Model Mélange Physical Models of Peptides and Proteins In the Model Mélange activity, you will visit four different stations each featuring a variety of different physical models of peptides or proteins. The purpose of this activity is two-fold: to explore levels of protein structure secondary, tertiary and quaternary structure. Primary structure will be explored briefly in the context of secondary structure. to explore different model formats. Each format provides useful, but different, information regarding the structure. The worksheets that follow will guide your exploration of each station. The CPK Color Scheme The standard colors of different atom types.established by Corey, Pauling (as in Linus) and Kolton. Carbon is grey (or black) Oxygen is red Nitrogen is blue Hydrogen is white Phosphorus is orange Sulfur is yellow Zinc is green Note that hydrogen atoms are typically NOT displayed in these models. There are two reasons for this: X-ray crystallography, which has been used to determine the structure of many of the models, does not resolve the position of hydrogen atoms. Because there are so many hydrogen atoms in many structures, if they were displayed, most of the rest of the structure would be obscured.
Station 1: Secondary Structure: α Helices There are three different depictions of the α helix at this station: 1. α helix construction kit: standard CPK colors, hydrogen bonds are depicted by metal posts with white balls. The green dots are on the central carbon atom (the α carbon) of each amino acid. This is the carbon atom to which the sidechain, amino and carboxyl groups are attached. 2. α helix without sidechains: CPK colors, green dot on α carbon. 3. α helix with sidechains: this is the same model as #2, but the sidechains have been added. Note that the backbone atoms are colored in light CPK colors (light grey is carbon, pale blue is nitrogen, pink is oxygen). Sidechains are in normal CPK colors. Alpha carbon atoms are identified by a green dot. Use these models to work through the activities below and answer the questions. 1. Identify the amino (N-terminal) and carboxy (C terminal) ends of the α helix. N terminus has a blue nitrogen atom. C terminus has a red oxygen atom (part of the carbonyl group). 2. How many amino acids are depicted in this structure? 14 3. How many hydrogen bonds stabilize this structure? 10 4. Numbering of a peptide begins at the N terminus and goes toward the C terminus, based on the order in which the peptide was made in the cell. Complete the following statement that describes the hydrogen bonds that stabilize the α helix: The carbonyl oxygen of amino acid number 1 is hydrogen-bonded to the amino nitrogen of amino acid number 5. 5. What are the four atoms that make up the repeating structural unit (backbone) of this model of the α helix? Nitrogen Carbon Carbon Oxygen
6. Describe (or draw) the path the backbone takes in 3D space. It looks like a spiral or spring. 7. The pitch of the helix is defined as the number of repeating units per turn. What is the pitch of the α helix? About 3.3 amino acids 8. What feature contributes most to the stability of the α helix? The hydrogen bonds between backbone atoms. 9. Beginning at the N-terminal end, what is the amino acid sequence of this peptide? NH 2 -Thr-Pro-Glu-Glu-Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp-Gly-Lys-COOH 10. Examine the model and predict which face of the helix is directed toward the center of the protein. (Recall that the protein folds in the aqueous environment of the cell.) List the amino acids on this face. Also list the amino acids which are on the face exposed to the outside of the protein. a. Amino acids directed toward the inside: Glu, Ala, Val, Leu, Trp b. Amino acids directed toward the outside: Glu, Lys, Ser, Thr, Trp c. What principles of chemistry influenced your selection of these amino acids? Polar amino acids will face outwards, toward the watery environment of the cell. Hydrophobic amino acids will face inward to avoid interacting with water. 11. If there is time, use the α helix construction kit to build a model helix A of β globin (see laminated sheet). 12. Summarize what you have learned about α helices. Answers will vary.
Station 2: Secondary Structure: β Helices There are three different depictions of the β sheet at this station: 1. β sheet construction kit: standard CPK colors, hydrogen bonds are depicted by metal posts with white balls. The yellow dots are on the central carbon atom (the α carbon) of each amino acid. This is the carbon atom to which the sidechain, amino and carboxyl groups are attached. 2. β sheet without sidechains: CPK colors, yellow dot on α carbon. 3. β sheet with sidechains: this is the same model as #2, but the sidechains have been added. Note that the backbone atoms are colored in light CPK colors (light grey is carbon, pale blue is nitrogen, pink is oxygen). Sidechains are in normal CPK colors. Alpha carbon atoms are identified by a yellow dot. Use these models to work through the activities below and answer the questions. 1. Identify the N-terminal and C terminal ends of the peptides that make up this threestranded β sheet. There are two strands. Both strands begin with a blue nitrogen atom (the N terminus). These near each other at one end of the protein. Both strands end with a red oxygen atom (part of the carbonyl group of the end amino acid). Because one of the strands is short, and the other strand forms a turn and doubles back on itself, the two C termini are at opposite ends of the model. 2. β sheets can be either parallel (all strands running in the same direction from N terminus to C terminus) or antiparallel (alternating strands are oriented in the opposite direction). Which type of β sheet is represented in this model? If you start at the N-terminus of the two strands, they are parallel to each other. (The N- termini are next to each other.) Because the middle strand forms a hairpin loop to form the third strand, these two strands are antiparallel. The N terminus and C terminus of this looped strand are next to each other. 3. Which type of β sheet could contain a hairpin (looped) structure? An antiparallel β sheet.
4. What role do sidechains play in the hydrogen-bonding that stabilizes protein secondary structure? Sidechains are not involved in the hydrogen bonds that stabilize the β sheet. 5. How many amino acids are there in the longest β strand in this model? 21 6. Beginning with the N-terminal end of the hairpin, what is the sequence of the first eight amino acids of this peptide? NH 2 -Pro-Ile-Leu-Val-Glu-Leu-Asp-Gly-COOH 7. What pattern do you notice about the orientation of each successive amino acid sidechain relative to the plane of the β sheet? They are on opposite faces of the plane of the β sheet. 8. How many hydrogen bonds stabilize this structure? 20 9. What are the four atoms that make up the repeating structural unit (backbone) of this model of the β sheet? Nitrogen Carbon Carbon Oxygen 10. Describe (or draw) the path the backbone takes in 3D space. It zig-zags up, down, up, down. 11. The pitch of the sheet is defined as the number of repeating units per turn. What is the pitch of the β sheet? There are two amino acids per turn a zig and a zag.
12. Examine the model and predict which face of the sheet is directed toward the center of the protein. (Recall that the protein folds in the aqueous environment of the cell.) List the amino acids on this face. Also list the amino acids which are on the face exposed to the outside of the protein. a. Amino acids directed toward the inside: Leu, Asn, Ile, Leu, Leu Ile, Val, Leu, Gly, Val, Thr, Val, Gly b. Amino acids directed toward the outside: Val, Arg, Glu, Leu Pro, Leu, Glu, Asp, Asp, Lys, Ser, Ser, Glu c. What principles of chemistry influenced your selection of these amino acids? Polar amino acids will face outwards, toward the watery environment of the cell. Hydrophobic amino acids will face inward to avoid interacting with water. 13. Summarize what you have learned about β sheets. Answers will vary.
Station 3: Tertiary Structure: Zinc Fingers This station focuses on tertiary structure utilizing zinc fingers. Zinc fingers share a common structure which includes 27 amino acids arranged in a two stranded β sheet and a single α helix. A zinc atom stabilizes the secondary structure and is coordinated by four amino acids a combination of cysteines and histidines. This particular zinc finger has two cysteines and two histidines. The zinc finger motif is often found in proteins that interact with DNA. [You can see a zinc finger in association with the DNA in one of the zinc finger models on the stand.] Perhaps you have heard the story of the seven blind men and the elephant? Each man felt a different part of the elephant and described it, then argued about which description was right. Another man resolved the argument by pointing out that each man had only seen a part of the elephant, and that an elephant included ALL of their descriptions. Similar to the elephant story, there are several models of zinc fingers at this station. Each depicts different aspects of the protein. When trying to tell a story about how a protein functions, scientists determine which features of the protein they want to emphasize, then choose the depiction that best illustrates those features. Match each physical model of the zinc finger to the correct format listed below, then list one positive and one negative feature of that format. α Carbon Backbone Format Advantage: It is easy to see secondary protein structure and the overall fold of a protein. Disadvantage: There aren t any details about the overall shape of the protein or the specific amino acids. All Atoms Backbone Format Advantage: You can see the hydrogen bonds that stabilize secondary structure. Disadvantage: There aren t any details about the overall shape of the protein or the specific amino acids.
All Atoms Format Advantage: You can determine the amino acid sequence from the structure and see how amino acid sidechains interact with each other. You can also trace the fold of the protein. Disadvantage: It is difficult to know which amino acids are important for protein function. Surface Format Advantage: You can see the overall shape of the protein and how it might interact with DNA or other molecules. Disadvantage: You can t see the fold of the protein or individual amino acids. α Carbon Backbone with Selected Sidechains Format Advantage: You can clearly see the fold of the protein and the important sidechains involved in protein function. Disadvantage: There is no information about amino acid sequence. If you only had one model format to use in your classroom, which format would you find most useful? Answers will vary.
Station 4: Quaternary Structure Some, but not all, proteins contain multiple peptides. This station allows you to explore several proteins that demonstrate this quaternary structure, as well as a few that lack quaternary structure. As you explore each of these proteins, see if you can determine: 1. How many subunits are in the protein? 2. Based on this information, does the protein have quaternary structure? 3. Are there α helices in the protein? (If so, how many helices?) 4. Are there β sheets in the protein? (If so, how many strands?) Aquaporin There is a single subunit, so this protein has no quaternary structure. There are 7 α helices and no β sheets. GFP (Green Fluorescent Protein) There is a single subunit, so this protein has no quaternary structure. There are 5 very short α helices and 11 β strands that form a β barrel. Hemoglobin Hemoglobin consists of 4 subunits (2 alpha globin and 2 beta globin), so it has quaternary structure. Each subunit has 7 α helices and no β strands. Beta-Globin This is a single subunit of hemoglobin, so it has no quaternary structure. It has 7 α helices and no β strands. Influenza HA (Hemagglutinin) This protein consists of three identical structures, each with two different protein strands, so it has quarternary structure. The shorter strand that mostly makes up the stalk of the protein has 4 α helices (two of them are very long) and 5 β strands. The longer strand that forms the bulb at the top of the protein has 4 shorter α helices and 30 β strands. MHC (Major Histocompatibility Complex) There are two strands in this protein, so it has quaternary structure. There are 2 α helices and 22 β strands. It is easy to see the 5 β sheets in this protein. F0 complex (from ATP synthase) This structure has 10 subunits (so it has quaternary structure), each consisting of 2 α helices.