Thermostability Expectations
Given the large decrease in predicted ΔG of folding from 8.44 kcal/mol to -36.83 kcal/mol, we expect the thermoengineered monomer to be more resistant to denaturation at higher temperatures. While we cannot predict the exact melting temperature of the protein, the increase in hydrophobic residues and removal of unstable noncovalent interactions should improve the stability of the molecule. The griffithsin homodimer's ΔG of folding was predicted to be decreased from 21.1 kcal/mol to -17.93 kcal/mol. Shown below are the two thermoengineered proteins colored by their B factor. When compared to the B factor models of the non-thermostable Griffithsin proteins on the "Trials" page, one realizes that the variants on this page have less unstable/red residues and have greater stabilizing interactions.
Thermostable GRFT constructs pictured above have amino acid residues with much lower B values than the normal protein shown previously, suggesting less amino acids are subject to thermal motion and have greater stability
Docking Expectations
Since the binding loops were preserved in the mutation of thermostable griffithsin, binding to gp120 was not altered between the variants and the thermostable proteins ligated gp120 at the same binding sites as non-thermostable griffithsin. The docking models of HIV and Zika antibodies with the thermostable monomer ligated to gp120 showed that the thermostable protein would be a good candidate for the lateral flow assay tests and forms stable interactions between the antibodies at predictable binding sites.
In the future, we hope to use these results to construct a cheap and reliable lateral flow assay which uses this thermoengineered GRFT to immobilize the gp120 protein found on the HIV viral envelope. In addition, we have proven in silico that different Zika and HIV antibodies are able to bind our thermostable GRFT without destroying the protein's structural integrity.References
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