Conditional mislocalization to regulate protein function

The ability to regulate protein function with small molecules provides great power to temporally, reversibly and dose‐dependently control cellular processes. One method of controlling function is through conditional regulation of protein localization. To accomplish this, a system has been developed that takes advantage of the small molecule rapamycin (rap) that binds both the FK506 Binding Protein (FKBP, 107 aa) and the FKBP‐rap binding domain of the cell cycle protein TOR (FRB, 89 aa) simultaneously. Through fusions to FKBP or FRB the localization of a protein of interest can be controlled by the presence or absence of rap. A major disadvantage of this system is that rap is toxic to eukaryotic cells, so a derivative was synthesized with a large, bulky group in the FRB‐binding region, abrogating binding of the small molecule to its native target TOR. For further technology development, a compensatory cavity in FRB must be generated to accommodate the bulky derivative. To this end, a yeast three‐hybrid assay has been developed to conditionally express GFP under control of an inducible promoter, in which the DNA‐binding and activation domains of a transcription factor are colocalized only in the presence of the rap derivative. Using this yeast three‐hybrid assay an FRB library has been screened in the presence of the derivative to find a protein with a compensatory hole. Subsequent FACS‐based sorting has selected for potential mutants capable of binding the derivative. This bio‐orthogonal ternary complex will be used to mislocalize target proteins in biological systems. This work is funded by NIH GM068589.