Introduction

Hydrogen bonding is a fundamental weak force of critical importance in maintaining the integrity and function of biological materials. The hydrogen bond is a special strong dipole-dipole interaction between an H atom bonded to a small highly electronegative element (O or N for biomolecules) and a small highly electronegative element (O and N) bonded to any other element. Recall that a hydrogen bond is not a "bond", it is a weak interaction. In this exercise you will investigate H-bonding between the heterocyclic bases (purines and pyrimidines) that is the basis of the structure of DNA as well as interactions that occur during the processes of translation, transcription and replication.

Specifically in this experiment you will do the following:

1. Create the 5 common purine and pyrimidine bases from "scratch."
2. Practice the fine art of rotating, translating and scaling molecular models.
3. Create a minimum of 6 combinations of base pairs and further learn the rules of hydrogen bonding.
4. Determine the calculated heats of formation of both the starting bases and the base paired complexes using molecular mechanics.
5. Make conclusions about the nature and strength hydrodgen bonding in base pairs.

Basics of Molecular Modeling using Scigress

The Scigress molecular modeling program can do many things. As with any tool, there are certain things that are done very well, other things that are done so so, and some things that cannot be done. Discovering the limitations of Scigress (and some shortcomings of molecular modeling in general), is one objective of this exercise. You will be using different parts of Scigress for each part of this exercise. Consult the accompanying Scigress manual for specific details. There is the hard copy version provided for this experiment. There is also a "soft copy" version in the form of a Help file that can be brought up on your screen as a separate window and consulted as needed. Whenever it is not clear what you need to do from the instructions in this exercise, go to help ; check by key words what you need to know.

In order to do molecular modeling, you have to generate a structure. This is done in the Scigress Editor. After a structure is drawn, many attributes of that structure can be obtained. These include bond angles and bond lengths, spectral characteristics and thermodynamics attributes. These can be obtained using different applications in the Scigress program. Each of these can be activated from the Applications menu in the Scigress Editor (side bar menu).

Open the Scigress Editor. Using the directions in the manual guide, make the five bases found in nucleic acids (A,C,G,T,U). The manual is written for the construction of benzene.  You will have to adapt the drawing instructions appropriately.  Pay attention to the atom selected, bond type and hybridization.  If you make a mistake you can select the mistake (right click for select menu) and delete as needed.  It is not important to be careful about how you make the molecule - the computer will clean it up for you at the end - just make sure you get the bonding in the ring correct and have all substituent large atoms added to the structure. In fact, for learning purposes, it will be better if you do not worry about the length of bonds and the orientation of atoms.  You do not need to add hydrogens, the computer will add all necessary hydrogens to satisfy the valence of each atom.

To obtain a valid structure for each base involves running a program that "knows" the rules of bonding. This program if under the Beautify menu. Basically what Beautify does is give the correct bond angles and bond lengths for the structure drawn. Select the entire structure and click on Beautify and select Comprehensive .  This routine will provide the corrected structure in conventional bond and ball formulation.

Rotate the molecule using the Trackball after you have clicked on the rotation (left side) key. Each base should come out to be planar. If it is not, you have made a mistake in the structure and need to go back. You should investigate other ways in which this structure can be represented by going to the View menu and selecting experimenting with the options available.  Change some of the selected options to see the effect it has on your view of the structure.  Make sure to check out the space filling view as well as other options. Spend some time rotating each structure to get an idea of the different orientations it would have in space. Doing this should also give you a sense of the parts of the molecule that will be interacting through H-bonding. The Hand icon can also be used to move the structure from left to right and up and down in the window (translation). Impotrant once you rotate the molecule return it to the orignal plane by the undo function (cntl Z)

Saving structures: You will have to save each base structure. After you have made your first base and beautified it, click on File and then Save. Before you save the individual file, create a folder; give this folder your name. Save the first structure as a file with its appropriate one letter shorthand in your new folder. After you have completed all 5 bases, go on to the animation in the next section. 

In order to make a base pair you need to bring together two bases and then form appropriate H-bonds. Recall the rules of H-bonding given above. You should make at least 6 base pairs (standard 3 and several non standard pairs (py:py, pu:pu, A-C etc)

Open ScigressEditor. In order to make a base pair start with one of the files you have already made for the desired base and open it.  Open the second base file.  Select the second base structure, copy and close the window.  Paste the structure into the first file.  In order to move one structure in relation to the other, you have to select one of the two molecules. This is done by selecting Molecules from the Select menu, clicking on one of the two bases (it becomes highlighted and will be the one you can move), and then selecting via Trackball from the Adjust menu. Moving molecules to dock with each other using the trackball can be tricky. Don’t get frustrated. You will quickly learn how to operate the Trackball - it's great for this. Do not use the right key - you want to maintain the same size for all your atoms!

Align the molecules such that appropriate H-bonds can be drawn. Check the structures to see if you have aligned the AT and CG pairs correctly. The combinations of the various optional base pairs are up to you.  Each base pair can have several combinations of H - bonding. You can even align base pairs such as to give "base pairing" that breaks the rules.

To make H-bonds use the drawing tool but connect the atoms with the appropriate bond type from the menu (think about it!)

After you have made the base pair, save it to your folder with an appropriate file name. Make sure you use the Save As key so you do not lose the structure of your starting base. Select the entire structure and select Beautify and then Comprehensive on the entire structure and then save again. To continue making other base pairs, either close the file or use it as the starting point of your next structure. Once H-bonds have been used to connect 2 molecules, the computer thinks the structure is one piece and rotates and translates it as one. To disconnect the H-bonding, use the Select Tool to highlight the H-bond then press Delete; the H-bond is gone. After you have cleared the H-bonding, you now have separate structures that can be selected, modified, moved or deleted using steps that you have already done.

At the end of this exercise you should have five files for each of the bases and at least six files for the base pairs. It is important that you have saved each of these after they have been "beautified". 

Determining the heats of formation of bases and base pairs

Molecular mechanics can determine the "relative stability" of different molecules. Molecular mechanics has certain underlying assumptions known as parameters. Given a certain set of parameters, a calculation can be done to determine different thermodynamic attributes of molecules. Relative heats of formation of many structures can be determined in this manner. The quality of the result obviously will depend upon how good the parameters are. To be honest, the types of parameters, their quality and their theoretical justification are all still being ironed out. In fact there are significant differences of opinion in this area. You will use the Project Leader component of Scigress to determine the relative "energy values" for each of the base pairs you have made.  Project Leader allows you to generate a task or evaluation table that will instruct the program to calculate values in empty cells. 

Open Project Leader from the Scigress folder. A Table will appear: untitled: Table 1. Give this Table an appropriate Title by selecting it and then editing. Notice the column Chemical sample in Column A. Double click on cell A1. A screen pops up labeled: select a chemical sample. Go to the folder you created and pick out the first base that you made (for instance A). Select it, then double click (or click and then select Add). The file name now appears in cell A1. Continue this process until you have entered all of your files into column A. Now double click on the box immediately below the B column heading. Several options will appear.  Choose property, thermodynamic, enthalpy, heat of formation,  DH_f at AM1 geometry. Click OK. Repeat this process by double clicking on the box immediately below the C column heading; this time select DH_f at PM3 geometry. Click OK. Highlight all the empty data cells (as you would using a spreadsheet). From the Evaluate menu, click on Cells. Scigress will now go do work calculating the heats of formation of each of your bases and base pairs using two different parameter sets.

Stabilization due to H-bonding

The stabilization due to H-bonding can be calculated using the following formula:

This is simply a restatement of the fact that the heat of a reaction is the difference between the heats of formation of the products and the heats of formation of the reactants. The "reaction" in this case is the formation of H-bonds. If the formation of H-bonds is favorable then the DH _{stabilization} should be negative; if it is not, it should be positive. Take the values in the AM 1 column and determine the DH_{stabilization} for each of the base pairs that you made. Do the same for the PM 3 column. Average the two values to get a value for the average stabilization. Pool similar data with the other members of your group. Put all this information in tabular form. This will constitute the results of your experiment

With all the information that you gathered come to some conclusions about base pairing and H-bonding. In your discussion you should consider all the factors that are variable in your experiment.

Consider the following questions.

 
• Do your results verify the difference between the strength of an O^.......H hydrogen bond and a N^.......H hydrogen bond?
• Do your results confirm the fact that the more H-bonds you have, the more stabilization you have?
• Do your results indicate that H-bonds are stronger than other attractions (dipole-dipole and van der Waals?
• Do these results by themselves suggest that the AT (or AU) and CG combinations are distinctly more favorable than other possible base pairing combinations?