Team:Manchester/Enzyme

To His-tag or not to His-tag
Now that βHACdH is ready to undergo simulations, we ran a simulation for 1 ns under the notion that we would be able to visualize motions around the N- and C-Terminals during the course of the simulation and therefore determine, which terminal would be more appropriate to add His-tags to. The conclusion for our simulation was that the N-Terminal is ideal for the addition of His-tags for several reasons. Firstly, the C-terminal is localized in close vicinity to the interaction domain of the βHACdH homodimer, therefore the addition of His-tags could possibly interfere with the dimer interaction^[6]. Our model also shows that the C-terminal is more dynamic compared to the N-terminal and there are several times in the simulation that the C-terminal interacts with the dimerization domain and may interfere with the folding and function of the protein^[4][5][6]. Therefore we concluded that the N-terminal would be ideal to add the His-tags to, as the N-terminal is less dynamic and will be less likely to interfere with folding and the protein function. With this in mind, our experimental team began to design the His-tagged FabA BioBrick (BBa_K1027003) to express a N-Terminal His-tagged βHACdH to use in the characterisation of βHACdH overexpression.

Figure 5. Overlay of structures from 1 ns βHACdH simulation. Images of overlaid from the following respective time points: 0 ps, 250 ps, 500 ps, 750 ps and 1000 ps with the following colours indicating each individual image: Green, Blue, Purple, Orange and Grey, respectively. Both the N-Terminal and C-Terminal, are specified (Dotted Box), with a zoom in on each respective terminal at an angle appropriate to visualise the positions of the terminals.