Intrinsically Disordered Regions in CAHS Proteins Govern Self-assembly and Enzyme Stabilization Against Multiple Stresses

Cytosolic Abundant Heat Soluble (CAHS) proteins are produced by extremotolerant tardigrades as a protective mechanism to stabilize cellular components, such as enzymes, in response to environmental stress. However, the molecular principles governing their protective function have only begun to be elucidated. Herein, we analyze nine CAHS proteins containing intrinsically disordered regions (IDRs) of different lengths to investigate their key role in self-assembly and enzyme stabilization. We show that CAHS proteins with a longer N-terminal IDR present a higher propensity for self-assembly at nano- and macro-scale. The propensity for self-assembly also correlates with their ability to stabilize enzymes, with CAHS proteins containing longer IDRs exhibiting up to 10-fold increase in enzyme stability when exposed to desiccation stress. Beyond desiccation, we reveal new stabilizing roles of CAHS assemblies under a broad variety of abiotic stresses, including extreme pH conditions, high temperatures, addition of organic co-solvents, long-term storage, and long operational times. This concept is further demonstrated in a bi-enzymatic cascade reaction composed of an ADH (Alcohol dehydrogenase) and a NOX (NADH oxidase) for the synthesis of a valuable natural product (peryllaldehyde). The addition of CAHS proteins resulted in a 3-fold increase in product formation compared to the bi-enzymatic cascade not supplemented with these proteins. Our findings not only reveal new structural insights into the functional roles of CAHS proteins but also introduce a new bioinspired platform to overcome the challenges of enzyme stability for biotechnological applications.