Figure 1. Y2H selected PAs bind to Pup with nanomolar affinity.

(a) Yeast-Two-Hybrid selection of PAs. 1) Pup, fused to the Gal4 DNA-binding domain (BD), is expressed in yeast containing several reporter genes that possess sites for the BD in their promoters. The reporter genes are not expressed if the activation domain (AD) is not present. 2) Pup, fused to the BD, is expressed concurrently with a randomized PA loop in a thioredoxin (Thio) scaffold¹³, fused to the AD. Binding between Pup and a PA activates transcription of reporter genes in cells, while reporter gene transcription is suppressed in cells where Pup and a PA do not interact. (b) PA sequences selected through Y2H screening and the Pup-binding affinity of each sequence. (c) Pup-PA binding isotherms. Changes in native PA fluorescence intensity are plotted vs. the concentration of Pup. Thio scaffold¹³ was used as a negative control and showed no changes in fluorescence upon titration with Pup. The data represent mean values and are fitted as described in Methods. All experiments were performed in triplicate.