Part BBa_K3515011 was the base for our improved part BBa_K3957000. The chimeric protein consists of the ligand binding domain of vitamin D receptor (VDR-LBD) with mNeonGreen and mCherry fluorophores. The protein database (PDB) file and model for the original part came from the Canadian team Queens. It is important to note that the protein was never expressed; in addition, we needed to add a polyhistidine-tag to extract and purify the protein. Thus, it was necessary to develop a new model. We decided to pursue homology modelling; the first web service used was Phyre2. It turned out that we were unable to use this service as it uses homology modelling based on the natural proteins database and it can only compare against a single protein. This makes Phyre2 useless to model chimeric proteins as the VDR-LBD is modelled as a fluorophore or the fluorophores are modelled as VDR. Therefore, we moved forward with a web service called i-Tasser, which worked well as it uses homology modelling based against a PDB file given to the program to compare against. Furthermore, we also used Chimera to confirm 1,25-dihydroxyvitamin D binding to the VDR region.
Figure 1. The BBa_K3957000 model. The fluorophores,
mCherry(C-terminus) and mNeonGreen (N-terminus), are the red and pale
green beta-barrels, respectively. The chromophores are visible in the
beta-barrels and 1,25-dihydroxyvitamin D can be seen bound to the
VDR-LBD region. Image made with PyMOL.
The VDR-FRET system works with mCherry and mNeonGreen since they are fluorescent proteins anchored to the C and N-termini of the VDR, respectively; they can absorb a specific wavelength of light as well as emit a different one. Having two fluorescent proteins allows for fluorescence resonance energy transfer (FRET), where one fluorophore absorbs a specific wavelength of light and emits a wavelength within the second fluorophore’s excitation range. The second fluorophore emits a wavelength which can then be detected. FRET signal strength is based on the proximity of the two fluorophores. With the binding of 1,25-dihydroxyvitamin D to the VDR region, a conformational change occurs. As a result of this, there will also be a change in the signal of FRET enabling a vitamin D biosensor. As a side note, when we sequenced our plasmid we found amissense mutation (Fig. 2).
Figure 2. The location of the missense mutation, His72 → Asp72, in the
mNeonGreen fluorophore is indicated in purple. Image made with PyMOL.
To verify the function of BBa_K3957000 we opted to perform molecular dynamics. We used GROMACS, but found that an ordinary laptop (Fig. 3&4) couldn’t process the 1 ms of molecular dynamics that was required. So, we used Computer Canada (Fig. 5), a supercomputer, in an attempt to achieve a proper timescale. Unfortunately, after multiple attempts, we were only able to obtain a maximum processing rate of 95 ns/day (with the ligand unbound), which is nowhere near what we would need to see an allosteric change (between 10 µs - 1 s1; we were aiming for 1 ms) in our system. It would take too long for the supercomputer to simulate this timescale.
Figure 3. 500 ps of VDR-FRET without ligand 1,25-dihydroxyvitamin D bound or chromophores. Molecular dynamics calculated by a laptop with GROMACS and visually simulated with PyMOL.
Figure 4. 500 ps of VDR-FRET with ligand 1,25-dihydroxyvitamin D bound and without chromophores. Molecular dynamics calculated by a laptop with GROMACS and visually simulated with PyMOL.
Figure 5. 10 ns of VDR-FRET without ligand 1,25-dihydroxyvitamin D bound or chromophores. Molecular dynamics calculated by Compute Canada with GROMACS and visually simulated with PyMOL.
1. Zwier, M. C.; Chong, L. T. Reaching Biological Timescales with All-Atom Molecular Dynamics Simulations. Current Opinion in Pharmacology 2010, 10 (6), 745–752. https://doi.org/10.1016/j.coph.2010.09.008.