title: An ALuc-Based Molecular Tension Probe for Sensing Intramolecular Protein-Protein Interactions.
abstract: Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. The present protocol demonstrates an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. A unique design of single-chain probes was fabricated, in which a full-length artificial luciferase (ALuc(®)) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. A molecular tension probe comprising ALuc23 greatly enhances the bioluminescence in response to varying concentrations of rapamycin, and named "tension probe (TP)." The basic probe design can be further modified towards eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "combinational probe." TPs may become an important addition to the tool box of bioassays in the determination of protein dynamics of interest in mammalian cells.
authors.affiliation: Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, 305-8569, Ibaraki, Japan. kimu-sb@aist.go.jp.
authors.forename: Sung-Bae
authors.initials: SB
authors.lastname: Kim
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Kanagawa, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Nishihara
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Kanagawa, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D008024:Ligands; D049410:Luciferases, Renilla / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D008024:Ligands; D015335:Molecular Probes; D049410:Luciferases, Renilla
keywords: Bioluminescence; Bioluminescent imaging; Combinational probe; Protein–protein interactions; Tension probe