Cooperative Binding Domains Enhance Sensitivity and Overcome the Hook Effect in Two-Component Protein Biosensors

Protein biosensors are important tools in research, bioengineering, and diagnostics. Unlike fully integrated single-component biosensors, two-component biosensors are modular and can be rapidly adapted to new targets by replacing binding elements. However, their utility is limited by a "hook effect," where high analyte concentrations lead to dissociation of the signalling complex and signal loss. We address this challenge by utilizing sequential mRNA display selection campaigns to develop high-affinity, cooperative FN3con (monobody) binding domains to Galectin-3, a protein biomarker of heart failure. Two-component biosensors equipped with such cooperative binders were devoid of the hook effect and demonstrated 5-fold higher sensitivity to Galectin-3 compared to systems based on non-cooperative binding domains. High-resolution crystal structures of cooperative and non-cooperative binding domains revealed how interactions between the two domains establish a cooperative Galectin-3 binding interface. The results and experimental approaches presented in this work enable the construction of high-performance biosensors for diverse applications.