Photoswitchable Control of Protein-Protein Interactions: Development of Light-Inducible Ligands

Abstract

At a time when the use of protein therapeutic molecules in medicine is growing rapidly, it is critical to evaluate these molecules and continue to improve them as new therapies enter the market. These proteins, ranging from antibodies for autoimmune disease to novel cancer therapies, have provided a tremendous benefit for patients; however, they can cause serious and life-threatening adverse effects as well. Therefore, research into how to mitigate such adverse effects is ongoing and important. One novel approach is the use of genetic engineering and light, coined optogenetics, to modulate protein structure and activity. This project investigates use of the LOV domain to confer light-inducible activity onto protein nanobodies, where light-driven nanobody-protein binding alterations are studied in yeast 2 hybrid (Y2H) and in vitro. Related mammalian cell results by my postdoctoral mentor, Agi of the Toettcher lab, are also reported. This project set out to achieve dual goals: 1. Construct a generalizable and light-inducible series of nanobodies screened by Y2H experiments 2. Compare in vivo and in vitro light-inducible nanobody binding via size-exclusion chromatography Overall, this thesis was able to achieve these goals and attain positive results, constructing a series of nanobodies displaying light-inducible activity in Y2H. However, the results from Agi’s mammalian screen contrast those observed in our Y2H experiments. The mammalian results were later validated with the in vitro experiments. Regarding the first goal, several GFP nanobodies containing the LOV domain were found to display significantly higher growth in the light than in the dark in the Y2H screen prior to initiation of this project. Based on these results, similar experiments were carried out for this project with mCherry and EGFR nanobodies. One nanobody in particular for each of these proteins displayed much higher growth in the light than in the dark for the Y2H screen. Importantly, the light-inducible LOV domain was inserted at the same site within the constant region of all three nanobodies, implying generalizability. Regarding the second goal, size-exclusion chromatography was used to study nanobody binding in the light and the dark. While few binding differences were observed for the control (no LOV domain) nanobody, mCherry-nanobody binding was observed in the dark for TK74 but not in the light. These results support those observed in the mammalian cell studies. Future investigation includes determining the reason behind observed differences in the Y2H and mammalian screens, as well as the development of monobodies for these experiments as an alternative to nanobodies.