P-glycoprotein is fully active after multiple tryptophan substitutions

► Substitution of the eleven native Trps in the P-glycoprotein multidrug transporter. ► Several Trps at the membrane interface are critical for substrate polyspecificity. ► Trps in the coupling helices are important for interdomain communication. ► Single Trp mutants can be combined while maintaining drug polyspecificity. ► Multi-Trp mutant protein is fully active with reduced intrinsic fluorescence.