Genetically Encoded Photoactivatable Proteins for Interrogating and Guiding Biological Function in 4D

Abstract

Proteins play a critical role in directing cell fate within living systems and their spatial and timed presentation results in distinct outcomes in various biological processes. In an aim to mimic complex protein signaling events, various techniques have been developed to control protein function in and out of biomaterials, as well as within living cells. This thesis focuses on exploiting chemical advances to genetically encode light-sensitive non-canonical amino acids into the active sites of proteins to near instantaneously control their activity in solution, living cells, and biomaterials. To accomplish this, this work introduces a method to spatiotemporally control the functional assembly of split proteins using a genetically encoded light-activated SpyLigation (LASL) reaction. For proteins not amenable to splitting, a second platform is introduced for directly photoactivating proteins through the incorporation of a photocaged amino acid at their catalytic sites. Photolithographic techniques were used to spatiotemporally control the activity of these proteins in hydrogels and in living cells. Both platforms are highly versatile, offering exciting potential in probing and directing cell fate for biological study and therapeutic development.