Yeast Two-Hybrid Screening of Photoswitchable Protein–Protein Interaction Libraries.

Although widely used in the detection and characterization of protein–protein interactions, Y2H screening has been under-used for the engineering of new optogenetic tools or the improvement of existing tools. Here we explore the feasibility of using Y2H selection and screening to evaluate libraries of photoswitchable protein–protein interactions. We targeted the interaction between circularly permuted photoactive yellow protein (cPYP) and its binding partner binder of PYP dark-state (BoPD) by mutating a set of four surface residues of cPYP that contribute to the binding interface. A library of ~ 10,000 variants was expressed in yeast together with BoPD in a Y2H format. An initial selection for the cPYP/BoPD interaction was performed using a range of concentrations of the cPYP chromophore. As expected, the majority (> 90% of cPYP variants) no longer bound to BoPD. Replica plating was then used to evaluate the photoswitchability of the surviving clones. Photoswitchable cPYP variants with BoPD affinities equal to, or higher than, native cPYP were recovered in addition to variants with altered photocycles and binders that interacted with BoPD as apo-proteins. Y2H results reflected protein–protein interaction affinity, expression, photoswitchability, and chromophore uptake, and correlated well with results obtained both in vitro and in mammalian cells. Thus, by systematic variation of selection parameters, Y2H screens can be effectively used to generate new optogenetic tools for controlling protein–protein interactions for use in diverse settings. Unlabelled Image • A library of ~ 10,000 variants of photoactive yellow protein (PYP) was evaluated for binding to a partner protein Binder of PYP-Dark (BoPD) using a yeast two-hybrid approach. • By varying parameters of the two-hybrid selection, distinct photoswitchable behaviors were recovered including PYP variants with altered affinity, kinetics, and apo-state binding. • Phenotypes observed in yeast two-hybrid assays correlate well with results obtained in vitro and in mammalian cells. [ABSTRACT FROM AUTHOR]