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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Veröffentlicht in:Bioconjugate chemistry Jg. 27; H. 2; S. 354
Hauptverfasser: Kim, Sung Bae, Nishihara, Ryo, Citterio, Daniel, Suzuki, Koji
Format: Journal Article
Sprache:Englisch
Veröffentlicht: United States 17.02.2016
Schlagworte:
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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Zusammenfassung: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.
Bibliographie:ObjectType-Article-1
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content type line 23
ISSN:1520-4812
1520-4812
DOI:10.1021/acs.bioconjchem.5b00421