Summary of the main results

Wild type simulations

First of all, the equilibration (stabilization of temperature, pressure and density) was accurate for both states of LOV2 domain.

• For dark state, see here

• For light state, see here

Secondly, we analyzed many important characteristics of the system:

1. RMSD was analyzed of all residues' alpha carbon, which shows us that the protein was stable and that our simulation was apparently trustable.

• For dark state, see here

• For light state, see here

2. RMSF was analyzed for residues' side chains. We were able to localize, helped by some differential analysis some residues that move much more than others, which would mean that these moving residues were possibly implicated in the movement transmission that induces the general conformational change of the protein upon light activation. The movement of these residues were not correlated. And further analysis demonstrate that we were not able to see the conformational change (see below).

• For dark state, see here

• For light state, see here

• For the differential analysis, see here

3. The angle between the main beta sheet and the J-alpha helix of LOV2 domain was computed, and we could not see any periodic main movement neither in the dark nor the light state.

• For the differential analysis of the angle, see here

4. Side chain dihedral angle of the reactive cystein (residue 450) was computed for both state.

Interestingly, in the dark state we were able to find that the sulfur atom of this cystein point 30% of the time toward the cofactor, FMN (molecule that reacts with the protein upon light activation) and 70% of the time toward the opposite side of the FMN.

• Important graphs and explanations, see here

These results were expected, because they confirm the in vitro results obtain by [Halavaty et al.], and this confirm once again that our simulation seems to be accurate.

In the light state, the cystein is covalently bonded with the cofactor, FMN, so the side chain dihedral angle was far more stable than in the dark state. This result was completely expected because after a covalent bonding with a big set of atom such as the FMN, the cystein's side chain is less free to move.

• Important graphs and explanations, see here

Simulations of non-light activated LOV2 domain with specific mutations

Based on the cystein's side chain movement analysis, residue mutations near the active site were designed. The goal was to "push" the side chain of the cystein more often in the direction of the FMN with steric interactions.

For a better view of the active site, see here

I427F

Here the isoleucine 427 was replaced by a phenylalanine.

This mutation gave very interesting results:

It changed the amount of time the cystein's side chain point toward the FMN from 30% in the wild type to ~57% in the I427F mutant.

• Important result, see here

So, an important hypothesis appears:

If the cystein's side chain points more often toward its reacting carbon in the FMN, there is more chances that upon light activation a covalent bond will be made. Moreover, if there is more steric obstruction toward the unbonding this newly formed covalent bond, this covalent bond will be stabilized, and it will finally leads to a general stabilization of the light activated state of the protein.

L453G

Here the leucine 453 was replaced by a glycine in order to let empty space for glycine's side chain to move toward the FMN.

This mutation gave less interesting results than the first mutation:

It changed the amount of time the cystein's side chain point toward the FMN from 30% in the wild type to ~31% in the I427F mutant.

• Important result, see here

So, an important hypothesis appears:

In that case, it seems that the cystein's side chain dosen't move a lot if empty space is available. This hypothesis sounds rational because the cystein in the wild type protein move in a more or less stable way in its available space. So, in increasing only the space available will make move the side chain slightly more but not significantly.