We believe that by adding a series of 9 GSG linkers to the prefoldin C-termini, we created more flexibility in the scaffold 6. However without the tag-catcher system and enzymes attached, we cannot know exactly how the molecule will move. The flexibility shown by the half hexamer does lend support to prefoldin's usability as protein scaffold.
SNOOP CATCHER YEAST FULL
When running the simulation on a half hexamer, we have to acknowledge that any results used to inform our use of the full scaffold are extrapolations. Generally speaking, the major errors that need to be corrected are erroneous ions and amino acids that need to be removed 4,5. Considering that we assembled the prefoldin-catcher-tag-enzyme complex manually in Chimera our structure likely had more errors. The major obstacle to running the simulations on the full scaffold was the need to edit the pdb files generated from crystal structures. We were able to run MD on half of the prefoldin hexamer PDB ID 1FXK. Running molecular dynamics served as an effective method of modelling the physical nature of our system. We used this distance to calculate the diffusion of substrate between the enzymes, and subsequent product yield at this distance in our mathematical model of enzyme kinetics and diffusion. From the graph, we estimate that over this short time period the subunits average a separation distance of about 50 angstroms. By measuring distance between the C-termini of the alpha and beta subunits, we are considering the distance between the point of attachment for the catcher-tag system. A still image of distance being measured between these residues is shown above (Figure 1). The residues between which distance was measured correspond to the C-termini of the alpha and beta subunits. This was required as many crystal structures are published with molecules left over from the crystallization, such as glycerol.įigure 3: The distance (angstroms) between a single alpha and beta subunit over time (picoseconds). Prior to running the simulation, the PDB file had to be manually corrected by removing unwanted ions and incorrect amino acids.
SNOOP CATCHER YEAST SOFTWARE
The amber software suite was used to run the molecular dynamics simulations, with a 1000 picoseconds simulation of the prefoldin molecule produced 4 5. Due to our inexperience with running MD we enlisted the help of Donald Thomas from UNSW’s School of Chemistry. We ran molecular dynamics on half of the alpha-beta prefoldin hexamer, with PDB ID 1FXK (Video 1). From this we aimed to estimate how close the enzymes could be clustered together when attached to the prefoldin complex. We aimed to determine how the various appendages of the prefoldin molecule move in space. We were also able to estimate physical parameters important for our mathematical model.
We believe that the simulation run on prefoldin paves the way for more complex simulations of the entire scaffold to be run in future. From this we were able to estimate the feasibility of being able to attach linkers, tags and other proteins to the prefoldin to form a scaffold. We ran molecular dynamics simulations of the prefoldin complex in order to visualize how flexible its components are 1. The exact combination of proteins used to build our scaffold has not been previously tried, and thus knowledge of its physical arrangement is limited. A protein’s structure is critical to its function, thus by modeling a protein’s structure, we can better understand its role 1.
Modeling the physical structure of a protein can reveal how its various domains and subunits arrange themselves in space.
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