Curated Optogenetic Publication Database

Abstract: Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.

Abstract: 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 BoPD (binder of PYP dark state) 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 the 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.