Part:BBa_K3032016

BphP1 (bacterial phytochrome photoreceptor)

BphP1 contain an N-terminal photosensory core domain (PCD) and C-terminal effector domain that regulates the biological response. BphP1 has phytochrome (PHY) motif with the chromophore biliverdin IXα (BV) covalently bound near the N-terminus. Most of the BphPs adopt inactive ground state with maximal absorption between 690 and 710 nm.

Canonical BphPs adopt an inactive ground (i.e., dark-adapted) conformation with maximal absorption between 690 and 710 nm. Upon exposure to red or short wavelength NIR light, these BphPs switch to the biologically active Pfr conformation, with maximal absorption between 750 and 760 nm. Canonical BphPs revert from Pfr to Pr in milliseconds after exposure to NIR light or in minutes to hours by thermal reversion in the dark. The bacteriophytochrome photoreceptor 1 (BphP1), found in several purple photosynthetic bacteria, senses NIR light and activates transcription of photosystem promoters by binding to and inhibiting the transcriptional repressor PpsR2.

We used truncated PpsR2 variant - QPas1 and fusing it to DNA binding domains of Gal4 from S. cerevisiae or LexA from Bacillus subtillis. DNA-binding domains not from E. coli and protein sensing NIR light opens a possibility for development of fully orthogonal optogenetic system.

Contribution

Group: iGEM21_LMSU

We performed the molecular dynamics simulation for BphP1 protein, since there was no data on the stability of the system with BphP1 dimers.

The molecular dynamics of dimers was calculated in the OPLS-AA/L force field for BphP1 dimer in BBa_K4044001. The BphP1 dimers molecular dynamics values were obtained in GROMACS program. The result is reliable if Epot is negative, and on the order of 10⁶-10⁷ for proteins in water, depending on the system size. During the energy minimization phase, the system maximum force should not exceed 1000 kJ mol^-1 nm^-1. Calculations of molecular dynamics and interaction kinetics demonstrated stability of BphP1 complex (RMSD < 0.5 nm for 100 ps, Epot = -1.6825×10⁷ kJ mol^-1, Etot = -1.411×10⁷ kJ mol^-1) which means the system with BphP1 dimer is stable and works correctly.

Molecular dynamics simulation for BphP1 dimer with unshortened amino acid sequence

RMSD for BphP1 dimer with unshortened amino acid sequence

The system's potential energy for BphP1 dimer with unshortened amino acid sequence

The system's total energy for BphP1 dimer with unshortened amino acid sequence

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1613
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 669
  Illegal AgeI site found at 2056
  
• 1000
  COMPATIBLE WITH RFC[1000]