CELL BIOLOGY 220 JMU

CELL AND MOLECULAR BIOLOGY - BIO 214

ASSIGNMENT OF THE WEEK: #2

Principles of Protein Structure

Scope of the assignment: This assignment is designed to introduce students to the basic concepts of protein structure by allowing then to look at and manipulate 3-dimensional models of several proteins. It is intended to support an introductory-level Biology text, not to stand alone. Technical problems concerning the assignment should be sent to Dr. Jon Monroe.

Introduction. Compared with nucleic acids and carbohydrates, proteins are macromolecules that display spectacular structural diversity. This is not surprising when one considers that a typical protein is composed of a chain of hundreds of amino acids, and that at each of those positions any of the 20 amino acids can be placed. Add to that linear complexity the many possible ways that the chain can be folded into a globular shape and one is quickly overwhelmed with the prospect of understanding these structures. Despite this mind boggling prospect, after viewing the three dimensional structures of several proteins, you will begin to see patterns emerge. Out of seeming chaos comes order, and out of order comes understanding and appreciation. Yipee!

In this exercise you will examine several proteins using a program called RasMol which allows one to not only visualize models of these complex structures, but also to rotate, and highlight various features of the proteins. The models are made by RasMol but are based on atomic coordinants stored in the Protein Data Bank (PDB) at Brookhaven National Laboratories. If you are linking to this page from the computer lab in Burruss 237 (JMU), skip to the section below titled "Answer form". If you want to do this exercise on a machine that is not located in Burruss 237, you will need to download a copy of RasMol from the RasMol home page. The data files used in this exercise can also be downloaded from "Molecules to Go" via an accompanying page. Be sure to rename the files after retrieving them to your computer. Please do not attempt to download structures to the computers in Burruss 237.

Answer form

Before beginning the exercise, link to the answer form and print the resulting page. Click the "back" button to return to this page. You may work in pairs turning in one form with both names.

Loading the RasMol program and loading files

If you are in Burruss 237 load the RasMol program by double clicking on the file "rw32b2a.exe". To load the first file pull down the "file" menu at the top of the RasMol window and select "open". You should see several .pdb files that you will use in the tutorial. Feel free to look at the others as well. Open "crambin.pdb". You should see an image appear in the black RasMol window. Before beginning the tutorial realize that each time you want to open a new file you will need to "close" the current file (see the "file" menu), and repeat the steps described above. Position the RasMol and Browser windows so that you can easily click back and forth between the two.

Protein 1 - crambin

Crambin is a seed storage protein from a plant called abyssinian cabbage (Crambe abyssinica). This protein serves to store amino acids for the new seedling. It is relatively small, as proteins go, but helps to illustrate many of the common features of protein structure.

A. When the crambin structure is loaded rotate the structure by moving the mouse while holding the left mouse button down. You can slide the structure up or down, etc., by moving the mouse while holding the right mouse button down. View the structure in all available RasMol display modes starting with the default display, wireframe. To change the view, pull down "display" and select "backbone", "sticks", "spacefill", "ball& sticks", "ribbons", "strands", or "cartoons". These various displays highlight different aspects of the proteins. Change the "display" back to "sticks". The default "color" used by RasMol is "CPK" in which the protein is colored by atom (carbon=grey, oxygen=red, nitrogen=blue, sulfur=yellow, and hydrogen=white)

Question 1. How many sulfur atoms are in crambin?
Question 2. How many pairs of sulfur atoms are there?

B. Change the "display" to "backbone" to highlight only those bonds in the backbone of the molecule (side groups are not visible).

Question 3. How many "ends" does the crambin backbone have?

C. Using the display in "cartoons" mode, change the "colours" from the default which is "CPK" to "structure" in which alpha helices are pink, beta sheets are yellow, and beta turns are blue.

Question 4. How many regions of alpha helix are there in crambin?
Question 5. How many regions of beta sheet are there in crambin?
Question 6. Are the beta sheets parallel or antiparallel (hint - are the arrows pointing the same direction or in opposite directions?

D. Visualize the 27 hydrogen bonds that help to stabilize the secondary and tertiary structure of crambin click on the button at the bottom of the screen called "RasMol Command Line". In the command line window type "hbonds" (no quotes). Return to the main window. The hydrogen bonds are best seen in "display = strands", "colour = structure". A single hydrogen bond in this view will be colored according to the structure that it is in. Rotate the image to determine the locations of these bonds.

Question 7. How many hydrogen bonds are located within the larger alpha helix?
Question 8. How many hydrogen bonds link this alpha helix to the rest of the molecule?

E. Visualize the disulfide bonds by selecting the command line window and typing "ssbond". To more easily see these you may wish to turn off the hydrogen bonds by typing "hbond off". In the main window (black), find the disulfide bonds that occur between the side groups of cysteine residues. Notice how they help "knit" distant parts of the amino acid chain together stabilizing the tertiary structure. Return the "display" to "sticks" to see the cysteines.

Question 9. How many disulfide bonds are there in crambin?

Protein 2 - hemoglobin

First close the crambin file and then load the next protein, "hemoglobin.pdb", the same way you loaded the crambin file. Hemoglobin is the oxygen carrying protein that colors our blood red (when oxygenated) or blue (when deoxygenated).

A. Visualize the protein in "display = backbone" and "colour = chain" to color each different amino acid chain a different color.

Question 10. How many amino acid chains are present in hemoglobin?

B. Visualize hemoglobin in "display = strands", "colour = structure".

Question 11. Is hemoglobin composed primarily of alpha helices or beta sheets?

Protein 3 - fibronectin

Load the next protein, fibronectin, by returning to the At Ease window and clicking on the "fibronectin.pdb" button. Fibronectin is an extracellular protein in animals that is involved in cell adhesion and blood clotting.

Question 12. How many amino acid chains are present in fibronectin?
Question 13. Is fibronectin composed primarily of alpha helices or beta sheets?
Question 14. What type of bond (hydrogen or disulfide) holds the beta sheet structure together?

Protein 4 - collagen

Next, load the protein "collagen". Collagen, like fibronectin, is an extracellular structural protein that acts like rope to hold animal cells together. This is actually a model of a synthetic form of collagen with a very simple repeating pattern of amino acids.

A. Change the "display" to "sticks". Zoom in on the protein by holding the shift key down while moving the mouse (with mouse button depressed) toward you.

Question 15. How many amino acid chains are present in this model of collagen?

B. Change the "display" back to "wireframe" and label each amino acid by selecting "option = labels". The amino acids are abbreviated as three letters, the numbers refer to the position of the amino acid on each chain, and the letters A, B or C refer to the chains.

Question 16. Of the 20 possible amino acids, how many are present in this model of collagen?
Question 17. What is the repeating pattern of these amino acids?
Question 18. Do the proline side groups (rings) point inward (to the center of the axis of the triple helix), or do they point out? You can change the "display" to "backbone" then back to "sticks" to see where the side groups of proline are.

Protein 5 - cro

Next, load the protein "cro.pdb". Cro is a DNA binding protein that regulates the expression of gene involved in the infection of E. coli by the virus, Lambda.

Question 19. What, besides protein, is modeled in this structure?
Question 20. How many amino acid chains are present in this model of cro?
Question 21. What type of secondary structure (alpha helix or beta sheet) is in closest contact with the DNA?

If you are in the Burruss computer lab (room 237) please unload the software you have been using and leave the computer on. Thanks!

If you wish to further explore protein structures using this program, you can find a few more models in the "cell220" folder. Alternatively you may download any of the thousands of known protein and nucleic acid structures to your personal computer from the Molecules To Go page maintained by the NIH.