Fig. 2.

Construction of protease-based peptide receptors. (A) The fusion of an affinity clamp with an autoinhibited TVMV can occupy four major conformational states, which include subequilibria that respond to the addition of a ligand in either switch-ON or switch-OFF fashions. (B) Summary of linker compositions L1, L2, and L3 that connect the different functional elements. (C–E) Time-resolved protease activity measurements of key mutants that respond to the addition of ligand in switch-OFF, neutral, and switch-ON fashions. (F) Apparent K_ds for the stronger and weaker binding ligand peptides determined by plotting the gradient of TVMV-based activity against the ligand concentration. Protease activities are normalized over the concentration of the ligand-sensing TVMV protease. Protease activities were assayed with 5 μM TVMV substrate peptide.