Circularly permutated bioluminescent probes for illuminating ligand-activated protein dynamics.
Engineered bioluminescent enzymes provide a creative approach for illuminating the intracellular molecular events. We demonstrate a new strategy for molecular imaging of bioactive small molecules using circularly permutated luciferases (cpLuc), derived from Gaussia princeps (GLuc), Photinus pyralis (FLuc), and Pyrearinus termitilluminans (CBLuc): (i) the luciferases were first dissected into two fragments, (ii) the original N- and C-termini were linked with a GS linker and the new termini were created in an appropriate site, and (iii) the new ends were correspondingly linked with proteins of interest, e.g., a ligand-binding domain (LBD) and an LBD-recognition protein. When the ends of the cpCBLuc were linked with LBD of estrogen receptor (ER) and Src homology 2 domain of Src (SH2 of Src), the estrogen can trigger an intramolecular ER LBD-Src SH2 binding. This assembly subsequently provokes an approximation of the adjacent fragments of cpCBLuc recovering the enzyme activity. These probes were surprisingly stable in the mammalian cells and largely exhibited a decreased background luminescence (e.g., 1000 times in case of cpGLuc) and improved signal-to-noise ratios, compared with the non-CP indicators. This study offers a new strategy for luciferase-aided probing systems. Our study is the first to fabricate circular permutation (CP) in luciferases for tracing the molecular dynamics of proteins.
Kim
Sung Bae
SB
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba 305-8569, Japan.
Sato
Moritoshi
M
Tao
Hiroaki
H
D000595:Amino Acid Sequence; D000818:Animals; D028023:Arecaceae / Q000201:enzymology; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D001517:Coleoptera / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D008027:Light; D008156:Luciferases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D049409:Luciferases, Firefly / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D015202:Protein Engineering; D013379:Substrate Specificity
D008024:Ligands; D008156:Luciferases; D049409:Luciferases, Firefly