Photoactive Split Green Fluorescent Protein: Engineering a New Optogenetic and Imaging System

Abstract

Visible light is a non-invasive reagent with the potential to control molecular processes with high spatiotemporal resolution, offering a unique approach to biotechnology. Many light-sensitive small molecules and proteins have been used to photochemically control, or “cage”, biologically active molecules. Initially suppressed, the molecule's activity is recovered with light. As versatile in vivo tools, these photocaged molecules have applications in drug delivery, gene regulation, biosensing, and imaging. However, current photocaging systems are limited by large size, restrictive cellular localization, toxic excitation wavelengths, and the need for exogenous chromophores. While green fluorescent proteins (GFPs) are well-established imaging tools, recent findings indicate split GFPs have the potential to function as genetically-encoded photocaging reagents without the limitations of other systems [1]. To optimize the light sensitivity of split GFP photocaging with directed evolution, we designed and tested a functional assay that directly links split GFP's photoresponse to transcription of a reporter gene. We then developed a complex, two-part high-throughput screen based on reporter gene expression to select for improved light sensitivity among split GFP variants. A new multipurpose protein engineering platform called μSCALE (microcapillary single-cell analysis and laser extraction) allowed for the rapid visualization of mutant libraries and subsequent isolation of variants with the desired phenotype [2]. In addition to the practical applications of an improved photocage, characterization and mutational analysis of the evolved, light-activated split GFP variants provide insight into the coupling between the chromophore excited state and protein dynamics, which broadly impacts our understanding of diverse photobiological systems. 1. Lin, C.-Y. et al. Proc. Nat. Acad. Sci. 2017, 114 (11), 2146-2155. 2. Chen, B. et al. Nat. Chem. Biol. 2016, 12 (2), 76–81.