Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

ABSTRACT: Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been

SUBMITTER: Colee CM 

PROVIDER: S-EPMC10956436 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature