Extremotolerant organisms from all domains of life produce protective intrinsically disordered proteins (IDPs) in response to desiccation stress. In vitro, many of these IDPs protect enzymes from dehydration stress better than U.S. Food and Drug Administration‐approved excipients. However, as with most excipients, their protective mechanism is poorly understood. Here, we apply thermogravimetric analysis, differential scanning calorimetry, and liquid‐observed vapor exchange (LOVE) NMR to study the protection of two model globular proteins (the B1 domain of staphylococcal protein G [GB1] and chymotrypsin inhibitor 2 [CI2]) by two desiccation‐tolerance proteins (CAHS D from tardigrades and PvLEA4 from an anhydrobiotic midge), as well as by disordered and globular protein controls. We find that all protein samples retain similar amounts of water and possess similar glass transition temperatures, suggesting that neither enhanced water retention nor vitrification is responsible for protection. LOVE NMR reveals that IDPs protect against dehydration‐induced unfolding better than the globular protein control, generally protect the same regions of GB1 and CI2, and protect GB1 better than CI2. These observations suggest that electrostatic interactions, charge patterning, and expanded conformations are key to protection. Further application of LOVE NMR to additional client proteins and protectants will deepen our understanding of dehydration protection, enabling the streamlined production of dehydrated proteins for expanded use in the medical, biotechnology, and chemical industries.

Extremotolerant organisms and industry exploit sugars as desiccation protectants, with trehalose being widely used by both. How sugars, in general, and the hydrolytically stable sugar trehalose, in particular, protect proteins is poorly understood, which hinders the rational design of new excipients and implementation of novel formulations for preserving lifesaving protein drugs and industrial enzymes. We employed liquid-observed vapor exchange nuclear magnetic resonance (LOVE NMR), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA) to show how trehalose and other sugars protect two model proteins: the B1 domain of streptococcal protein G (GB1) and truncated barley chymotrypsin inhibitor 2 (CI2). Residues with intramolecular H-bonds are most protected. The LOVE NMR and DSC data indicate that vitrification may be protective. Combining LOVE NMR and TGA data shows that water retention is not important. Our data suggest that sugars protect protein structure as they dry by strengthening intraprotein H-bonds and water replacement and that trehalose is the stresstolerance sugar of choice because of its covalent stability.

Protein‐based pharmaceuticals are increasingly important, but their inherent instability necessitates a “cold chain” requiring costly refrigeration during production, shipment, and storage. Drying can overcome this problem, but most proteins need the addition of stabilizers, and some cannot be successfully formulated. Thus, there is a need for new, more effective protective molecules. Cytosolically, abundant heat‐soluble proteins from tardigrades are both fundamentally interesting and a promising source of inspiration; these disordered, monodisperse polymers form hydrogels whose structure may protect client proteins during drying. We used attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, and small‐amplitude oscillatory shear rheometry to characterize gelation. A 5% (wt/vol) gel has a strength comparable with human skin, and melts cooperatively and reversibly near body temperature with an enthalpy comparable with globular proteins. We suggest that the dilute protein forms α‐helical coiled coils and increasing their concentration drives gelation via intermolecular β‐sheet formation.

Water is key to protein structure and stability, yet the relationship between protein–water interactions and structure is poorly understood, in part because there are few techniques that permit the study of dehydrated protein structure at high resolution. Here, we describe liquid-observed vapor exchange (LOVE) NMR, a solution NMR-based method that provides residue-level information about the structure of dehydrated proteins. Using the model protein GB1, we show that LOVE NMR measurements reflect the fraction of the dried protein population trapped in a conformation where a given residue is protected from exchange with D2O vapor. Comparisons to solution hydrogen–deuterium exchange data affirm that the dried protein structure is strongly influenced by local solution stability and that the mechanism of dehydration protection exerted by the widely used protectant trehalose differs from its mechanism of stabilization in solution. Our results highlight the need for refined models of cosolute-mediated dehydration protection and demonstrate the ability of LOVE NMR to inform such models.

No abstract