Part:BBa_K4728008
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).
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 657
Illegal NgoMIV site found at 672 - 1000COMPATIBLE 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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