An ALuc-Based Molecular Tension Probe for Sensing Intramolecular Protein–Protein Interactions
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.
Key wordsBioluminescence Protein–protein interactions Tension probe Bioluminescent imaging Combinational probe
This work was partly supported by JSPS KAKENHI Grants Number 26288088, 16K14051, and 15KK0029.
- 4.Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D (2004) Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci USA 101:12288–12293Google Scholar
- 11.Kim SB, Otani Y, Umezawa Y, Tao H (2007) Bioluminescent indicator for determining protein–protein interactions using intramolecular complementation of split click beetle luciferase. Anal Chem 79:4820–4826Google Scholar
- 16.Banaszynski LA, Liu CW, Wandless TJ (2005) Characterization of the FKBP center dot Rapamycin center dot FRB ternary complex. J Am Chem Soc 127:4715–4721Google Scholar
- 17.Wear MA, Walkinshaw MD (2007) Determination of the rate constants for the FK506 binding protein/rapamycin interaction using surface plasmon resonance: An alternative sensor surface for Ni -nitrilotriacetic acid immobilization of His-tagged proteins. Anal Biochem 371:250–252Google Scholar