title: Mechanism-based design of a photoactivatable firefly luciferase.
abstract: We developed a photoactivatable firefly luciferase (pfLuc) whose activation can be controlled by light. A photocaged Lys analogue was site-specifically incorporated into fLuc to replace its key catalytic Lys residue, Lys529, rendering fLuc inactive until light-triggered removal of the caging group. This photoinduced gain of luminescence provides a facile approach for assessing the photolysis efficiency of this valuable photosensitive Lys analogue within the context of its carrier protein in vitro and in living cells. We further took advantage of the spatial and temporal activation feature of pfLuc for intracellular measurement of labile ATP levels without impairment of cellular physiology.
authors.affiliation: Shenzhen Key Lab of Nano-Micro Material Research, School of Chemical Biology and Biotechnology, Shenzhen Graduate School of Peking University, Shenzhen, 518055, China.
authors.forename: Jingyi
authors.initials: J
authors.lastname: Zhao
authors.forename: Shixian
authors.initials: S
authors.lastname: Lin
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Peng R
authors.initials: PR
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D048888:Fireflies; D057809:HEK293 Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D055668:Photochemical Processes
chemical_list: D002352:Carrier Proteins; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly; D008239:Lysine