Light switchable protein engineering
Photoactive yellow protein engineering
Frameshifted and circular permutants
PYP is a very malleable protein. We have made circular permutants, versions with loop insertions, and a version with a duplicated beta strand. These provide novel scaffolds for tool design and new insights into protein folding in a general sense.
Opto-DN-CREB
In collaborative work the Karanicolas lab, we designed a PYP fusion to a dominant negative version of the CREB transcription factor. In the dark the dominant negative is distorted and inactive allowing CREB to interact with its endogenous promoter. Conversely under blue light PYP undergoes conformational changes allowing the dominant negative CREB to inhibit CREB mediated initiation of transcription. This was developed in vitro, then tested in cells, then ultimalety in mice by the Josselyn lab. In mice, spatiotemporal control of CREB actitivy with light allows control of fear learning.
Duplicating a beta strand in PYP creates a light switchable metamorphic protein.
In the dark opto-DN-CREB (cyan) is inactive and CREB (green) binds DNA; Under blue light opto-DN-CREB (white) binds CREB and sequesters it.
BoPD - binder of PYP dark-state
In collaboration with the Uppalapati group at the University of Saskatchewan we used phage display to develop binders to both the light and dark state conformations of photoactive yellow protein (PYP) and the light-oxygen-voltage (LOV) photoswitchable proteins. Our results show that phage display can be used to generate binding partners to the different conformations of photoswitchable proteins with no known binding partners as in the absence of structural information. These work in vitro and in cells.
See a video of PYP/BoPD in action>>
LOV protein engineering
We have also used LOV domains as photoswitchable units in designed optogenetic tools. We developed small protein binding partners to LOV dark and light states using phage display. By developing a yeast-based screening system, we discovered LOV-based inhibitors of eukaryotic translation initiation.
GAF protein engineering
GAF-only domains or cyanobacteriochromes offer a spectacular array of photoswitchable domains that respond to all colours of light from UV to far-red. These domains bind bilin pigments such as PCB or biliverdin. Using our phage display approach in collaboration with the Uppalapati lab, we have discovered selective binding partners for green, orange, red, and far-red states of CBCRs.
BoPD (ribbons) bound to the dark state of PYP
A LOV-4EBP hybrid for controlling translation initiation
Green, orange, red and far-red tools built from cyanobacteriochromes