Using Molecular Dynamics (MD), the time evolution of biomolecular systems, such as cryptochrome proteins, can be studied, on time scales up to microseconds. The time evolution of proteins give insights into properties such as activation mechanisms, binding of ligands, stability of structure predictions and other dynamic processes that occur on the microsecond time scale.
The most common way to evaluate the stability of a dynamical protein simulation, is to look at the Root Mean Square Displacement (RMSD) of the protein backbone. The RMSD is an indication of the change in structure, with respect to the initial configuration, a large RMSD means that the protein has undergone large structural changes, while a small RMSD indicates smaller changes, usually an RMSD around 3 Å is to be expected for a protein on the size of cryptochrome. An example of a small and large RMSD can be seen below.
For a homology model of European robin cryptochrome 1, the RMSD has been calculated over a 1 microsecond simulation, see below, and stabilises around 3 Å, which indicates that the model does not undergo any major rearrangements and that it has become stable after around 500 ns of simulations.
Once a stable model has been obtained, more advanced properties can be investigated.
One example of such a property could be the force required to pull FAD from the binding pocket in cryptochrome from different organisms, or finding the interaction energy between FAD and the cryptochrome protein. This can be achieved by using steered molecular dynamics simulations, where an additional force is ascribed to an atom in the FAD molecule, and by evaluating the interaction energies over the dynamic simulation where the structure is in equilibrium, in this case the last 500 ns. The interaction energy between FAD and avian cryptochrome, plant cryptochrome and insect cryptochrome can be seen in figure A below, as well as the force required to pull FAD from the cryptochromes, figure B.
These calculations show that the interaction between FAD and the protein is very similar in the three different organisms, however, the force required to pull FAD from avian cryptochrome is much larger than the force required in plant and insect cryptochrome, indicating that there are physical hinderances in avian cryptochrome that makes it difficult for FAD to enter or leave the binding pocket.