Activity: Nanoscale Interactions in
Hemoglobin Function and Disease
Steve Oden, MathScience Innovation Center
Developed with funding from the MathScience Innovation Center
Questions
How is hemoglobin function influenced by its shape? Like all proteins,
hemoglobin is a nano-scale molecule whose function is dictated by its
structure. Innovative techniques for modeling molecular structure can reveal
new insights into hemoglobin structure. Tertiary and quaternary folding of a
protein results from hydrophobic and ionic interactions among amino acid side
chains (= R-groups). Hemoglobin’s function as an oxygen courier is an
emergent property of the protein’s quaternary structure. The oxygen
dissociation curve illustrates the shape-influenced affinity of hemoglobin for
oxygen: the affinity increases as more oxygen is bound and decreases as more
oxygen is released. The Bohr shift illustrates the change in affinity as pH
changes: affinity increases in oxygen-rich tissues where pH is higher and
decreases in oxygen-poor tissues where pH is lower. R-group interactions also
are responsible for the compromised function of sickle-cell hemoglobin. Sickle
cell anemia is due to a single point mutation that changes a hydrophilic amino
acid to a hydrophobic one on the beta chains of hemoglobin. This results in a
hydrophobic interaction with R-groups in a neighboring alpha chain.
Virginia
Standards of
Learning
2010 Biology (BIO.3, BIO.5)
21st Century
Curriculum
Nanotechnology: Forces; Models & Simulations
Materials
Part I. Shape-Mediated Oxygen Affinity of Hemoglobin
 Oxygen dissociation graph axes
 Data table of O2 saturation of hemoglobin vs. PO2 of tissues
 Tetrad of O2
 Scotch tape
 Scissors
 Data Table
Part II. Hydrophobic Interactions in Sickle Cell Hemoglobin
 Laptops – one per group of 2 students
 Program “Cn3D” installed on each laptop
 Protein files: normal and sickle hemoglobin
Nanoscale Interactions in Hemoglobin
Function and Disease
http://MathInScience.info
© MathScience Innovation Center, 2005
Safety
Directions
Exercise caution when using scissors.
Part I.
1. Use four pieces of transparent tape to connect the 4 “O2” squares as shown:
2. Bend the top left and bottom right corners of the tetrad toward each other and
connect with a piece of tape.
3. Repeat Step 2 for the other tetrad corners so that they are connected on the
opposite side from the first 2:
4. Each square of the tetrad represents one O2 molecule. Remove one square from
the tetrad. In the table below, record how many “bonds” you had to sever to
remove this first square and calculate the number of bonds remaining.
Table 1.
# Bonds Remaining
% Tetrad Remaining
6
100
5
4
Nanoscale Interactions in Hemoglobin
Function and Disease
http://MathInScience.info
© MathScience Innovation Center, 2005
3
2
1
0
5. Graph the data in Table 1 on the following axes:
6. How does the number of bonds required to remove the first “O2” compare to the
number required to remove the last O2?
7. What does this suggest about the ease with which hemoglobin unloads oxygen?
8. The data in the following table shows the oxygen saturation of hemoglobin as the
surrounding oxygen concentration changes. This represents the change in oxygen
concentration of the tissues as the blood moves through the body. At low PO2, the
tissues are far removed from the lungs and deficient in oxygen.
Table 2. % Saturation of hemoglobin vs. PO2 of surrounding tissues.
Nanoscale Interactions in Hemoglobin
Function and Disease
http://MathInScience.info
© MathScience Innovation Center, 2005
9. Graph the data in Table 2 on the following axes:
10. How does this graph illustrate cooperative binding of oxygen by hemoglobin?
Nanoscale Interactions in Hemoglobin
Function and Disease
http://MathInScience.info
© MathScience Innovation Center, 2005
11. How does this graph relate to the one you drew in Step 5?
12. Compare the images of hemoglobin in Figure 1. How does the deoxygenated
molecule differ from the oxygenated one? What does this suggest about the role of
shape in oxygen dissociation?
13. What might be causing the change in shape as oxygen saturation changes?
Part II.
1. Double-click on the file “normal hb” to load the normal hemoglobin structure.
2. Maximize the window and move the primary sequence window down toward the
bottom of the screen.
3. Go to the “View” menu to learn how to turn the image and to zoom in on it.
4. Under the “Style” menu, go to “Edit Global Style” and remove the helix objects.
5. How many peptide chains or subunits comprise this hemoglobin molecule?
6. In the primary sequence window, find amino acid #6 in chain “B” and doubleclick it to highlight it. What is this amino acid?
7. In the “Select” menu, click on “Annotate” and then on “New.”
8. Name it “Normal.”
9. Click “Edit Style”.
10. Click on the “Protein Backbone” radio button.
11. Move over and choose “Ball and Stick.”
12. Move over and choose “Hydrophobicity.
13. Click on the “Labels” tab and click the protein backbone spacing to 1.
14. Click on the “Details” tab and click the space fill size to .6.
15. Close out the global style windows by clicking OK and Done.
16. Click between lines in the primary sequence window to remove the highlights.
What color is the amino acid side chain you edited?
17. The color indicates whether the side chain is hydrophobic or hydrophilic. What
property does the color of this side chain (glutamic acid) suggest?
Nanoscale Interactions in Hemoglobin
Function and Disease
http://MathInScience.info
© MathScience Innovation Center, 2005
18. What is the function of the Fe atoms?
19. Close Cn3D. Save your file, if you wish.
20. Double-click on “sickle hb” to load the structure for sickle cell hemoglobin.
21. Follow the instructions in Step 4 to remove the helix objects.
22. Chain B and chain H are both beta chains in the hemoglobin molecule. Identify
the other beta chains and the alpha chains.
23. The mutation causing sickle cell changes amino acid #6 on the beta chains of
hemoglobin. Double-click on amino acid #6 in the H chain to highlight it. What is
this amino acid?
24. Repeat Steps 7–15 to create a new annotation. Instead of “normal,” name this
annotation “sickle.”
25. Remove the highlights in the molecule by clicking between the lines in the
primary sequence window. How does the color of the side chain of amino acid #6
differ from that of normal hemoglobin?
26. Based upon the color, is this side chain hydrophilic or hydrophobic?
27. Because this amino acid, valine, has a hydrophobic side chain it will interact with
neighboring side chains differently than the side chains of glutamic acid in normal
hemoglobin.
28. Double-click on the #6 valine in chain H to highlight it.
29. Go to the “Show hide” menu and select “Select by distance.” (This will let us
choose what things to show and how far away from our highlighted amino acid we
want them to be.)
30. Choose “Other residues” and select 4 angstroms.
31. You should see some other parts of the hemoglobin turn yellow. These are
molecules located within 4 angstroms of the valine.
32. Go back to “Style” and “Annotate.” Make a “new” annotation and name it “other
residues.” Edit the style as before: Protein sidechains, Space fill, Hydrophobicity;
Labels = 1, Space fill = .6. Done, OK, Done.
33. Click between the lines in the primary sequence window and then click on the
blue bar and slide the viewer down and out of the way.
34. Rotate the model and zoom to a little to view the visible R-groups. Do you see any
R-groups that might interact with one another? Which ones? Why do you think
Nanoscale Interactions in Hemoglobin
Function and Disease
http://MathInScience.info
© MathScience Innovation Center, 2005
they might interact?
35. Some protein folds are held together by hydrophobic interactions among proximal
R-groups. Does this appear to have happened in this molecule?
36. Locate, highlight, and annotate the mutated amino acids in the other beta chains.
Might you expect additional hydrophobic interactions between these amino acids
and other hemoglobin molecules?
37. In sickle cell anemia, hemoglobin molecules will clump together and form fibers.
Why does this not happen in normal hemoglobin?
38. This clumping only occurs as the hemoglobin becomes less saturated with oxygen.
Referring back to what you learned in Part I, why do you think this happens?
Nanoscale Interactions in Hemoglobin
Function and Disease
http://MathInScience.info
© MathScience Innovation Center, 2005