Coding

Part:BBa_K4728008

Designed by: Igor Serafini   Group: iGEM23_Queens-Canada   (2023-10-12)

PhaF mutant

This part is an improvement upon the wildtype phasin PhaF, BBa_K4728007.

Phasins play a crucial intracellular role in facilitating the interaction between PHB (polyhydroxybutyrate) deposits and the cell through electrostatic interactions. It’s N-terminus consists of four distinct segments: Bi1, Bi2, Bi3, and Bi4, with Bi1 exhibiting the highest affinity for phasin binding [2]. We focused on a specific favored residues (26-32) and, after thorough analysis, opted to incorporate it into the adjacent 33-39 residues, which lacked a defined function. This modification resulted in an increased binding capacity and enhanced amphiphilicity, which was confirmed in a protein docking simulation. Our tests aimed to 1) evaluate its role in PHB binding, and 2) compare its binding capacity with our wildtype PhaF.

PhaF's C-terminal domain is highly disordered, compared to the N-terminal PHB-binding portion (Figure 1, 2).

Fig.16
Figure 1. Disordered protein analysis conducted on Dispredict_v1 (Iqbal & Hoque, 2015).
Fig.17
Figure 2. PhaF structure generated by alphafold2, in red, is the N-terminus region, while in blue it's the C-terminus.

We employed AutoDock Vina for our docking studies, leveraging genetic algorithms and local optimization for precise ligand-receptor interaction prediction. This provided deeper insights into the wildtype and mutant interaction. The docking results highlighted significant differences in binding affinity. The Mutant:Monomer exhibited a binding affinity of -2.8 kcal/mol, while for Wildtype:Monomer it was -2.0 kcal/mol, both at an RMSD score of 0.0.

VMD of PhaF mutant
Figure 3. Molecular dynamic simulation of a PhaF mutant, truncated to the N-terminal first 52 amino acids.
Figure 4. PHB monomer binding to Bi1 domain of mutant, binding affinity is indicated, it interacts with LEU, GLY & TRP.

Due to time constraints, we weren't able to conduct Molecular dynamic (MD) simulations accounting for both our wildtype and mutant in relation to the PHB membrane. However, our proposed model holds promise in light of our docking results. Below we have constructed a prepared minimized CHARRM-GUI model for our potential MD.

Figure 5. Proposed model for our membrane protein interaction, built in CHARRM-GUI


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 657
    Illegal NgoMIV site found at 672
  • 1000
    COMPATIBLE WITH RFC[1000]


References

Iqbal, S., & Hoque, M. T. (2015). DisPredict: A Predictor of Disordered Protein Using Optimized RBF Kernel. PLoS ONE, 10(10), e0141551. <a href="https://doi.org/10.1371/journal.pone.0141551" target="_blank">https://doi.org/10.1371/journal.pone.0141551

Maestro, Beatriz, et al. “A New Family of Intrinsically Disordered Proteins: Structural Characterization of the Major Phasin PhaF from Pseudomonas Putida KT2440.” PLoS ONE, vol. 8, no. 2, 15 Feb. 2013, p. e56904, https://doi.org/10.1371/journal.pone.0056904. Accessed 7 Feb. 2023.

Jendrossek, Dieter, and Daniel Pfeiffer. “New Insights in the Formation of Polyhydroxyalkanoate Granules (Carbonosomes) and Novel Functions of Poly(3-Hydroxybutyrate).” Environmental Microbiology, vol. 16, no. 8, 21 Jan. 2014, pp. 2357–2373, https://doi.org/10.1111/1462-2920.12356. Accessed 18 Nov. 2020.

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