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A yeast sensor of ligand binding

Abstract

We describe a biosensor that reports the binding of small-molecule ligands to proteins as changes in growth of temperature-sensitive yeast. The yeast strains lack dihydrofolate reductase (DHFR) and are complemented by mouse DHFR containing a ligand-binding domain inserted in a flexible loop. Yeast strains expressing two ligand-binding domain fusions, FKBP12-DHFR and estrogen receptor-α (ERα)-DHFR, show increased growth in the presence of their corresponding ligands. We used this sensor to identify mutations in residues of ERα important for ligand binding, as well as mutations generally affecting protein activity or expression. We also tested the sensor against a chemical array to identify ligands that bind to FKBP12 or ERα. The ERα sensor was able to discriminate among estrogen analogs, showing different degrees of growth for the analogs that correlated with their relative binding affinities (RBAs). This growth assay provides a simple and inexpensive method to select novel ligands and ligand-binding domains.

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Figure 1: DHFR constructs.
Figure 2: Growth of P66LdhFK in the presence of FK506.
Figure 3: Responses of yeast expressing dhER or P66LdhFK to estrogen or FK506.
Figure 4: ERα-binding domain variants.
Figure 5: Chemical screen.

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Acknowledgements

We thank Jurgen Dohmen, Stephen Michnick, Randall Morse, and Carol Sibley for providing constructs and strains. We also thank Paul Clemons and Stuart Schreiber for the protocol for extraction of FK506, Eleanor Hankins for helpful advice, Matt Kennedy for help with Figures 2B and 5B, and Mike DeVit, Tony Hazbun, Tina Guina, Visvanathan Ramamurthy, and David Baker for useful comments. This work was supported by National Institutes of Health grant 1F32GM20532-01 to C.L.T. S.F. is an investigator of the Howard Hughes Medical Institute.

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Correspondence to Stanley Fields.

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Tucker, C., Fields, S. A yeast sensor of ligand binding. Nat Biotechnol 19, 1042–1046 (2001). https://doi.org/10.1038/nbt1101-1042

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