Modeling

To do

- Model allosteric interactions between LOVTAP & TrpR

What will be done:

- Model of LOVTAP in dark phase

- Model of LOVTAP in light phase

- Characterize how the J-alpha helix changes

- Model sturctural changes that enhance the switch feature of LOVTAP e.g. in dark phase: really weak interaction between LOVTAP and the corresponding DNA sequence, in light phase: strong binding of LOVTAP on DNA.

Modeling reference

LOVTAP simulation

We will follow the following article protocol:
Freddolino, P.L., Dittrich M., Schulten K., Dynamic Switching Mechanisms in LOV1 and LOV2 Domains of Plant Phototropins. Biophysical Journal, 91, 3630-3639, 2006 ([http://www.ncbi.nlm.nih.gov/sites/entrez?db=pmc&cmd=search&term=PMC1630464 Pubmed])

VMD informations

VMD is used to visualize molecules.

• A tutorial for VMD can be found [http://www.ks.uiuc.edu/Training/Tutorials/ here]. VMD is quite user friendly.

NAMD informations

NAMD performs minimization and equilibration.

• A tutorial is on the same page as for VMD, [http://www.ks.uiuc.edu/Training/Tutorials/ here].
• [http://www.ks.uiuc.edu/Research/namd/2.7b1/ug/ NAMD 2.7b1 User's Guide]

Run a simulation

A simulation is composed of different steps. Here are a few links that deal with heating and stabilization.

• [http://www.ks.uiuc.edu/Research/namd/tutorial/NCSA2002/hands-on/ Building Gramicidin A: Equilibration]: protocol uses a single .conf file, heating process is too fast.
• [http://www.biochemistry.uab.edu/robinson/documents/howto/modeling/NAMD_notes.htm NAMD notes from Robinson Lab]: a really nice heating process, but involves different .conf files, which is really painful.

Implementation of the simulation

LOV domains are the light-sensitive portion of phototropins. They absorb light through a flavin cofactor, photo-chemicaly form a covalent bond between the chromophore and a cysteine residue in the protein, and proceed to mediate activation of an attached kinase domain.

We generated all the files needed to do the simulation, thanks to the tutorial of NAMD, which can be found on the following page:
http://www.ks.uiuc.edu/Training/Tutorials/

Generating input files

First we need a compatible .pdb in addition to parameter and topology files. Steps to generate all the input files are explained in detail on this page How to generate input files. This is a kind of summary of the tuto.

.conf parameters

We should explain here what are the keywords we use in the .conf.

Run a complete simulation

We start from .pdb, .psf, .rtf generated in the previous section. Complete process is on a separate page How to run a simulation.

Molecular dynamics theory

The protein moves thanks to different forces, which can be separated in bonded forces and non-bonded forces. The bonded forces correspond to

• the Lennard-Jones potential, which take into account the Van der Waals forces. It represents the non-bonded forces and the total potential energy can be calculated from the sum of energy contributions between pairs of atoms.

Lennard Jones potential

• another force is the well-known Coulomb force

Coulomb force

To envisage

- Molecular mutationnal assay

Already done

Here is our first movie from the modeling, showing the behavior of the protein in the dark state condition: Dark State

After having modified some parameters in the parameter files, here is our second movie, concerning the light state of the protein this time, with the FMN: Light State with FMN without water