(307f) Directed Evolution of Photoswitchable Enzymes

Protein light switches—or optogenetic actuators—provide a powerful means of placing enzymes under optical control. Most contemporary strategies for using optogenetic actuators, however, suffer from several limitations: (i) They disrupt the structure of the enzyme and can thus interfere with native enzyme-substrate interactions. (ii) They yield dynamic ranges (DR, the ratio of activity in the off- and on-states) that are low and/or difficult to enhance through systematic optimization. (iii) They are modulated by blue or green light, which have heightened phototoxicities, suffer from short biological penetration depths, and, because of their spectral similarity, limit actuation to individual signaling events. (iv) They tend to be limited to specific subsets of enzymes (notably, kinases or recombinases). In this presentation, I will discuss our efforts to address these shortcomings with synthetic biology and directed evolution. In brief, we have developed an approach for using microbial systems to evolve light-sensitive enzymes, and we are using it to extend optogenetic actuation to new enzymes and new colors of light. Our initial efforts focused on protein tyrosine phosphatases, a broadly influential class of signaling enzymes. In early work, we developed a photoswitchable variant of PTP1B with a low—yet strangely influential—dynamic range (2.5-fold). Our approach, which exploits a selection-counterselection screen to find photoswitchable variants, has allowed us to identify beneficial mutations in photoswitchable variants of PTP1B and promising starting points for the assembly of red-light-sensitive enzymes. Broadly, the constructs developed with our platform will support (and, as I will mention, are supporting) detailed studies of intracellular signaling networks in living cells.