Team:Duke/Model
Modeling Overview
In order to develop a rapid diagnostic lateral flow assay (LFA) using griffithsin (GRFT), the thermostability of GRFT must be improved while preserving its biological activity and structural integrity. Proteins with greater thermostability are more resistant to conformational changes at higher temperatures and thus, have longer shelf lives. Thermostability can be improved by altering a protein’s noncovalent and disulfide bonds through random and site specific mutagenesis. Instead of blindly altering the protein structure and measuring the melting temperatures of each GRFT variant, we chose to use the YASARA modeling package to selectively alter amino acids with the goals of minimizing the ΔG of folding and preserving structural similarity of both the monomeric (3ll2) and oligomeric homodimer (2HYQ) forms of GRFT. After engineering the two thermostable variants of GRFT through computational directed evolution, the biological activity of these proteins were evaluated through the docking package ZDOCK.
Modeling Packages
Yasara is a modeling package which has many useful functions for protein engineering. In addition to imaging the protein of interest, Yasara is useful for repairing the unfolded structures, mutating multiple protein residues, and predicting the stability of formed variants. For the purposes of this project, Yasara was used to calculate the B values of protein residues and predict the structures of proteins based on certain residue changes. After using the B values to determine which residues are unstable, the position scan function in Yasara's FOLDX suite was used to mutate each amino acid to the other 20 and predicting the change in stability between each variant and the original. The mutations that increased the stability were stacked upon each other to create more thermostable Griffithsin protein.
ZDOCK is a docking package from the University of Massachusetts that predicts the interactions between different proteins and small molecules. The ZDOCK algorithm predicts the top 2000 positions where the ligand or small molecule should bind noncovalently to the protein of interest. These predictions are ranked by other algorithms RDOCK and CHARMM by their energetics and structural stability. The output of this model gives the PDB or protein data bank files of the top 10 predictions. For the purposes of this project, the thermostable monomer and dimer predicted by Yasara were docked with gp120, the envelope glycoprotein on HIV. The top three structures were then docked with different Zika and HIV antibodies to predict the interactions and structure of immobilization in the lateral flow assay. The comparison of how the griffithsin and its thermostable variant bind to the antibodies and envelope proteins will suggest if the mutated residues impacted biological activity of the GRFT.
Lastly, PyMOL, a common protein visualization system, perform basic molecular dynamic simulations and image the docking interactions. For the purposes of this project, PyMOL was adopted to make movies of the protein structure and color the molecule to make understandable models for presentation.
