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HisH System Setup

Exercise 1: Creating the covalently bound substrate

Open a terminal window, and move into the directory for this tutorial by typing:

> cd forcefield-tutorial/forcefield-files

To get an introduction to the system, load in the structure HisH_glutamine.pdb into VMD. To do this, at the unix prompt type:

> vmd HisH_glutamine.pdb

The above command will launch VMD and simultaneously load in the molecule we specify. Now take some time to investigate HisH. First we will display the glutamine substrate. Make one atom selection for the ligand glutamine by selecting Graphics: Representations from the VMD Main menu and typing:

> segname LIG

in the Selected Atoms box. You can change the Drawing Method of the selection if you like (CPK is a nice view). Now let's look at the catalytic triad. To do this, click on Create Rep and type:

> segname HISH and resid 84 178 180

This creates a new representation for the catalytic triad of hisH. For this selection, change the Drawing Method to licorice.

the solvent accessibility of the glutamine docking site.}
\end{minipage} }

Zoom in on the catalytic triad. Looking at the mechanism depicted in Fig. 1, identify which carbon of the substrate glutamine will covalently bind to the cysteine sulfur. Measure the distance between the two atoms. Identify which nitrogen group will be hydrolized off by this enzyme to become free, reactive ammonia.

Now we are going to perform ``open-heart surgery'' on the active site. The first step to making a covalently bound substrate is to append the glutamine substrate to the active site cysteine in hisH. This will take several steps in VMD and then the use of a text editor.

To begin, open a TK Console window (under Extensions in the VMD Main window)and perform the following selections and commands:

> set sel [atomselect top "resid 84"]
> $sel set resname CYG
> set sel2 [atomselect top "segname LIG"]
> $sel2 set segname HISH
> set all [atomselect top "all"]
> $all writepdb hisH_cyg.pdb

After you have created the new pdb file, please quit VMD.

Let's take a look at what you've done. To do this, at the unix prompt in the working directory, type:

> more HisH_glutamine.pdb

Look at this file; see where the ligand glutamine is specified and look at the resname of the first residue 84. Now do the same for your hisH_cyg.pdb file. What did the above commands do to the file?

Now, we want to open the hisH_cyg.pdb file in a text editor. To do this, open a new terminal window and move into the working directory. Type:

> cd forcefield-tutorial/forcefield-files

Then type:

> nedit hisH_cyg.pdb

(You may use a text editor of your choice instead of nedit)

Our CYG residue still exists as two pieces; we want to combine them and will do so right now. Scroll down to residue 84. Notice that this residue (atoms 670 to 675) has all the atoms of a cysteine residue, but now has the resname field CYG. Now scroll to the end of the file; the last residue appearing is the glutamine molecule (atoms 1628 to 1637). Notice that it now has the segname HISH and it has a resname field called CYG. Copy and cut out this part of CYG by cutting all the lines specifying atoms 1628 to 1637. Be sure you cut out the entire line, not just a part of it! Leave the END statement intact.

We need to insert this cut text (i.e. the substrate) into the active site cysteine. To do this, scroll back up to the real residue 84 and paste the rest of the CYG residue in after atom 673 (the sulfur of CYG).

Now it is time to hydrolyze the ammonia molecule! To do this, delete out the entire line specifiying atom name NH2 (atom 1630).

Finally, save this modified file as hisH_cyg_mod.pdb in the folder:

It's time to admire your handiwork. Open the hisH_cyg_mod.pdb file in VMD, and highlight residue 84 (hint: it helps if you set the representation to CPK). You just created the covalently bound substrate! (See Figure 3 for comparison - what you made should look identical to the picture.) When you are finished admiring your fine work, close VMD.

Figure: The novel CYG residue in the hisH active site. This residue was created by merging a glutamine and a regular cysteine residue.

We have renamed the entire residue CYG, so it is easy to select and identify this novel residue throughout your simulations. Although we have parameters for both cysteine and glutamine, this residue will require some parameterization because they are connected in a new way.

What part(s) of this novel residue will need parameters?}
\end{minipage} }

next up previous
Next: Testing out your new Up: Parameterization Tutorial Previous: Biological Background and Chemical