Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells

Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in whi...

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Published in:	Bioconjugate chemistry Vol. 27; no. 2; p. 354
Main Authors:	Kim, Sung Bae, Nishihara, Ryo, Citterio, Daniel, Suzuki, Koji
Format:	Journal Article
Language:	English
Published:	United States 17.02.2016
Subjects:	Amino Acid Sequence Animals Biomechanical Phenomena Cercopithecus aethiops COS Cells Humans Luciferases, Renilla - chemistry Luciferases, Renilla - metabolism Luminescent Measurements - methods Molecular Probes - chemistry Molecular Probes - metabolism Molecular Sequence Data Protein Binding Protein Interaction Mapping - methods Protein Structure, Tertiary Renilla - chemistry Renilla - enzymology Tacrolimus Binding Proteins - chemistry Tacrolimus Binding Proteins - metabolism TOR Serine-Threonine Kinases - chemistry TOR Serine-Threonine Kinases - metabolism
ISSN:	1520-4812, 1520-4812
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Abstract	Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.
AbstractList	Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects. Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.
Author	Kim, Sung Bae Nishihara, Ryo Citterio, Daniel Suzuki, Koji
Author_xml	– sequence: 1 givenname: Sung Bae surname: Kim fullname: Kim, Sung Bae organization: Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST) , 16-1 Onogawa, Tsukuba 305-8569, Japan – sequence: 2 givenname: Ryo surname: Nishihara fullname: Nishihara, Ryo organization: Department of Applied Chemistry, Faculty of Science and Technology, Keio University , 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan – sequence: 3 givenname: Daniel surname: Citterio fullname: Citterio, Daniel organization: Department of Applied Chemistry, Faculty of Science and Technology, Keio University , 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan – sequence: 4 givenname: Koji surname: Suzuki fullname: Suzuki, Koji organization: Department of Applied Chemistry, Faculty of Science and Technology, Keio University , 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
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SubjectTerms	Amino Acid Sequence Animals Biomechanical Phenomena Cercopithecus aethiops COS Cells Humans Luciferases, Renilla - chemistry Luciferases, Renilla - metabolism Luminescent Measurements - methods Molecular Probes - chemistry Molecular Probes - metabolism Molecular Sequence Data Protein Binding Protein Interaction Mapping - methods Protein Structure, Tertiary Renilla - chemistry Renilla - enzymology Tacrolimus Binding Proteins - chemistry Tacrolimus Binding Proteins - metabolism TOR Serine-Threonine Kinases - chemistry TOR Serine-Threonine Kinases - metabolism
Title	Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells
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