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Why did we do Physical Modeling?

Modeling the physical structure of a protein can reveal how its various domains and subunits arrange themselves in space. A protein’s structure is critical to its function, thus by modeling a protein’s structure, we can better understand its role¹. The exact combination of proteins used to build our scaffold has not been previously tried, and thus knowledge of its physical arrangement is limited. We ran molecular dynamics simulations of the prefoldin molecule in order to visualize how flexible its components are¹. 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 believe that the simulation run on prefoldin paves the way for more complex simulations of the entire scaffold to be run in future. We were also able to estimate physical parameters important for our mathematical model.

Aim of Modeling

We aimed to determine how the various appendages of the prefoldin molecule move in space. From this we aimed to estimate how close the enzymes could be clustered together when attached to the prefoldin complex.

Molecular Dynamics

We ran molecular dynamics on half of the alpha-beta prefoldin hexamer, with PDB ID 1FXK (Video 1). Due to our inexperience with running MD we enlisted the help of Donald Thomas from UNSW’s School of Chemistry. The amber software suite was used to run the molecular dynamics simulations, with a 1000 picosecond simulation of the prefoldin molecule produced^{4; 5}. Prior to running the simulation, the PDB file had to be manually corrected by removing unwanted ions, and incorrect amino acids. This was required as many crystal structures are published with molecules left over from the crystallization, such as glycerol.

INSERT VID/GIF of MD from Daniel’s page

Video 1: Molecular dynamics simulation of the half-hexamer of prefoldin. The half hexamer contains 1 beta and 2 alpha subunits of the prefoldin hexamer. The two alpha subunits flank the central beta subunit.

From this simulation it is clear the prefoldin complex is flexible. The beta subunits can be seen branching away from the central alpha subunit, reaching a distance of 60 angstroms, whilst also collapsing in to a distance of 25 angstroms (Figure 1). Due to the flexible nature of prefoldin we believe it would be possible to attach a range of different enzymes to the scaffold, without being limited by the size of the enzyme to be attached.

Figure 1: The straight line distance between the C-termini of the alpha and beta subunits was measured in PyMol. The image on the left shows the subunits as they expand apart, the image on the right shows the subunits as they close together.

Furthermore, our actual system has a series of 9 GSG linkers attached to the C-termini, to which the catcher part of the Spy and Snoop Tag-Catcher systems is attached. The longer GSG linker should allow for even more flexibility of the scaffold, allowing large enzymes to be attached⁶. The C-termini coil of one of the beta prefoldin subunits can be seen flexing and coiling in the simulation (Figure 2). Thus we believe that the prefoldin molecule has sufficient flexibility at the coil for the tag-catcher, and enzymes to be attached.

Insert Figure titled “abpfd randomcoil conformations” in the google drive where Daniel uploaded MD

Figure 2: The C-termini coil can be seen retracted on the left image, whilst extended on the right image. This indicates flexibility at the linker regions on the prefoldin molecule.

We believe that these simulations show how the prefoldin molecule can be employed as a scaffold. In future we aim to run more complex simulations involving the tag-catcher system, and enzymes attached to prefoldin.

How this Informed the Mathematical Modeling

From the results of molecular dynamics, and the published crystal structure of the prefoldin hexamer we were able to estimate what distance the enzymes would be when clustered on the prefoldin complex³.

Figure 3: Please Write (***)

The above figure is drawn to represent the physical and spatial relationship between enzymes A and B on the prefoldin scaffold. Setting up three equations as follows: