Molecular Modeling, Laboratory 2

Laboratory Exercise #2, Using A Molecular Mechanics Force Fields

For this laboratory we will model a simple cyclic peptide and carry out a computational investigation of its structure using minimizations with different force field selections.

Few small peptides have stable conformations in solution. Indeed, few of them crystallize. This makes it difficult to test protein force fields with convenient sized molecules. However, there is a growing body of structural data on small cyclic peptides (see the reference list at the end of this page).

These small cyclic peptides are attractive as models for testing molecular mechanics programs in that they are naturally restrained in conformational space and have fairly well described structures. Both Hall an Pavitt (1984) and Lii and Allinger (1991) have used comparisons between the minimized models of various of these peptides and the crystal structures as a criterion for testing various force fields. In their studies, the modeling was carried out in a crystal environment with simulated interactions of crystal packing. In our exercise, we will carry out the calculations in vacuo and can not expect the high degree of accuracy seen by Hall and Pavitt (1984) and Lii and Allinger (1991).

These two exercises use use a small cyclic hexapeptide, cyclo (-Ala-Ala-Gly-Gly- Ala-Gly), which should have a stable conformation. The structure of this peptide was reported by Hossain and van der Helm (1978).

PREPARATIONS:

In preparation for this exercise, I suggest you create a subdirectory called "lab2", change to that directory and operate the insightII program from there.
You will need a copy of the crystal structure of cyclo (Ala-Ala-Gly-Gly-Ala-Gly) for comparison to your models. Copy it to your directory (e.g. cp /11/users/BCMB8200/lab2/hexpexp.pdb .).
You will also need to copy the lab report template, lab-report2.html, to your working directory (e.g. cp /11/users/BCMB8200/lab2/lab2-tmp.html .) and rename it as you want (e.g. mv lab2-tmp.html jsmith-2.html).
OPEN THE LAB-REPORT FILE IN THE JOT TEXT EDITOR AND ENTER YOUR NAME WHERE SPECIFIED.
You may also find it handy to have a second Unix Wsh window active on the same working directory.

SECTION 1:

1) Build cyclo (-Ala-Ala-Gly-Gly-Ala-Gly)
A. Build the linear peptide (Biopolymer menu, Residues, Append)

From the MSI icon (upper left corner) select the Biopolymer Module.
From the Biopolymer menus select the Residue Menu
Select Append from the Residue Menu.
Give your peptide a name, say "MODEL1"
Carefully select

Ala twice,
Gly twice,
Ala once,
Gly once

Then use the Cancel button to quit adding residues.
B. Close the cycle (Biopolymer menu, Modify, bond, click on ...)

From the Modify Menu (Biopolymer module), select BOND
Use the target cursor to click on the N-terminal nitrogen and the C-terminal Carboxy carbon.
NOTE: Be sure to click on the N and C atoms and NOT their attached hydrogens!
NOTE: the long bond drawn will look double (like a double 
bond), since it is also an amide bond with partial double
bond character.
2) PICK A FORCE FIELD:
Use the "hostname" command (at the Unix prompt) to find out which machine you are using (e.g. mgl9). Divid the numeric part of the host name by 3. If the remainder is 0, use the cvff force field. If the remainder is 1, use the AMBER force field. If the remainder is 2, use the cff91 force field. Enter the force field name into the Web-report in place of the string of X's (XXXXX).
2) Initial minimization:

A. Minimize the structure with your force field.

From the MSI icon, select the Builder Module NOTE: AT THIS POINT THE "Potentials" menu may automatically appear. If so, select the "Cancel" buttion and go on.

from the Builder Module, select the Forcefield menu
from this menu, select "SELECT"
click on the appropriate force field (amber.frc, cff91.frc, or cvff.frc)
click ON the "Clear_Charges" button
Then "Execute"
from the Forcefield menu, select "Potentials"
Make sure that each "Fix" button is active, then
click execute
From the Build Module Menu, select Optimize

accept the default selections and select "Execute"
From the Unix window look at the output (File: model10.out). Toward the bottom of the file you will find a line that says either "No convergence after 1000 iterations" or reports the time and results of some lesser number of iterations. IF THE MINIMIZATION HAS NOT CONVERGED, DO IT AGAIN UNTIL IT DOES.

B. Save the final minimized structure (Molecule, Put, Biosym format, ...)

From the top menu line, select the Molecule Menu

select the PUT item
select PDB format
In the file name box enter a name, e.g. MODEL1
Either select Execute or type the ENTER key after entering the name.

NOTE: If you save your structures in PDB format, you may not be able to continue your calculations easily if you are interrupted or the program crashes. TO RELOAD A STRUCTURE, USE THE MOLECULE MENU, THE "GET" SELECTION AND THE "ARCHIVE" FILE TYPE.

C. Document the force field:

From the Forcefield Menu, select Tabulate
Select Output_Non_Bonds and Output_Internals (should already be selected)
Give the program a dummy table name (say Table1)
From the Spreadsheet that appears, select SAVE and give the output the output file name "forcefld.html"
From a unix window, select the jot editor to edit this table
...% jot forcefld.html

add the following lines to the top of the file

<HTML>
<BODY>
<PRE>

add the following line to the end of the file:

</PRE>
</BODY>
</HTML>

Save your changes and exit jot.

D. Document the partial charges used:

Select the Molecule Menu from the top menu line
Select "Tabulate"
Be careful to select ONLY the following buttons:

Name
Partial_Charge
Create_Table

You many accept the default table name
Select "EXECUTE"
View the table and then save it as a text file, e.g. chargetab.html.
As with the force-field table, edit chargetab.html to add the minimal 3 needed lines at the top and bottom.

E. Compare the model structure to the X-ray structure:

First, load the X-ray structure

Molecule menu
select "Get"
select the "hexpexp.pdb" file

From the "Transform" Menu

select "Superimpose"
select first "MODEL1"
select next "HEXPEXP"
Make sure that the "Heavy" button is active
select the "End_Definition" button
select the "Execute" button
Record the RMSD in your Web-Report at in place of the Y's (YYYY)

Turn the models so that the alignment is nicely visible and save the image in the file "model1.rgb"
Molecule Menu
Color
Specification -> By_Atom

File Menu
SGI_RGB
RGB color space
Current_Window Image size
File name model1.rgb

Convert image to a gif file:

from a Unix window on the working directory
use the togif command
e.g. togif model1.rgb model1.gif
deleted the rgb file (rm command)

Delete the HEXPEXP structure from the display

select the object menu
select "Delete"
select "HEXPEXP" and "Execute"

Section 2:

In a similar manner to that illustrated by the step in Section 1, do each of the following and enter the RMS for all heavy atoms when the structure you generate is compared to the crystal structure in the hexpexp.pdb file. Fill out your web report with the information requested using a free form (what-you-see-is-what-you-get) between the <PRE> and the ≶/PRE> tags in the template.

Compare the structure after you further minimize it without charges (select Optimize again from the Builder menu, but click OFF the charges button).

Add constraints to the two internal hydrogen bonds that are found in the crystal structure (Gly3 amide H to Gly6 carbonyl O; Gly3 carbonyl O to Gly6 amide H)

Select the Discover module from the MSI icon menu
It will help to have the atoms colored "BY_ATOM" and the residues labeled by "Monomer/Residue"
Orient the structure so that you can clearly see the amide H and carbonyl O atoms of residues 3 and 6.
select the "Constraint" menu
select "GenericDIS" from this menu
Add the following constraints:

...Upr_Bnd = 2.6
...Lwr_Bnd = 2.0
...Upr_K = 100000
...Lwr_K = 100000
...Max_Frc = 1000000

Click on the atoms of one of the hydrogen bonded pair (NOTE: the input box must be selected first).
Enter the constraint for the second hydrogen bonded pair by again selecting that pair of atoms.
Read the current distance of these two bonds from the display and enter them into your Web-report.
Select the Parameters menu
Select the Minimize menu item
Be sure that the "Charges" button is active. If not, click it and the Execute button before selecting Cancel.
Select the Run menu and the "Run" menu item
As before, save the structure and compare it to the experimental structure.

NOTE: Whenever you load the comparison structure, it is best to delete it afterwards to avoid making a mistake and changing it.

Section 3:

Document the comparison between your "best structure" based on the heavy atom RMSD and the experimental structure using such data as torsion angles, hydrogen bond distances, etc.
As before, be sure you save your files, clean up your directory and submit the report to Dr. Wampler via E-mail.

References:

Francart,C., Wieruszeski, J. M., Tartar, A., and Lippens, G. (1996) J. Am. Chem. Soc. 118, 7019-7027.
Hall, D., and Pavitt, N. (1984) J. Comp. Chem. 5, 441.
Hossain, M. B., and van der Helm, D., (1978) J. Am. Chem. Soc 100, 5191.
Karle, I.L., Gibson, J. W., and Karle, J. (1970) J. Am. Chem. Soc. 92, 3755.
Kostansek, E. C., Thiessen, W. E., Schomberg, D., and Lipscomb, W. N. (1979) J. Am. Chem. Soc 101, 5811.
Karle, I. L. (1978) J. Am. Chem. Soc. 100, 1286.
Kopple, K. D., Wang, Y.-S., Cheng, A. G., and Bhandary, K. K. (1988) J. Am. Chem. Soc. 110, 4168.
Lii, J-H., and Allinger, N. L. (1991) J. Comp. Chem. 12, 186-199.
Lin, M. F., Chan, M. F., Balaji, V. N., Castillo, R. S., and Larive, C. K. (1996) Inter. J. Pept. Prot. Res. 48, 229-239.
Siahann, T. J., & Lutz, K. (1994) J. Pharm. Biomed. Anal. 12, 65-71.
Weisshoff, H. Wieprecht, T., Henklein, P., Frommel, C. Antz, C., and Muge, C. (1996) Febs Letters 387, 201-207.