High-throughput characterization and engineering of brighter luciferases

Project Summary Bioluminescence Imaging (BLI) is a non-invasive method that enables the monitoring of in vitro and in vivo biological events, cells, and molecules. In BLI, the bioluminescent enzyme luciferase is used as a reporter. The detection limit, versatility, and temporal and spatial resolution of BLI assays consistently exceeds fluorescence-based methods in the context of deep tissue or whole animal imaging applications. Thus, BLI is a critical imaging tool for whole animal imaging applied to both the study of diseases as well as in the evaluation of potential therapies. The most frequently used enzyme for BLI is Firefly luciferase (F-Luc), the luciferase enzyme from the North American firefly (Photinus pyralis). Currently, F-Luc is highly effective for imaging single events or molecules; however, engineering of F-Luc is needed to improve imaging applications where multiple events are simultaneously monitored. In our prior R15 award, we developed novel biochemical methods that can rapidly assess the effect of hundreds to thousands of amino acid changes on F-Luc’s function and then applied those discoveries to create improved F-Luc mutant enzymes. In this proposal, we apply our previously developed methodology to ask a new biochemical question that will lead to improved engineered enzymes. Specifically, while it is well known that the C-terminal domain (CTD) of F-Luc is functionally important to the protein, it has been rarely applied for engineering improved enzymes. We hypothesize that this is due to the flexibility of this region of the enzyme, which makes it challenging to know which regions of the protein are best to mutate to create improved variants. Here, we propose to systematically probe this region of the protein to identify amino acid positions that are important for key parameters; we will then directly apply this to create improved F-Luc probes. In Aim 1, we will discover amino acid changes that inactivate the protein, improving our biochemical understanding of the functional role of the C-terminal domain. In Aim 2, we will discover amino acid changes that change the color of F-Luc emission and leverage our discoveries to create an improved variant of a current leading multicomponent imaging probe. In Aim 3, we will discover amino acid changes that improve the light output of another multicomponent imaging probe, Pistachio. In addition to creating improved probes for multicomponent imaging, this work will shed light on the function of this understudied domain and demonstrate that the C-terminal domain can be used to engineer improved probes.