Part:BBa_K3429013

TasA-EreB fusion protein

Profile

Name	TasA-EreB fusion protein
Base pairs	2335
Molecular weight	78.48 kDa
Origin	Bacillus subtilis and Escherichia coli, synthetic
Properties	Fusion protein of B. subtilis extrapolymeric matrix protein TasA and E. coli erythromycin esterase type II EreB. TasA is utilized for protein immobilization of macrolide transforming enzyme EreB in B. subtilis biofilms.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1206
  Illegal XhoI site found at 1616
  Illegal XhoI site found at 2206
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1333
  

Structure

The protein's structure was predicted using the RosettaCMapplication. Detailed information on the algorithm of RosettaCM can be found in the modelling section for EreBon our iGEM Wiki page.

Figure 1: EreB TasA fusion protein. TasA was fused N-terminally to EreB with a short linker peptide as described by Huang et al. Structure was obtained by homology modelling using RosettaCM.

Figure 2: A: RMSD vs SCORE plot of the fusion protein. RMSD was calculated using the python package Biotite and total score values outputted by Rosetta were used as score. B: Ramachandran plot of the diheadreal angles of the best scoring candidate. C: Ramachandran plot of the top 5 highest scoring structures.

To investigate functionality of the protein domains and structure stability of the fusion proteins MD simulations were carried out using GROMACS and the Charmm27 forcefield with explicit TIP3P water. MD is a useful tool to validate the proteins folding in aqueous environment and study the enzyme’s movements in a time-dependent physical forcefield of Newtonian equations of motion. The structure’s energy is minimized in a first step. Afterwards the system gets equilibrated in a NVT and NPT simulation step. All of these steps are carried out using a system restraint file to maintain the target’s structure during the preparation steps. After equilibration is finished the main simulation for 100 ns can be started. If the MD simulations output conserved tertiary structures of the protein domains we assumed functionality of the immobilization induced by TasA matrix protein. Also, preserved structure indicates successful azithromycin transformation by EreB.

Root-mean-square deviation (RMSD), small root-mean-square fluctuation (RMSF) and radii of gyration (Rg) were plotted to analyse the simulation and validate the structure’s stability. Converging RMSD and Rg are primary indicators for stable protein structures. Analysis of the EreB-TasA fusion protein shows fluctuation of both values around a certain value showing a clear trend of convergence. RMSF analysis displays low derivation of the internal residues, especially those relevant for catalytic activity suggesting stable protein domains and could thus signify preserved catalytic activity and enzyme immobilization, respectively. It was already shown that the active side residues of unfused EreB protein show low atomic fluctuation in the EreB MD simulation. Also low Rg values are an indicator for preserved secondary structure, so we can assume that the protein did not denaturate during the simulation.

Figure 3: A: RMSD plot of the TasA-EreB fusion protein structure obtained by CM. The red plot represents RMSD and the black plot the RMSD calculated against the crystal structure. After 65 ns both the values start fluctuating about a nearly constant value suggesting convergence, a main indicator for a stable protein structure. B: Radius of gyration (Rg) plot. Convergence can be assumed as the value only fluctuates around a constant Rg. C: RMSF plot for every residue in the protein’s 3D structure. For RMSF determination the C alpha values were observed.

For further analysis principal component analysis (PCA) was performed to analyse internal movement of the protein. By PCA we are able to filter global collective movement from local movement to study the enzymes dynamics. The MD simulation run’s covariance matrix was generated and diagonalized using the GROMACS covar tool. The resulting eigenvectors were visualized using the GROMACS anaeig tool and are here presented as 3D visualization of the first eigenvectors showing strongest protein fluctuation. As visible in the simulation the protein shows internal movements of outer residues but functional internal domains stay structurally preserved as already visible in the RMSF graph. Especially the azithromycin binding pocket and residues E43, H46, R55, R74, H285 and H288 of EreB enzyme domain (highlighted in the simulation) important for catalytic activity remain stable and show weak movement. The most relevant principal mode (PM) shows increasing distance of the EreB and TasA protein domain during the simulation. This way accessibility of both domains increases and the function of the linker peptide as dynamic connection of both domains can be assumed. In summary the PMs describing internal movements show reinforces our method of immobilizing the transforming enzymes with extrapolymeric matrix protein TasA and suggests our assumption of correct protein folding after fusion based on the publication of Huang et al. (2018). ^[25]

Figure 11: Visualization of the first four essential modes' extremes. The covariance matrix was analysed using the GROMACS anaig tool and compared to the CM modeling derived structure candidate S_0484.pdb. Only backbone atoms were considered. TasA domain is coloured in light blue, the linker peptide in red and the EreB domain in blue. Top left: Fist (most relevant) PM Top right: second PM containing Bottom left: third PM Bottom right fourth PM

[edit]

Categories

Parameters

None