Secondary structure and stability of a gel-forming tardigrade desiccation-tolerance protein.

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 ß-sheet formation.

Water's Variable Role in Protein Stability Uncovered by Liquid-Observed Vapor Exchange NMR.

Water is essential to protein structure and stability, yet our understanding of how water shapes proteins is far from thorough. Our incomplete knowledge of protein-water interactions is due in part to a long-standing technological inability to assess experimentally how water removal impacts local protein structure. It is now possible to obtain residue-level information on dehydrated protein structures via liquid-observed vapor exchange (LOVE) NMR, a solution NMR technique that quantifies the extent of hydrogen-deuterium exchange between unprotected amide protons of a dehydrated protein and D2O vapor. Here, we apply LOVE NMR, Fourier transform infrared spectroscopy, and solution hydrogen-deuterium exchange to globular proteins GB1, CI2, and two variants thereof to link mutation-induced changes in the dehydrated protein structure to changes in solution structure and stability. We find that a mutation that destabilizes GB1 in solution does not affect its dehydrated structure, whereas a mutation that stabilizes CI2 in solution makes several regions of the protein more susceptible to dehydration-induced unfolding, suggesting that water is primarily responsible for the destabilization of the GB1 variant but plays a stabilizing role in the CI2 variant. Our results indicate that changes in dehydrated protein structure cannot be predicted from changes in solution stability alone and demonstrate the ability of LOVE NMR to uncover the variable role of water in protein stability. Further application of LOVE NMR to other proteins and their variants will improve the ability to predict and modulate protein structure and stability in both the hydrated and dehydrated states for applications in medicine and biotechnology.