Abstract
Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- CC
- coiled coil
- CCPO
- coiled-coil protein origami
- fLuc
- firefly luciferase
- rLuc
- renilla luciferase
- nLuc
- N-terminal half of firefly luciferase
- cLuc
- C-terminal half of firefly luciferase
- TALE
- DNA-binding domain of TALEN
- TALE10x
- a TALE binding element concatenated ten times
- Kd
- dissociation constant
- RLU
- relative light units
- Tm
- denaturation temperature
- s.d.
- standard deviation
- CD
- circular dichroism
- MRE
- mean residual ellipticity
- ITC
- isothermal titration calorimetry
- SEC
- size-exclusion chromatography
- SEC-MALS
- SEC-multi angle light scattering