Photo Control of Protein Activity Using Genetically Encoded Photoswitches

Many biological processes are governed by protein activities that are spatially and temporally regulated. Where (i.e.: in a particular cell type or subcellular organelle) and when (i.e.: in relation to a cue, or for what duration) a protein is active can dictate its biological role. Photo-control, in which a target protein is made sensitive to light, can be used to precisely match the spatial and temporal patterning of protein activity in living cells. However, photo-control of protein activity often requires extensive protein engineering. This thesis describes three unique approaches to achieve photo-control of protein activity, as well as biophysical data to guide design of new optogenetic tools based on a novel red light-switchable protein. Chapter 2 describes the design and characterization a photo-switchable fusion protein, in which a coiled coil forming motif (Ehelix) is inserted into a circular permutant of photoactive yellow protein (c-PYP). c-Ehelix-PYP forms domain-swapped dimers that bind tightly to a peptide motif, whereas the monomers are inactive, enabling a novel mechanism for photo-control. Chapter 3 describes a blue light controlled dominant negative inhibitor to the transcription factor CREB (opto-DN-CREB). opto-DN-CREB was designed by fusing a transcription factor inhibitor (A-CREB) to PYP and making complementary mutations to PYP using the software suite ROSETTA. We demonstrate that opto-DN-CREB binds selectively to CREB in the dark, and that photo-control of CREB activity in cells is possible. Chapter 4 uses phage display to select for light-switchable protein interactions. Small proteins that bind selectively to the light or dark states of PYP and Avena sativa light-oxygen-voltage domain (asLOV) were selected, characterized extensively in vitro. They were then used to control subcellular localization of a fluorescent protein reporter in eukaryotic cells. Chapter 5 describes the biophysical characterization of a small, soluble red light-switchable protein (GAF3). NMR characterization shows that GAF3 undergoes conformational changes upon red light irradiation and suggest key residues involved. Together, this work adds to the available optogenetic toolkit and to our fundamental knowledge in the engineering of photo-switchable proteins.

Department dc:contributor.department

Chemistry

Year dc:date.issued

2018

Author dc:creator

• Reis, Jakeb

Advisor dc:contributor.advisor

• Woolley, Andrew

• Optogenetics
• Protein design
• Protein folding
• Protein structure

Handle dc:identifier.uri

http://hdl.handle.net/1807/95724

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/95724