Inspiration
The coiled-coil is one of the most important aspects of our project. It is what enhances our proteins beyond than being just pretty constructs, but functional units in a working biosensor. Using 35 residue coils with five-heptad repeats containing a hydrophobic core and electrostatic tail interactions, our two coils readily associate together spontaneously. As we were unable to gain access to a laboratory over the summer, it was imperative that we tested the stability of our coils one way or another. Upon our exploration of the molecular dynamics suite GROMACS, we discovered a technique known as Umbrella Sampling and began conducting simulations.
Umbrella sampling is a technique used to calculate potential of mean force (PMF) to study protein binding-unbinding interactions. The PMF can then be used to derive the binding energy (ΔGbind). Umbrella sampling is conducted by generating an initial series of configurations corresponding to a location where one of the coils is harmonically restrained at increasing center-of-mass (COM) distance from the other coil through an umbrella biasing potential. The restraint placed upon one of the coils promotes it to sample the configurational space in a defined region along a reaction coordinate defined between the two coils. The result of this sampling is a derivation of the ΔGbind between the two coils.
Methods
Step 1. Generate the Protein Topology.
After importing the coiled-coil construct files into GROMACS, the protein topology was generated. The topology contains the defining characteristics of a protein within a simulation, including nonbonded (atom types, charges) and bonded (bonds, angles, dihedrals) parameters. It also contains a force field, which is a collection of equations and associated constants that attempt to recapitulate the physical characteristics of a protein. The PLS-AA/L all-atom force field was used for our models.