Coding

Part:BBa_K3032017

Designed by: Denis Baronas   Group: iGEM19_Vilnius-Lithuania   (2019-10-13)
Revision as of 20:07, 21 October 2021 by Alexandr56 (Talk | contribs)


Gal4_Q-PAS1 repressor

Gal4_Q-PAS1 repressor is a fusion protein which consists of Gal4 domain (from Saccharomyces cerevisiae) and Q-PAS1 (from Rhodopseudomonas palustris). This repressor binds to promoter containing Gal4 DNA binding sequence and dissociates when it is illuminated with NIR light. Bphp1 (BBa_K3032016) and ho1 (BBa_K3032015) proteins required for system to work.

Contribution

Group: LMSU iGEM 2021

We performed the molecular dynamics simulation for Gal4-Q-PAS1 chimeric protein, since there was no data on the stability of the system with Gal4-Q-PAS1 monomers and dimers.

The molecular dynamics was calculated in the OPLS-AA/L force field by LMSU team because LMSU team created an optogenetic system based on this protein and optimized the Gal4-Q-PAS1 monomers and dimers. Once we were sure that the obtained structures deviate insignificantly when calculating the molecular dynamics in water (RMSD < 0.5 nm for 100 ps), and the monomers remain stable, we proceeded to construct dimers.

Molecular dynamics simulation for Gal4-Q-PAS1 monomer
RMSD for Gal4-Q-PAS1 monomer

The Gal4-Q-PAS1 dimers molecular dynamics values were obtained in GROMACS program. The result is reliable if Epot is negative, and on the order of 10^6-10^7 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 Gal4-Q-PAS1 dimer (RMSD < 0.5 nm for 100 ps, Epot = -5.07×e+6 kJ×mol-1, Etot = -4.25×e+6 kJ×mol-1). At the same time, the BphP1-BcLOV4 lowest total score, calculated using PyDockWEB, for this model is -124.942 kJ×mol-1. Total score is calculated based on electrostatics, desolvation energy and limited van der Waals contribution.

Molecular dynamics simulation for Gal4-Q-PAS1 dimer
RMSD for Gal4-Q-PAS1 dimer
The system's potential energy for Gal4-Q-PAS1 dimer
The system's total energy for Gal4-Q-PAS1 dimer

Our findings confirm that the systems with Gal4-Q-PAS1 monomers and dimers are stable and work correctly.

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 577
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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