to denature the protein, ΔG°′ den (6). Therefore, these experiments provide a thermodynamically rigorous measure of equilibrium global protein stability. Using this information, we quantified the stability of GB1 at the residue level in Escherichia coli (6) via NMRdetected backbone amide hydrogen/deuterium exchange (16).Thermodynamic cycles (17) can be used to quantify the energetics of interactions between proteins in specific protein complexes (17,18) and between side chains in globular proteins (19,20). Briefly, the individual effects of two single-site amino acid changes are compared with the combined effect of both mutations. If the sites interact, the sum of the contributions from the single-site changes will not equal the contributi...

Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMRdetected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.M acromolecules in Escherichia coli reach concentrations of 300-400 g/L and occupy up to 40% of the cellular volume (1), but proteins are normally studied in buffer alone. The effects of crowding arise from two phenomena, hard-core repulsions and nonspecific chemical (soft) interactions (2-9). Hard-core repulsions limit the volume available to biological macromolecules for the simple reason that two molecules cannot be in the same place at the same time. This press for space favors compact states over expanded states. The second phenomenon arises because crowders not only exclude volume, but also participate in chemical interactions. Even though individually weak, the high concentration of macromolecules can lead to a large net effect. Repulsive nonspecific interactions reinforce the hardcore repulsions, whereas attractive nonspecific interactions oppose them. We use the term "nonspecific attractive interactions" to distinguish these from specific chemical interactions, such as ligand binding.Our aim is to understand how the crowded and heterogeneous, intracellular environment affects the equilibrium thermodynamic stability of globular proteins. Globular proteins are marginally stable in buffer at room temperature (10), with Gibbs free energy differences of 5-15 kcal/mol between the efficiently packed native (N) state and the ensemble of higher-energy denatured (D) states ðΔG o′ D Þ (11). Crowding effects arise from entropic and enthalpic contributions; hard-core repulsions are entropic, whereas the consequent nonspecific chemical interactions are also enthalpic. Hard-core repulsions always increase ΔG o′ D for globular proteins because D occupies more space than N (12-14). However,...