Uncovering the molecular grammar of IDPs in desiccation tolerance.

Abstract

Tardigrades, aka water bears, are remarkable for their ability to survive many environmental stresses, including desiccation (extreme drying). Desiccation tolerance in tardigrades depends on the accumulation of several families of intrinsically disordered proteins (IDPs), of which Cytosolic Abundant Heat Soluble (CAHS) proteins are essential to survive desiccation. Beyond tardigrades, CAHS proteins can also improve desiccation tolerance in heterologous systems and prevent desiccation-induced protein damage in vitro. However, what sequence features drive CAHS protein function is unknown. Understanding the CAHS sequence-ensemble-function relationship will shed light on the molecular mechanisms by which tardigrades survive extreme stresses. Using a model CAHS protein, CAHS D, we show that helix formation is important for its protective capacity. Utilizing a rational mutagenesis approach, we find that an internal linker region is the protective portion of CAHS D. We find that within this linker region specific sequence features such as charge identity, hydrophobicity, and amino acid organization dictate specific biophysical properties, such as helix formation, and are correlated to the desiccation protection in vitro. However, small helical peptides by themselves cannot confer the same levels of protection as CAHS D. This indicates that along with helicity, other features likely play a role in determining the protective capacity of CAHS D. These findings support the existing theory of helicity provided protection by different stress-tolerant LEA proteins. Understanding CAHS D's sequence-structure-function relationship will not only provide insights into how IDP sequences dictate their conformational dynamics and functional properties, but can also be useful for real-world applications like designing stress-tolerant crops or helping increase the shelf-life of sensitive biomedical materials in a dry state.