Why was this model created
When diving into the literature for the 7-hydroxymethyl chlorophyll a reductase (7-HCAR) enzyme, we discovered something shocking about its purification. Not one person has been able to His-tag purify 7-HCAR so far. The inability to use his-tag purification would introduce additional costs to our use of the protein, so we looked into ways in which we could solve this problem. In an attempt to better understand the phenomena hindering the purification of 7-HCAR, we ventured into electrostatics interaction modelling. The electrostatics modelling showed that the charge differences between the protein and the his-tag may cause unforeseen consequences. To address these consequences, we sought to develop a new spacer to sit in between the protein and the His-tag that would be able to withstand these forces.
Figure 1: vacuum electrostatic model of 7-HCAR. Blue denotes regions of electropositivity, while red denotes regions of electronegativity.
A Snapshot of what we accomplished
Using the electrostatics interactions model, we generated an “ideal” spacer that would be able to fold in such a way that it can be used for purification. Using a feedback loop oscillating between structural prediction models and wetlab review, a final spacer with the desired folding was developed. This spacer is now known as the ICARUS spacer. The ICARUS spacer has been successfully implemented for the purification of the 7-HCAR protein. Along with its use with 7-HCAR, ICARUS has been successfully used on the protein pheophytinase. Structural models have indicated that ICARUS maintains its desired function on the other two proteins in the chlorophyll degradation pathway, which are yet to be synthesised.
What models were employed
We began developing ICARUS with an electrostatic mesh model based around the 7-HCAR protein with an attached unfolded spacer-His tag complex. This model calculates the electrostatic charge at every possible solvent interaction point, thereby generating a mesh of charges (Baker et al. 2001). From this mesh, the long range forces acting within the protein could be quantified. The electrostatic mesh was generated using the Adaptive Poission Boltzman Solver (APBS) package within Pymol 3.7.
This mesh is what alerted us to the possible electronegativity of the protein core causing purification issues. In the image below, the blue box shows the positively charged 6xHist tag on the unfolded spacer while the red box captures the extremely electronegative core.
Figure 2: Electrostatic model of 7-HCAR with a 6xHis-tag.
From this model we hypothesised that the attractive forces between the core and tag were responsible for the inhibited purification. To address this we worked with our wet lab to determine a spacer design that would fold ideally for these circumstances. Multiple homological models were generated to slowly understand ways to induce turns and helices with desired charges.
The team decided to use a twelve long-six long-twelve long Alanine-Serine spacer as the body of the spacer as they are uncharged and also showed to form nice helices. To incorporate charged turns into the protein, the team included a proteolytic site and a series of Aspartic acids, with the first turn being the neutral proteolytic site and the second turn being the negatively charged row of Aspartic acids. The entire spacer and His-tag were flanked with two SacII restriction sites to facilitate the removal of the spacer and tag at the DNA level if necessary.
Figure 3: Schematic diagram of ICARUS spacer
To ensure that the protein design was not biased by its repeated use of homology modelling, another method of structural prediction was used. Ab initio folding was conducted to determine the folded structure of the spacer. Folding was completed using Rosetta on the Robetta server at the University of Washington. The generated structure verified that the spacer would prevent electrostatic interactions between the 6xHis-tag and the protein. The protein below is the ab initio folded ICARUS spacer colour coded to resemble the diagram for the ideal spacer.
Figure 4: 3-D model of ICARUS spacer
After modelling the spacer on its own we decided to use Rosetta once again to gain an understanding of the folding possibilities for the four proteins of the chlorophyll degradation pathway. These protein structures include structures for 7-hydroxymethyl chlorophyll a reductase(7-HCAR), pheophytinase(PPH), Mg2+-Dechelatase(SGR), chlorophyll-b reductase(CBR).
Figure 5: Top left CBR, Top Right 7-HCAR, Bottom Left CBR, Bottom Right PPH.
From these models we gained confidence that the inclusion of the Icarus spacer did not have any catastrophic effects. With these models completed, the team was comfortable with moving forward with the expression and purification of these four proteins.
For the Icarus spacer Models were generated with the following assumptions.
Assumption 1. For the extensive use of homology models in the design of the protein we assumed like with the use of all homology models that proteins with similar sequence will have similar functions.
Assumption 2. We assumed in this model that our hypothesis for the reason behind inhibited folding was correct. The assumption that the electrostatic interactions were responsible for the inhibition was integral to the development of our final spacer.
The impact of this model
After the ICARUS spacer had been generated it was attached to 7-HCAR, the protein for which it had been designed. With the attached ICARUS spacer, we were able to successfully purify the 7-HCAR protein using a Ni-NTA column.
Following our success with ICARUS, we looked to whether ICARUS would be applicable to other proteins, to see whether or not other teams with purification issues could also use ICARUS. To determine if this was possible, we attached ICARUS to pheophytinase, another protein that has historically been difficult to purify with a 6xHis-tag. Our team was successfully able to His-tag purify pheophytinase using the ICARUS spacer.
The ICARUS spacer can be found in the registry as its own part (BBa_K3114014). It can also be found in the registry connected to the parts for pheophytinase (BBa_K3114027), 7-HCAR (BBa_K3114025), Mg-Dechelatase (BBa_K3114026), and chlorophyll-b reductase (BBa_K3114024).
Where is Icarus going?
We hope to further characterize the utility of ICARUS through His-tag purification of the two remaining enzymes in the chlorophyll degradation pathway: Mg2+-Dechelatase and chlorophyll-b reductase. We are currently in the process of cloning these enzymes.
Another avenue we wish to explore with this spacer is the characterization of its efficacy with respect to other spacer systems. After characterizing and developing a more diverse platform for the use of the ICARUS spacer, our hope is that this spacer will be applicable to more iGEM teams in the future.
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