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Correlation between ligand-receptor affinity and the transcription readout in a yeast three-hybrid system.

The yeast two-hybrid assay has proven to be a powerful method to detect protein-protein interactions as well as to derive genome-wide protein interaction maps. More recently, three-hybrid assays have emerged as a means to detect both protein-RNA and protein-small molecule interactions. Despite the routine use of the two-hybrid assay and the potential of three-hybrid systems, there has been little quantitative characterization to understand how the strength of the protein interaction correlates with transcription activation. It is not known if the additional interaction in three-hybrid systems compromises the sensitivity of the system. Thus, here, we set out to determine the K(D) cutoff of a small molecule three-hybrid system and to determine if there is a correlation between the K(D) and the levels of transcription activation. A series of mutations to FK506-binding protein 12 (FKBP12) were designed to vary the affinity of this protein for the small molecule synthetic ligand for FK506-binding protein 12 (SLF). These FKBP12 variants were overexpressed and purified, and their K(D)'s for SLF were measured using a fluorescence polarization assay. Then the levels of transcription activation in a Mtx-DHFR yeast three-hybrid system were determined for these variants using a lacZ reporter gene. The K(D) cutoff of the Mtx yeast three-hybrid system is found to be ca. 50 nM. Further, the levels of transcription activation correlate with the strength of the binding interaction, though the dynamic range is only 1 order of magnitude. These results establish that the three-hybrid assay has the requisite sensitivity for drug discovery. However, the small dynamic range highlights a limitation to equilibrium-based assays for discriminating interactions based on affinity.