Part:BBa_K4044009

BphP1-BcLOV4_QPAS1-Gal4 optogenetic system

There are several states of the ontogenetic system. Under the influence of far red light, BphP1 part of the chimeric protein BphP1-BcLOV4 becomes active and binds to QPAS1-Gal4. This leads to the expression of target gene. Under the influence of red light, BphP1 is inactivated, QPAS1-Gal4 remains in the dimerized state. This leads to inhibition of transcription. At the same time, there may be residual activity of BphP1 and expression of target genes, which we do not need, therefore, under the influence of blue light, the entire BphP1-BcLOV4 complex binds to the lipid membrane, which completely eliminates the possibility of undesirable expression.

File:T--LMSU--eng13.png/800px-T--LMSU--eng13.png

Scheme of composed optogenetic system with BphP1 and BcLOV4

The molecular dynamics of dimers was also calculated in the OPLS-AA/L force field. The BphP1 dimers molecular dynamics values were obtained in GROMACS program. We proved the stability of the BphP1-BcLOV4 and Gal4-Q-PAS1 structures binding upon 760 nm light stimulation. We compared these structures with the shortened variants of BphP1-BcLOV4 and Gal4-Q-PAS1 and turned out that both complexes were stable (RMSD < 0.5 nm for 100 ps).

File:T--LMSU--Modeling-Modeling-othBphP1-Gal4.mp4

RMSD for BphP1-BcLOV4 and Gal4-Q-PAS1 complex with full-length amino acid sequences

The system's potential energy for BphP1-BcLOV4 and Gal4-Q-PAS1 complex with full-length amino acid sequences

The system's total energy for BphP1-BcLOV4 and Gal4-Q-PAS1 complex with full-length amino acid sequences

However, the complex of chimeric proteins with a full-length amino acid sequence had a lower potential energy but higher PyDockWEB total score (S) (Epot = -8.11×10⁶ kJ mol^-1 and S = -35.312 kJ mol^-1, respectively) than the complex of shortened proteins (Epot = -7.185×10⁶ kJ mol^-1 and S = -36.828 kJ mol^-1, respectively). As expected, the Gal4-Q-PAS1 orientation relative to BphP1-BcLOV4 in two variants prevented the BphP1-BcLOV4 and Gal4-Q-PAS1 dimers formation. PyDockWEB total score is calculated based on electrostatics, desolvation energy and limited van der Waals contribution.

File:T--LMSU--Modeling-ourBphP1-Gal4.mp4

RMSD for BphP1-BcLOV4 and Gal4-Q-PAS1 complex with shortened amino acid sequences

The system's potential energy for BphP1-BcLOV4 and Gal4-Q-PAS1 complex with shortened amino acid sequences

The system's total energy for BphP1-BcLOV4 and Gal4-Q-PAS1 complex with shortened amino acid sequences

Our findings confirm that this optogenetic system can be implemented and can work more efficiently with full-length chimeric proteins.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 815
  Illegal PstI site found at 1112
  Illegal PstI site found at 1559
  Illegal PstI site found at 2608
  Illegal PstI site found at 2852
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 815
  Illegal PstI site found at 1112
  Illegal PstI site found at 1559
  Illegal PstI site found at 2608
  Illegal PstI site found at 2852
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 301
  Illegal BamHI site found at 1876
  Illegal BamHI site found at 4029
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 815
  Illegal PstI site found at 1112
  Illegal PstI site found at 1559
  Illegal PstI site found at 2608
  Illegal PstI site found at 2852
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 815
  Illegal PstI site found at 1112
  Illegal PstI site found at 1559
  Illegal PstI site found at 2608
  Illegal PstI site found at 2852
  Illegal NgoMIV site found at 457
  Illegal NgoMIV site found at 1208
  Illegal NgoMIV site found at 1705
  
• 1000
  COMPATIBLE WITH RFC[1000]