Name: ___________________________________ Period: ______ Date: __________
Unit 1 - Biochemistry
Module 2 - Protein Structure Visualization and Enzyme Exploration
Introduction
You will be working with pepsin, a protein found in the human stomach. Pepsin is an enzyme that
catalyzes the reaction to break down proteins into single or small chains of amino acids. As you go
through this activity, you will learn about different ways to visualize protein structure. You will also learn
how to find the active site of an enzyme such as pepsin. Remember, the structure of a protein
determines its function. Do you think that a part of a protein that is very important for function is likely
to change a lot (have variation) or stay the same (be conserved) over evolutionary time? Follow the
steps and answer the questions in this activity to find out!
Getting started
Go to [Link] the landing page of structural information about the
pepsin protein from the human stomach. On the left side, you have different information about the
protein, and on the right side, you will have the protein structure.
A. Can you zoom in on the left to see the sequence (amino acid letters) at the top? YES or NO
B. Are you able to move the protein around on the right? YES or NO
If you answered NO to A or B watch this video for help before continuing. Otherwise, move forward to
Question 1.
Questions
1. The protein is visualized in Cartoon representation. In the Cartoon representation, the secondary
structures are shown as spirals and arrows. What secondary structure do you think the spirals and
arrows in the protein structure represent?
2. On the left-side panel, find the row for the Active site. Hover over the two pink dots representing
the key amino acids located in the active site one at a time and find the amino acid letter and
position number that correspond to these pink dots. (If you need help to identify the active site,
watch this video.)
Amino acids in the active site are important for catalysis.
The left dot corresponds to amino acid _____ and position _____.
The right dot corresponds to amino acid _____ and position _____.
How many amino acids spaced apart in the sequence are the two key amino acids? _____ amino
acids.
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3. Do you think the key amino acids are close in the 3D structure? Why or why not?
4. Click on the square icon below the wrench on the right to maximize the protein viewer
window. Notice how you can hover over an amino acid in the sequence so that it turns pink and how
it also turns pink in the structure so you can see where in the structure it is located. Information
about the selected amino acid is also shown in the information window inside the viewer. (If you
need help to maximize the protein viewer window, watch this video.)
5. Select the two key player amino acids (the active site) in the sequence at the top and click on the
paintbrush to color them magenta. Click Apply Theme after selecting the desired color.
6. Where are the key players located in the 3D structure? Are they close in space?
Remember that you can spin the protein around or scroll to zoom in and out. Some angles will give
you a clearer view while others will be blocked by other parts of the protein. (If you need help to
color the key play amino acids, watch this video.)
7. You may notice that one of the two key players is located just at the tip of an arrow. What are the
arrows? What are the spirals?
arrows _________________________________ spirals ___________________________________
8. Turn your attention to the sidebar on the right. Next to A you can see that it says Cartoon, which
means that the protein is shown using the Cartoon representation. The Cartoon (also called ribbon)
representation and green color for the entire protein are the default starting representation. To
change the color of the entire protein, click the 3 dots, select Set coloring, select Residue Property,
and click Secondary Structure. (If you need help to color the protein, watch this video.)
What color are alpha helices? __________________________
What color are beta strands? ___________________________
The parts colored in darker purple denote a different type of helix so we can refer to these and
the alpha helices together as helices.
The parts colored white are loops that connect the different secondary structure elements and
add structural flexibility to the protein.
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9. Describe the structure based on how the secondary structure elements are organized. Note that
the icon above the wrench lets you take a good screenshot that you can use as an aid to describe the
structure. (If you need help to find the screenshot button, watch this video.)
Paste the screenshot:
10. The cartoon representation is easy to understand, but other representations may be better for
certain tasks. You could display the protein in a surface representation. Follow the steps: in the A
row next to Cartoon Representation, click the eye icon to hide this view (the protein will go away;
it is ok). Then, select the 3 dots in the A row, select Add Representation and choose the Molecular
Surface representation. The protein will now appear using the new representation. (If you need help
showing surface representation, watch this video.)
Describe the protein structure:
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Take a screenshot of the Surface Representation and paste the picture here:
11. Color the molecular surface representation by accessible surface area to help identify the active site
that is in a deep blue-green pocket. Follow the steps: select the 3 dots in the A row, select Set
Coloring, select Residue Property and, select Accessible Surface Area. (If you need help coloring by
accessible surface area, watch this video.)
Describe what you see now:
The protein surface will be colored in a rainbow scale where orange = more accessible, and blue =
less accessible. Look for the active site in the structure by hovering or clicking on the key player
amino acids in the sequence at the top.
When you have found it, take a screenshot of the protein showing the active site in the blue-green
pocket with Aspartate 32 highlighted.
Paste the Accessible Surface Area picture here:
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12. Think Critically: Pepsin is an enzyme, a protein with a catalytic function. The active sites in enzymes
tend to be similar in sequence and structure across different species. You can compare the sequence
similarity between the human pepsin you have been working with and the pepsin enzymes from a
few species you may recognize.
Species Dog Alligator Frog Shark
Percent Similarity 81.4% 69.8% 68.0% 59.7%
Table 1. Percent identical amino acids in human pepsin vs pepsin in dog, alligator, frog, and
shark.
Where in the pepsin structure do you expect to see more sequence variation across species (many
amino acid differences in the sequence) and sequence conservation across species (few amino acid
differences in the sequence), respectively? Why? Add your prediction of whether you think the
locations in the table are generally conserved or variable.
My predictions
Location Conserved Variable
Active site
Surface (amino acids on the
outside of the protein)
Buried (amino acids on the inside
of the protein)
Secondary structure (amino acids
in alpha helices or beta strands)
Loop (amino acids not in alpha
helices or beta strands)
13. Check your Thinking:
ConSurf is a bioinformatics tool that shows a protein colored by variation (green) and conservation
(purple) based on scores calculated from large sequence datasets of evolutionarily related proteins.
Look at the structure of pepsin colored by ConSurf here. What ConSurf color are the key players in
the active site? What does that tell you about the level of conservation or variability found at the
active site?
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14. Challenge Question: Look at the structure of pepsin colored by ConSurf here. Describe the overall
observation of the level of conservation and variability of a) alpha helices, b) beta strands, and c)
loops. Which elements appear the most and the least conserved?
15. Look at the structure of pepsin colored by ConSurf here. Describe the pattern of conservation and
variability seen on the surface of the protein. How does this pattern compare to the conservation
seen at the active site, which is also found on the surface?
16. Compare the structures of pepsin colored by ConSurf here. Describe the pattern of conservation and
variability seen in the buried amino acids. How does that compare to the pattern of conservation
seen at the surface?
17. Based on your predictions and exploration of pepsin, complete the table below (copy your
predictions from question 12).
My predictions What I observed
Location Conserved Variable Conserved Variable
Active site
Surface (amino acids
on the outside of the
protein)
Buried (amino acids
on the inside of the
protein)
Secondary structure
(amino acids in alpha
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helices or beta
strands)
Loop (amino acids
not in alpha helices
or beta strands)
18. How do your predictions compare to what you found? Based on the results, can you determine
which locations appear more important for the function of the protein? Explain.
