Quench dynamics module

Conformational search on a prodrug

 

Objective: to create a model of a Leu-enkaphalin model drug delivery system. The prodrug is a cyclized peptide that is more membrane permeable. To understand its relative membrane permeability first we must search the thermodynamically accessible states. To accomplish this we heat the prodrug to a high temperature and allow it to execute these dynamics at temperature high above isomerization barriers for the molecule. The molecule samples states according to the partition function. By quenching (selecting and annealing by progressive cooling followed by energy minimization) snap shots of different structures are obtained. Their relative energies map out the thermally accessible conformations of the prodrug.

 

Criteria for analysis:

    1. Energy distribution of conformers.
    2. Structural analysis of conformers - dihedral angles in key regions that indicate different ring geometries.
    3. Accessibility as a function of the production dynamics temperature.

 

Method: Build a small polypeptide with a coumaric acid linker that allows cyclization of the peptide. The more hydrophobic cyclized molecule can penetrate the membrane and is only hydrolyzed to release the Leu-enkephalin analog drug inside the cell. The cyclized peptide is called a prodrug. The prodrug is subjected to energy minimization followed by equilibration at a high temperature (e.g. 500 K). The prodrug strcutures are then selected at given time intervals during the production dynamics. The selected structures are progressively cooled (while performing dynamics) and then energy minimized to represent a locally minimum structure. In the analysis you will look at the distribution of energies.

 

 The prodrug has the following appearance

 

To construct the prodrug you must make a five amino acid polypeptide and a linker (coumaric acid). The coumaric acid linker must be attached to both the N-terminus and the C-terminus of the polypeptide to make the prodrug.

We begin with the construction of the prodrug linker. We will demonstrate use of the Builder to make the coumaric acid linker. To create the linker it is easiest to begin with ethylene. In the Builder go to the FRAGMENT_WINDOW and select Ethylene by clicking on it. Ethylene will appear in the center of the screen. The Bond menu will appear subsequently. Select one of the hdyrogen atoms on the ethylene group. Then click on Benzene in the FRAGMENT_WINDOW. A carbon-carbon bond will be formed between the ehtylene and the benzene.

Select the vicinal hydrogen (cis to the one just selected for phenyl ring attachment). This should be the A atom. Then select methane (B atom). This may require multiple clicks on methane and some patience for the bond to actually form. Finally, you can bond both the methane and the ortho hydrogen on the phenyl ring to hydroxy to make a structure that looks like

 

To create the appropriate aldehyde you must oxidize the primary alcohol. This can be accomplished simply by change the parameters (atom type) as the bonding designation (and valence) are changed. To alter the bond order select the Modify/Bond menu. Under Bond Operation choose the selection Modify_Order. The target order is set below as Single, Partial Double, Double, and Triple. After selecting Double as the appropriate choice you click on the A atom (the carbon) and the B atom (the oxygen) to make

which is the desired product. 

If you are having difficulty with the FRAGMENT_WINDOW (and it occurs with some frequency that the small fragments are difficult to select for bonding), there is an alternative procedure. Starting with the styrene molecule above use the Atom/Replace command to change the ortho hydrogen to oxygen. A periodic table will appear. Select O on the table and that should appear as the Element Type on the menu. Then click on the ortho hydrogen on the phenyl ring. It should turn from white to red and there is an O label showing that the atom has been transformed. To correct the valence by adding hydrogens you may use the Modify/Hydrogens command. After application the hydroxy group should be protonated. You may do the same change to build the chain of cinnamaldehyde from

Once you have changed the hydrogen to carbon you can obtain new branch points for a growing chain using the Modify/Hydrogens command to add three hydrogens to the methyl group you have made.

The building of a molecule using the Builder or Biopolymer module requires a little skill and you may need to do this a couple of times to get the molecule that we want.

Before going to the next step of building the polypeptide move your coumaraldehyde linker from the center to the side. Recall that you must select with the middle mouse button and drag the structure off to the side. This will make room for the selection of an amino acid.

To attach the linker to the N-terminus of the polypeptide we use the Builder once again. Use the Fragment/Get command and select Tyrosine from the FRAGMENT_WINDOW to create a tyrosine molecule. The FRAGMENT_WINDOW contains a number of Fragment Libraries, including amino acids. To see all of the Fragment Libraries click on the Fragment Libraries button in the lower left hand corner of the FRAGMENT_WINDOW. You will see amino acid residues appear below the standard groups such as hydroxy, amino etc. Each of the amino acid residues is represented not by zwitterion (H3N+-Ca-R-COO-) but rather by the amino aldehyde (H2N-Ca-R-CH=O) . By selecting tyrosine you have selected the first residue in a polypeptide. You will now see your linker and the newly selected Tyr.

To bond these two together use the Bond Menu that is automatically presented to you following the selection of the first residue. Select the aldehydic hydrogen in the linker by clicking directly on it (this is the A atom). Then select the amino group of the Tyrosine residue in the center of the screen as the B atom. The tyrosine will move into position so that it is bonded to the coumaric acid (though probably not with the correct dihedral angles in the polypeptide backbone). To create the second bond select the aldehydic hydrogen of the Tyr residue as the A atom.

NOTE: You might see an entry in the A atom after finishing this procedure.

For example, the A atom is light blue and reads TYR$:1:HN2. Here the TYR$ represents the fragment type, 1 represents the residue number and the last entry is the atom designation.

Ignore this reading and click on A atom followed by the atom you need to complete the growing chain. It should be the aldehydic hydrogen and so A atom should read:

TYR_B0:1:HC

 

Once you have selected the A atom, then select a second residue (Gly in this case) from the FRAGMENT_WINDOW. The B atom will be one of the hydrogens on the amino group of the selected residue. The two residues should be automatically connected (though probably not with the correct dihedral angles in the polypeptide backbone). Continue this procedure with the other amino acid residues. The sequence from N-terminus to C-terminus is

YGGFL

One all of the five amino acids have been joined (usually in an extended conformation) we can save the file as an intermediate step.

To save the file use the Molecule/Put command. If you want to save it as a car file you will need assign potentials first. However, you can also save it as a pdb file by selecting the PDB option in the Molecule pulldown menu.

Finally, comes the step of linking the C-terminus of the growing polypeptide chain with the hydroxyl group in the ortho position of the phenyl ring of the coumaric acid linker. To accomplish this you may need to leave the Bond menu of the Fragment/Get subcommand and use Modify/Bond. The create a bond you simply leave the Bond Operation on Create and then select the appropropriate aldehydic carbon (on the C-terminal Leucine) and the oxygen of the OH group on the ortho position of the phenyl ring.

Note that the bond you are forming in the final step is an ester unlike the amides that you have formed up to this point.

Until now TYR_B0 is the object name given to the growing polypeptide. If you want to change the object name, go to the Object/Rename menu. You will see the name TYR_B0 (all capitals) for your object (or some other name). In the new name menu type LEU_ENK. Then select execute. The polypeptide you have made will now be named leu_enk.

If you attempt to save this file using the Molecule/Put command you are likely to run into difficulty because the potentials have not been set. If you want to save your work as *.car and *.mdf files, then you must use the Forcefield/Select option followed by the Forcefield/Potentials option in the Builder. In the Forcefield/Select menu there are several possible forcefields. We shall use the standard CVFF forcefield. That is also the default selection so all you need to do is select execute once the menu appears. 

The prodrug is the starting point for extended high temperature dynamics and quenching.

The stages are

  1. energy minimization.
  2. production dynamics at a high temperature.
  3. sampling - selection of frames for annealing.
  4. annealing - cooling while executing molecular dynamics.
  5. energy minimization of structure.
  6. tabulation of statistical data for a large number of structures

To carry out these steps you need a *.car file (containing molecular coordinates) and *.mdf file (a molecular data file that contains atom types) and an input file. In the previous modules we have generated the input file in the DISCOVER3 module. For the simulated annealing calculation we will use an input file that has been written in BTCL language. For example, if your *.car and *.mdf file are both named leu_enk.car and leu_enk.mdf, respectively, then you need an input file named leu_enk.inp. To examine the input file use the "vi" editor.

You may rename the standard input file for simulated annealing to this name and then run DISCOVER3.

If you are generating more than 100 structures then the run will take longer than 30 minutes. In this case you will need to submit a batch job to the queue. To do this use the script run_DISCOVER.