HONORS OPTION: GEN CHEM 1

Fall 2008

Chemistry and Medicine

Last update: 09/16/2008

Part 1: Structure makes a difference!

Some small molecules: water and ethanol

Part 2: Intermolecular Forces (IMFs) - from water to proteins

Hydrogen bonds - H bond: Water Jmol | liquid to solid transition Jmol
To bury or not. That is the question. A micelle
A typical protein: Triose Phosphate Isomerase
An alpha helix with H bonds
To bury or not. That is the question. A protein
A complex of two proteins: Thrombin and Hirudin

Part 3: HIV Protease: an Enzyme at Work - Video

Part 4: Molecular Modeling of the HIV Protease/Inhibitor Complex

In this exercise, you will study the structures of HIV protease and an inhibitor which binds to the protease. You will use a powerful molecular modeling program called VMD, which was developed at the University Illinois (Champaign/Urbana). For more information, see the website below

VMD Homepage, University Illinois

OpeningVMD

On a networked PC, select Start, All Programs, Academic Software, Chemistry, VMD 1.8.6. Three windows will open for you.

VMD Main - contains menu bar for running VMD
VMD 1.8.6 OpenGL Display - displays molecules
VMD - contains text and place for command line input

Loading a Molecule

Our first step is to load our molecule. A pdb file, 1HSG.pdb, that contains the atom coordinates of HIV protease and an inhibitor is provided with the tutorial.

1: Choose the File $\rightarrow$ New Molecule... menu item Fig 1(a) in the VMD Main window. Another window, the Molecule File Browser (b), will appear in your screen.
2: Select the drop down in the Look in: line. Then select Admain$ on 'Ad' (S:). The 1HSG.pdb file is located in the S:\Chemistry\VMD\VMDBCHM\CH123. Keep clicking until you get there, then select the file.; Note that when you select the file, you will be back in the Molecule File Browser window. In order to actually load the file you have to press Load (d). Do not forget to do this!

**Figure 1:** Loading a Molecule.

Now, HIV protease is shown in your screen in the OpenGL Display window. You may close the Molecule File Browser window at any time. You will see many red dots around the protein. These represent the oxygen of water molecules that co-crystallized with the protein that was used to determine the x-ray structure.

Displaying the Protein

In order to see the 3D structure of our protein we will use the mouse and its multiple modes.

1: While holding the left button pressed over the protein in the OpenGL Display, move the mouse and explore what happens. This is the rotation mode of the mouse and allows you to rotate the molecule around an axis parallel to the screen Fig. 2(a).

**Figure 2:** Rotation modes.

2: If you press the second button and repeat the previous step, the rotation will be done around an axis perpendicular to your screen (b) (For Mac users, the second button is equivalent to press the command key while holding the mouse button pressed).
3: In the VMD Main window, look at the Mouse menu (Fig 3). Here, you will be able to switch the mouse mode from Rotation to Translation or Scale modes.

4: The Translation mode will allow you to move the molecule around the screen while holding the first button pressed.

**Figure 3:** Mouse modes.

5: The Scale mode will allow you to zoom in or out by moving the mouse horizontally while holding the first button pressed.

It should be noted that the previous actions performed with the mouse do not change the actual coordinates of the molecule atoms.

Another useful option is the Mouse $\rightarrow$ Center menu item. It allows you to specify the point around which rotations are done.

6: Select the Center menu item and pick one atom at one of the ends of the protein. (The cursor should display a cross.)
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Exploring Different Drawing Styles

VMD can display your molecule using a wide variety of drawing styles. Here, we will explore those that can help you to identify different structures in the protein.

1: Choose the Graphics $\rightarrow$ Representations... menu item. A window called Graphical Representations will appear and you will see in yellow Fig 4(a) the current graphical representation used to display your molecule.
2: In the Draw Style tab (b) we can change the style (d) and color (c) of the representation. In this section we will focus in the drawing style (the default is Lines).
3: Each drawing style has its own parameters. For instance, change the Thickness of the lines by using the controls on the right bottom part (e) of the Graphical Representation window.
4: Now, choose from Drawing Method the VDW (van der Waals) menu item. Each atom is now represented by a sphere. In this way you can see more easily the volumetric distribution of the protein.

**Figure 4:** Graphical Representations window.

5: In order to see the arrangements of atoms in the interior of the protein, use the new controls on the right bottom part of the window (e) to change the Sphere Scale to 0.5 and the Sphere Resolution to 13. Be aware that the higher the resolution you choose, the slower the display of your molecule will be.
6: Note that in the Name coloring method, each atom has its own color, i.e: O is red, N is blue, C is cyan and S is yellow.
7: Press the Default button. This allows you to return to the default properties of the drawing method.

The previous representations allows you to see the micromolecular details of your protein. However, more general structural properties can be seen by using more abstract drawing methods.

8: Choose the Tube style under Drawing Method and observe the backbone of your protein. Set the Radius at 0.8.
9: By looking at your protein in the tube mode, can you distinguish how many helices, b sheets and coils are present in the protein?

The last drawing method we will explore here is called Cartoon. It gives a simplified representation of a protein based in its secondary structure. Helices are drawn as cylinders, b sheets as solid ribbons and all other structures as a tube. This is probably the most popular drawing method to view the overall architecture of a protein.

10: Choose the Cartoon style and set the Beta Sheet Thickness as 3, the Helix/Coil Radius as 1.5.
11: Identify now how many helices, betasheets and coils are present in the protein.

Exploring Different Coloring Methods

1: Now, let's modify the colors of our representation. Choose the ResType coloring method Fig. 4(c). This allows you to distinguish non-polar residues (white), basic residues (blue), acidic residues (red) and polar residues (green).
2: Select now the Structure coloring method (c) and confirm that the cartoon representation displays colors consistent with secondary structure.

Exploring Different Selections

Let's look at different independent (and interesting) parts of our molecule.

1

Combinations of Boolean operators can also be used when writing a selection. The review below is from Wikipedia

"Venn diagram showing the intersection of sets "A AND B" (in violet/dark shading), the union of sets "A OR B" (all the colored/shaded regions), and the exclusive OR case "set A XOR B" (all the colored regions except the violet/only the lightly shaded regions). The "universe" is represented by all the area within the rectangular frame." AND limits the search while OR broadens it.

3

**Figure 6:** Graphical Representations window and the *Selections* tab.

Multiple Representations

The button Create Rep Fig 7(a) in the Graphical Representations window allows you to create multiple representations and therefore have a mixture of different selections with different styles and colors, all displayed at the same time.

1: Be sure that the current representation is in CPK style and coloring method Name
2

**Figure 7:** Multiple Representations of Ubiquitin (not the protein we are studying).

4: Repeating the previous procedure, create the following three new representations:

To Display	Drawing Style	Coloring Method	Selection
c. Protein without water	Cartoon	Structure	protein
d. Protein Surface w/o water	Surface	Pos	protein
e. inhibitor alone	VDW - van der Waals	Molecule	(not protein) and (not water)
f. Protein and inhibitor	CPK	Name	all and (not water)

Double click on the representations to toggle them off and on. Display just the Protein Surface (d) and the Inhibitor Alone (e). Toggle (e) on and off to see how the inhibitor fits snuggly into a cavity or "active" site in the protein. Likewise click display (c) and (e) alone. Again toggle on and off the inhibitor (e). Experiment with other options.

5

A closer look at the inhibitor binding site

The complex you have seen above, no matter how you render it, is still quite complicated. We have used a program called MOE to create a file (HIVMOECONTACTpolarH.pdb) that shows only the inhibitor and the amino acids around it that make contacts. Load the file now.

Choose the File $\rightarrow$ New Molecule... menu item Fig 1(a) in the VMD Main window. Another window, the Molecule File Browser (b), will appear in your screen.
Select the drop down in the Look in: line. Then select Admain$ on 'Ad' (S:). The HIVMOECONTACTpolarH.pdb file is located in the S:\Chemistry\VMD\VMDBCHM\CH123. Keep clicking until you get there, then select the HIVMOECONTACTpolarH.pdb fil

What properties must the inhibitor and binding pocket of the enzyme have in common to allow inhibitor binding? You discussed these earlier today and you will cover them more in depth in General Chemistry II (Chapter 10, Section 10.2). In essence there are intermolecular forces - forces between molecules that are distinguished from covalent bonds within a molecule) that allow reversible binding. In general, there are 3 types of intermolecular forces between the inhibitor and protease (the two different molecules) that may be involved here:

ion...ion forces: between a positively or negatively charged ion in the protease binding pocket and the oppositely charged ion in the inhibitor (in this case there are none)

H -bonds: electrostatic interactions between a slight positive charge on a H atom on one molecule and a slight negative charge on a N or O atom on the other molecule. H atoms are slightly positive, and N and O atoms are slightly positive if they are one of the atoms in a polar covalent bond.

Hydrophobic (water fearing): You know that certain types of liquids (oil, gasoline) do not dissolve in water. The molecules that make up these liquids do not usually contain polar covalent bonds, and hence do not interact strongly with water through H bonding. The prefer to interact with themselves, causing them to "shun water" and be insoluble in water. Hydrophobic interactions between molecules occur because nonpolar parts of one molecule prefer to interact with nonpolar parts of another molecule. This was the basis for the interaction of your nonpolar dyes in Lab 1 with the nonpolar beads.

These same types of interactions allow binding of the inhibitor to HIV protease. Their binding surfaces are complementary. The list below shows the contacts between HIV protease and the inhibitor. Try to identify them in the active site file you just loaded.

Important Note: The first pdb file you loaded did not contain H atoms, since they are too small to be detect when the x-ray structure of the molecules was determined. In the second pdb file showing the active site, "polar" H atoms have been added to the protein and inhibitor by another software program called MOE. The chart below shows the H bonds occurring between N and O atoms, not between Hs on N and O atoms and N and O atoms. In the cases listed, a H atom is assumed to be linked by a covalent bond to one of the listed O or N atoms.

In the cells below, listings for the amino acids have the 3 letter name and number of the amino acid with the next letter representing the atom (O is oxygen, etc). For the inhibitor, MK1902 is the name of the inhibitor and the next letter stands of the atom followed by the number of that atom in the inhibitor.

Atom number	Type contact	Protease chain	Amino acid(#), atom	Atom on inhibitor
23	H-BOND	A	ASP25.OD1	MK1902.O2
37	H-BOND	B	ASP25.OD1	MK1902.O2
38	H-BOND	B	GLY27.O	MK1902.N4
39	H-BOND	B	ASP29.OD2	MK1902.O4
101	HYDROPHOBIC	A	LEU23.CD2	MK1902.C16
102	HYDROPHOBIC	A	VAL32.CG2	MK1902.C6
103	HYDROPHOBIC	A	ILE47.CD1	MK1902.C7
104	HYDROPHOBIC	A	ILE50.CG1	MK1902.C29
105	HYDROPHOBIC	A	VAL82.CG1	MK1902.C20
106	HYDROPHOBIC	A	ILE84.CD1	MK1902.C14
148	HYDROPHOBIC	B	VAL32.CG2	MK1902.C27
149	HYDROPHOBIC	B	ILE47.CD1	MK1902.C27
150	HYDROPHOBIC	B	ILE50.CG1	MK1902.C7
151	HYDROPHOBIC	B	ILE84.CD1	MK1902.C28

You can visualize the H bonds in VMD by:

selecting Graphics from the VMD main window
selecting Representations,
creating a new representation called H bonds.
select drawing method: H bonds
increasing distance cutoff to 4.0.
select Mouse, Labels, Atoms 1 and see if you can identify the atoms involved in H bonds listed in the table above.

Now create a new representation to change the rendering of the inhibitor to better identify nonpolar:nonpolar (hydrophobic) interactions

selected atoms as hetero
drawing method VDW
material as transparent
coloring method as element
select Mouse, Labels, Atoms 1 and see if you can identify the C atoms involved in nonpolar interactions listed in the table above.

Hope you found this evening interesting and applicable to your interests.

Submitted Images from Fall 2008