Guided Inquiry Tutorial on Protein Structure

Amino acids are the building blocks of proteins (the figure below is the amino acid methionine). Each amino acid is made up of the atoms Hydrogen (H), Carbon (C), Oxygen (O), Nitrogen (N), and Sulfur (S).

There are 20 naturally occurring amino acids. The figure shows the structure of the free amino acid methionine. Free indicates that the amino acid is not covalently bound into a protein. In the figure:

• The small black spheres are H atoms
• The gray spheres are C atoms
• The red spheres are O atoms
• The blue spheres are N atoms
• The yellow spheres are S atoms
• The rods represent chemical bonds

There is one N atom in methionine, since we see only one blue sphere. Similarly, there is only one S atom in methionine, since we see only one yellow sphere.

Question A: How many C atoms are there in methionine?

Answer:

Each amino acid is divided into the backbone and the side chain. All of the amino acids have the same atoms in their backbone. They have different atoms in their side chains. You can see a list of all twenty amino acids at this link or “The Amino Acids” link.

In the figure, we again show the free amino acid methionine (MET), this time without the hydrogen atoms. The backbone and the side chain are labeled, with the side chain atoms transparent. To see an animation of MET and to see some of its properties, click on this link or “Methionine” in the list of amino acids.

Question B: Looking at the figure, or at the animation, we can see that the backbone of free methionine has, not counting H atoms:

1. Three carbons and one sulfur
2. Two carbons, one nitrogen, and two oxygens
3. Two carbons, one sulfur, and one oxygen.
4. One carbon, one nitrogen, and two oxygens

Answer:

Proteins are formed when the backbones of two amino acids are joined together by what is called the peptide bond.

To form a peptide bond between two amino acids, an O in the backbone of the first amino acid is replaced by an N in the backbone of the second amino acid, releasing a water molecule (H2O). The two amino acids are then bound together by the remaining CO-NH link, which is called the peptide bond.

The figure shows six amino acids bound by peptide bonds into a short protein. The animation at this link, also available from “The Peptide Bond” page, shows these six amino acids as they join together.

Question C: How many water molecules are formed when the six amino acids form the short protein shown?

Answer:

We want to explore the structure of an important protein, human deoxy hemoglobin, using the StarBioChem Viewer. There should be a shortcut to this program on your computer desktop.

The Protein Data Bank ID sequence for human deoxy hemoglobin is 1A3N. In this problem we will look at the overall shape of this protein, and also look at its ordered sequence of amino acids. This ordered sequence is called the primary structure of 1A3N. We will also look at atoms in the protein which are not part of an amino acid; these non-amino acid atoms are grouped into structures called heteroatoms. We first need to load hemoglobin.

Loading 1A3N:

• Left click on the “File” tab in the upper left corner of the Viewer
• Choose “Open/Import” from the pull down menu using the left mouse button.
• In the dialog box, select 1A3N by left clicking on name of the protein. It is the first one on the list.
• Load 1A3N by going to the bottom of the dialog box and left clicking on the “Open” button.

When 1A3N loads, you will see an image of the protein showing all of the chemical bonds, but no atoms or other structures.

Changing Viewpoint in the View Window

• TO ROTATE the protein, left-click and drag the mouse.
• TO MOVE UP/DOWN RIGHT/LEFT, right-click and drag the mouse
• TO ZOOM, Alt-left-click and drag the mouse (or middle-click and drag if you have three buttons on your mouse)

If the structure disappears, and anything weird happens, you can reset by left clicking on the “View” tab and choosing “Reset Molecule”.

The first thing you will need to do is to zoom out so you can see all of the protein.

Take a minute to look at the structure of this protein just looking at this “bonds only” view from various angles. Answer the following question.

QUESTION A: Which of the following is true about the shape of human deoxy hemoglobin?

Check one of the options below:

• It is a spherical ball of amino acids with no other structure
• It has a hole right through the middle
• It is shaped like a dumbbell

This protein has many many amino acids, and we want to show you how to find a list of those. The “Structure/primary” panel contains this list, numbered according to their sequence in the protein. To see this list,

• Open the “Structure” panel by left clicking anywhere in the bar (on the right) labeled “Structure”.
• Left-click on the “primary” tab to select it
• You will see a numbered list of the amino acids in this protein. Scroll down using the right scroll bar to see them all.
• There may be more than one numbered list of amino acids.
• If so those amino acids correspond to different chains (discussed later), and there will be a different color for every chain.

Question B: How many amino acids are there in the first chain of human deoxy hemoglobin?

Answer:

In addition to the amino acids, proteins also have structures called heteroatoms. The heteroatoms in 1A3N are called hemes, and carry oxygen in the blood stream.

To see the heteroatoms in 1A3N, do the following:

• Reset 1A3N to its initial configuration by selecting “Reset Molecule” in the “View” menu.

• Open the “PDB Tree” panel by left-clicking anywhere in the bar labeled “PDB Tree”.
• Double left-click on the box labeled “1A3N” to open it.
• Click on “Hem_142” to select it. Ctrl-left-click on the remaining “Hem_147”, “Hem_142”, and “Hem_147” in turn to select all of them as well. Be sure you “Ctrl-left-click” or you will deselect the heteroatoms you have already selected. You should now have four heteroatom boxes selected.
• Open the “View Controls” panel by left-clicking in the bar labeled “View Controls”.
• In the View Controls panel, set the “Unselected” transparency slider to 0.2. You can do this either by moving the slider or entering 0.2 in the transparency box.
• Rotate the view in the View Window to see how the hemes are oriented.
• Check off “Draw” in the “Atoms” box to see what atom types are present.

Question C: Do the oxygen atoms in the hemes lie on the surface of hemoglobin or are they buried deep inside the protein?

Check one of the options below:

• They are buried deep within the protein
• They lie on the outside close to the surface of the protein

The secondary structure of a protein refers to how the backbones of the amino acids are arranged in space in characteristic patterns. We want to explore the secondary and higher structure of 1A3N using the StarBioChem Viewer, the same program as in the previous problem.

There are over 500 amino acids in 1A3N, and with this amount of information it is hard to see much of the secondary structure in the protein. We can see more of the underlying structure if we choose a part of the structure and make the other parts invisible.

Selecting A Helix

• Reset 1A3N to its initial configuration by selecting “Reset Molecule” in the “View” menu.
• Open the “Structure” panel and click on the “Secondary” tab. Check the box next to “31” under “Helix Selection”

• Open the “View Controls” panel.

• In the “View Controls” panel, set the “Unselected” transparency slider to 0. You can do this either by moving the slider or entering 0 in the box next to the slider.

• Set the “Sidechain” transparency slider to 0.
• Left-click on the “Draw” box under “Atoms” to turn on drawing of the atoms.

You should see something like the figure to the right. This figure shows that these particular backbone atoms are arranged in what looks like a helix. We call this an  helix. Be sure to rotate the  helix in the View Window to look at this from a variey of viewpoints (in particular, try to look right down the axis of the helix). If you have forgotten how to rotate etc., go back to page 4 of these instructions.

The figure only shows the backbone atoms.

• Go the the “View Controls” window and move the slider for the “Sidechain” transparency back and forth, to turn the side chains on and then back off.

Question A: How are the side chains of the amino acids that make up the  helix oriented with respect to the inside and outside of the helix?

Check one of the options below:

• Some of the side chains point in toward the interior of the  helix and some point out away from the interior of the  helix.
• All of the side chains point in toward the interior of the  helix.
• All of the side chains point out away from the interior of the  helix.

We highlight the  helices and other secondary structures by tracing their shapes with visual guides called ribbons. The ribbons are there to guide the eye so that you can pick out patterns that might be hard to discern in a protein with a large number of residues.

Turning On the Ribbon for Helix A31

• Open the “Structure” panel.
• Select “Secondary”.
• Check the “Track Selection” box, the “Helices” box, and make sure you still have the box next to “31” under “Helix Selection” checked.

You should now see the ribbon for Helix 31 of 1A3N. Let’s look at some more of these structures.

Turning On All the Secondary Structure Ribbons for 1A3N

• Open the “Structure” panel. Select “Secondary”.
• Uncheck “Track Selection” (the box should be empty).
• Check the box next to “All Ribbons”.

You will now see all the ribbon structures for 1A3N. You may have to zoom out to see them all. Click on the different ribbon types (helix, sheet, and coil) to see what they look like.

Question B: Which one of the following secondary structures does 1A3N NOT have? Check one of the options below:

• random coils (unstructured)
• β sheets
• α helices

We now want to look at larger scale structure in the protein. Quaternary structure is the interaction of multiple protein chains.

Quaternary Structure by Color

• Reset 1A3N to its initial configuration by selecting “Reset Molecule” in the “View” menu.
• Open the “Structure” panel and left click on the “quaternary” tab

• Under “Color by:”, left click in the box labeled “Chain”
• Open the “View Controls” and check both “Draw” and “Space-fill” under “Atoms”.
• At the bottom of the “View Controls” panel, left click on the “Show All” button.

Question C: Describe the quaternary structure of hemoglobin.

Check one of the options below:

• Pentamer (5 chains)
• Tetramer (4 chains)
• Trimer (3 chains)
• Dimer (2 chains)
• Monomer (1 chain)

We want to explore what mutation in 1A3N causes sickle cell anemia, using the StarBioChem Viewer, the same program as in the previous problem.

A single amino acid mutation in hemoglobin causes sickle cell anemia. Affected red blood cells have a sickle shape caused by the formation of long chains of mutated hemoglobin. The mutation that causes the disease is a substitution of an acidic, polar glutamic acid (Glu_6) in the B and D chains by a nonpolar amino acid, valine.

Question A: Choose from the possibilites below about the properties of the amino acids glutamic acid (GLU) and valine (VAL).

Check one of the options below:

• Glutamic acid is polar, valine is polar
• Glutamic acid is polar, valine is nonpolar
• Glutamic acid is polar, valine is acidic
• Glutamic acid is polar, valine is basic

Let's locate Glu_6 in the B chain of 1A3N and look to see how it is situated in the protein. This will help us understand how replacing Glu_6 with a valine causes such problems. Displaying the Glu_6 in the B Chain:

• Reset 1A3N to its initial configuration by selecting “Reset Molecule” in the “View” menu. It may take a little while to reset the molecule at this poit, so be patient.
• Open the “Structure” panel and left-click on “primary”. Scroll down to the B chain, and left-click on “Glu_6”

• Go to the “View Controls” panel. Set the transparency of “Unselected” down to 0.2. Remember if you enter 0.2 in the box you must HIT ENTER for it to take effect.
• Left-click on the “Draw” box under “ATOMS”
• Rotate the view until Glu_6 is in the foreground of the View Window (e.g. close to the front).

Question B: Where is Glu_6 located in hemoglobin?

Check one of the options below:

• Glutamic acid is polar, valine is polar
• Glutamic acid is polar, valine is nonpolar
• Glutamic acid is polar, valine is acidic
• Glutamic acid is polar, valine is basic

Believe it or not, replacing Glu_6 in the B chain with a valine, and Glu_6 in the D chain with a valine, causes the hemoglobin to stick to other similarly mutated hemoglobin proteins. In the mutation, the exposed nonpolar valine is very hydrophobic (or water hating) and wants to be away from the polar solvent. In order to “hide” from the water, this valine associates with a nonpolar cluster of residues from a nearby hemoglobin. This nonpolar cluster of residues to which valine “sticks” is called a hydrophobic pocket, and consists of the amino acids Phe_85 and Leu_88.

Displaying the Mutated Residue and the Hydrophobic Pocket to Which It “Sticks”

• Reset 1A3N to its initial configuration by selecting “Reset Molecule” in the “View” menu.
• Open the “Structure” panel and left-click on “primary”. Scroll down to the B chain, and left-click on Glu_6. Scroll down further in Chain B and “Ctrl-left-click” on Phe_85 and then “Ctrl-left-click” on Leu_88. Make sure you are “Ctrl-left-click” ing and not just left-clicking, otherwise you will deselect the earlier amino acids.
• Scroll down to the D chain and “Ctrl-left-click” on Glu_6, Phe_85 and Leu_88 in succession. You should now have six residues selected.
• Go to the “View Controls” panel, set the transparency of “Unselected” to 0.2, and check “Draw”.
• Rotate the view until your six residues are in the foreground of the View Window (e.g. close to the front).

Question C: The two mutated residues and the two hydrophobic pockets have the following positions on hemoglobin:

Check one of the options below:

• It is located close to the middle of the protein
• It is located on the surface of the protein with its sidechains oriented toward the inside
• It is located on the surface of the protein with its sidechains oriented toward the outside

In this problem we want you to use the StarBioChem Viewer to study the structure of a protein you select from a list of proteins. The protein you select will collect points according to the criteria enumerated below. Generally, high values correspond to rare proteins, while lower values indicate more common proteins.

If you have closed your viewer, start the viewer again. Use the “File/Open” pull down menu to load any protein (other than 1A3N) in the list.

What is the 4 digit Protein Data Base accession ID of the protein you chose? (e.g. 1ZBN)

PDB ID:

If your protein only has one chain, what is the length of that chain, in numbers of amino acids? If your protein is composed of more than one chain, use the number of amino acids in the longest one. Here is the point scale for the length. Note that is not linearly dependent on the number of amino acids–rather it reflects the rarity of the length of the protein you have chosen.

• 8 points: 1000 or more amino acids:
• 7 points: 800-1000 amino acids
• 6 points: 600-800 amino acids
• 5 points: 500-600 amino acids
• 4 points: 0-100 amino acids
• 3 points: 100-200 amino acids
• 2 points: 400-500 amino acids
• 1 point: 200-400 amino acids

What is the number of points for the length of your protein? Keep track of the points for each section and a running point total of your own.

Part B points Total points

Proteins contain non-amino acid groups like the heme in 1A3N, or metal ions. These are called heteroatoms and appear as separate groups in the “PDB Tree” panel. How many heteroatoms does your protein have? Here is the point scale for the the number of heteroatoms:

• 5 points: 5 or more heteroatoms
• 4 points: 4 heteroatoms
• 3 points: 3 heteroatoms
• 2 points: 2 heteroatoms
• 1 point: 1 heteroatoms
• 0 point: no heteroatoms

What is the number of points for the heteroatoms? Also keep track of the points for each section and keep a running point total of your own.

Part C points Total points

What is the secondary structure of your protein? Here is the point scale for the secondary structure:

• 5 points Only  sheets and/or random coils
• 3 points Only  helices and/or random coils
• 1 points Mixed  helices and  sheets and/or random coils

What is the number of points for the secondary structure? Also keep track of the points for each section and a running point total of your own.

Part D points Total points

What is the quaternary structure of your protein? By quaternary structure, we mean the number of chains. Here is the point scale for the quaternary structure:

• 5 points: 5 chains or more
• 4 points: 4 chains
• 3 points: 3 chains
• 2 points: 2 chains
• 1 point: 1 chain

What is the number of points for the quaternary structure? Also keep track of the points for each section and a running point total of your own.

Part E points Total points

Measure the approximate radius of a sphere that encloses your protein. Do this in the following way:

• Reset your protein by selecting “Reset Molecule” in the “View” menu
• Open the Measure Tools panel and check “Enable Radius”
• Move the radius slider until the sphere encloses most of your protein.

Note the radius value at that point and allocate points according to:

• 5 points: Greater than 100 A
• 4 points: 75 to 100 A
• 3 points: 50 to 75 A
• 2 points: 25 to 50 A
• 1 point: 0 to 25 A

What is the number of points for the size of your protein? Also keep track the points for each section and a running point total of your own.

Part F points Total points

Add up the total number of points for your protein and enter it in the box below. We will compare these values at the end of the session. Generally, high values correspond to rare proteins, while lower values indicate more common proteins.

What is the total number of points for your protein? Also give this number to an instructor.

Total points__

You are finished with the formal part of this exercise. If you want to explore other proteins on the list under the “File/Open” button, by all means do so.