A major obstacle our creation of an effective biosensor would be the matter of immobilization. We had modelled and understood the significance of protein stability, and had devised and modelled a coiled-coil protein for the physical immobilization. Yet, we had to explore options as to how we would combine our fluorescent biosensor protein constructs with the coiled-coil, and have the entire system bind to a physical biosensor casing. After significant discussion with experts in protein immobilization at Queen’s University, we ultimately decided upon cysteine binding chemistry as our anchor point – completing the system.
Cysteine Placement Strategy
Anchoring the coil to the protein and the coil to the gold surface of our biosensor is a cysteine residue – forming a bond with gold and a disulfide bridge with the fluorescent binding protein. Only one exposed surface cysteine can be on the fluorescent binding protein to ensure controlled binding with the coil in the proper orientation. To close to the active site or fluorophores and you can have disaster. On an intrinsically disordered region and the protein may not undergo a conformational change. We’ve done intensive homology searches, static and dynamic modelling, and met with some of the leading protein engineering experts in Canada to make these mutations. Read our step-by-step construct development protocol for more information.
Figure 1. Cysteine immobilization modifications of binding proteins. Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a blue cartoon preset with cysteine residues in red cartoon, respectively, using PyMOL. A. Phosphate binding protein with a Cys 256 modification (red). B. Potassium binding protein with a Cys 26 modification (red). C. Parathyroid hormone receptor, with a Cys 48 residue (red). D. Glucose/Galactose binding protein with a Cys 190 modification (red). E. Alpha-klotho (FGF23 receptor) with a Cys 664 modification (red).
To begin validating our cysteine placement strategy, we performed nanoscale molecular dynamic simulations (Click here for how) on FBP-Pi as a reference construct. Using the Visual Molecular Dynamics (VMD) software we observed the nanosecond trajectory of the construct with our specified cysteine (139 CYS) highlighted purple.
Figure 2. Nanosecond trajectory of FBP-Pi with cysteine modification (purple).
This specific video was generated to visualize the trajectory of our directed cysteine mutation at position 139. In order for our immobilization technique to work successfully, the cysteine had to be facing the surface, pointed outwards and at a location opposite from the active site. As can be seen by the purple residue in focus, our cysteine modification fulfills these requirements and is likely placed optimally. In order to quantify what we observed visually, we analyzed the RMSD of Cys 139 (Figure 3).
Figure 3. Root-mean-square-deviation (RMSD) of Cysteine 139 in FBP-Pi.
Knowledge of the RMSD of cysteine 139 over the nanosecond simulation informed us towards some of the intrinsic stability properties of this mutation – specifically how prone it is to fluctuations in orientation. We can see that the mutation is more or less stable over the 1000 picosecond simulation course, with the exception of the spike in RMSD at around 850 picoseconds. While this is slightly concerning, the fact that it stabilizes at around 900 picoseconds mitigates these concerns.
The finalized construct (without the coiled-coils) can be seen below (Figure 4).
Figure 4. Rotation movie of FBP-Pi construct with Cysteine modification highlighted.
This is rotation movie of our finalized FBP-Pi construct with After running nanosecond molecular dynamics on FBP-Pi, an output file of the stabilized and equilibrated was given to use by GROMACS which we input into PyMOL. Within PyMOL we highlighted the 139-cysteine residue and viewed the construct at different angles to confirm that it was in the correct position and orientation. As can be seen in Figure 4., the mutated cysteine appears to be in an ideal location – opposite to the active site, and on the outward facing surface of an alpha helix. This suggests that our directed cysteine modification was successful.
To further enhance our verification of directed cysteine modification, we are focusing on developing a software to expedite the process. Currently we have developed MutaGuide (link), however this software lacks the ability to incorporate molecular dynamics simulations, which are crucial for assessing factors such as RMSD, RMSF, and gyration.
As computational power was a limitation over the summer, we were unable to perform protein-docking simulations to assess the binding of the coiled-coil to our directed cysteine modification. This would have provided us valuable information towards the binding interactions of the two components, such as thermodynamic stability, binding affinity, and dissociation constants. Protein-docking simulations would be the next step to confirm whether our directed cysteine modification was effectively performed.
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- J.A. Lemkul (2018) "From Proteins to Perturbed Hamiltonians: A Suite of Tutorials for the GROMACS-2018 Molecular Simulation Package, v1.0" Living J. Comp. Mol. Sci. In Press. GitHub.
- The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC, 2019, www.pymol.org.
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