Effect of Trifluoroethanol on a Tardigrade Desiccation-Tolerance Protein

Protein-based drugs revolutionized medicine, yet require a cold-chain, low temperature transport and storage. Dry formulations offer a room temperature alternative. Tardigrades, a phylum of microscopic animals capable of surviving complete desiccation, offer a promising route towards this goal. My project focuses on a particular tardigrade desiccation-tolerance protein, cytosolic abundant heat soluble (CAHS) D. CAHS D protects client proteins from inactivation in vitro but the mechanism is unknown. My graduate student mentor and I showed that pure CAHS D forms a concentration-dependent thermoreversible gel cross-linked by intermolecular β-sheets, and we posit the gel matrix acts as a molecular shield upon desiccation. Here, trifluoroethanol (TFE) was used to mimic the effect of dehydration on CAHS D. Circular dichroism spectropolarimeter data indicated that low concentrations of CAHS D gained α-helix in TFE. At higher CAHS D concentrations, the protein went through liquid, gel, aggregate and phases, the latter with liquid-gel phase separation at increasing % TFE. Using attenuated total internal reflectance Fourier transformation infrared spectroscopy, I showed that gelation was due to intermolecular interactions between β-strands, which are not significant enough at low CAHS D concentration. I suggest that only at high CAHS D concentration did TFE mimic water deficiency, strengthening CAHS D gelation to let it act like a ‘molecular shield’ against water deficient environment.Bachelor of Scienc