Fig. 1: Protein structure-guided design of high-affinity PYR1-based cannabinoid sensors.

a, The 19 side chains of residues in PYR1’s binding pocket targeted for double-site mutagenesis (DSM) are shown along with ABA (yellow) and HAB1’s W385 ‘lock’ residue and water network (3QN1). b, Sensor evolution pipeline. The PYR1 library was constructed by NM^12,15 in two subpools, one using single-mutant oligos and another using double-mutant oligo pools. The combined pools were screened for sensors using Y2H growth selections in the presence of a ligand of interest. c, Representative screen results. The DSM library was screened for mutants that respond to the synthetic cannabinoid JWH-015 yielding five hits that were subsequently optimized by two rounds of DNA shuffling to yield PYR1^JWH-015, which harbors four mutations. The yeast two-hybrid (Y2H) staining data show different receptor responses to JWH-015 by β-galactosidase activity.