Water is essential for active life, yet some organisms, such as tardigrades, can survive prolonged periods of drying-induced dormancy. Cytoplasmic abundant heat-soluble (CAHS) proteins are disordered proteins that undergo a phase transition from the solution to gel state. CAHS proteins help tardigrades survive extreme drying, increase hyperosmotic stress tolerance in heterologous systems, and preserve the function of labile enzymes during drying in vitro. It has been speculated that the ability of CAHS proteins to form gels might be mechanistically linked to their protective capacity. However, recent evidence suggests that while gelation enhances hyperosmotic stress tolerance, it is not required for this phenomenon. Still, the extent to which gelation is necessary for other CAHS-based protective functions, such as enzyme protection during drying, is unknown. Here, we show that rather than the solution or gel state of CAHS proteins being the sole protective phase, each phase is optimized to protect different enzymes during drying. Using in vitro assays that provide clear functional readouts and allow for precise control over CAHS and client enzyme ratios, we show that the gelled state of CAHS D, a model CAHS protein, promotes the protection of the enzyme lactate dehydrogenase during drying. We find that the opposite is true for the enzyme citrate synthase, with variants of CAHS D that do not gel providing optimal protection to this enzyme. Correlative analysis between protective capacity and sequence/ensemble features of CAHS D variants supports the notion that phase is a major driver of differential enzyme protection. Finally, we show that enhanced water binding is an emergent property of gelation that positively correlates with the protein's ability to protect LDH. These results demonstrate a link between the phase of CAHS proteins and their protective function, providing insights into how CAHS proteins help tardigrades counteract the spectrum of stresses encountered during different stages of drying. Broadly, this study advances our understanding of desiccation tolerance, while providing insights into engineering strategies to tune protein-based excipients to protect specific clients. This study contributes to a broader discussion in the protein field about the functionality of phase behavior and states.
Nguyen et al. (Wed,) studied this question.