Student Handout 1 A dual coloring scheme allows students to first 3dmoleculardesigns.com Introduction - Page 1

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1 Proteins are large, linear polymers of amino acids that spontaneously fold into complex 3D shapes. Although protein structure appears to be very complex, the chemical properties that determine protein structure are relatively simple. The Amino Acid Starter Kit allows your students to explore how the unique chemical properties of the 20 amino acids determine the final shape of a protein. Two Student Handouts lead your students through the hands-on activities. Student Handout 1 A dual coloring scheme allows students to first - group the different sidechains into one of five categories: hydrophobic (nonpolar), hydrophilic (polar), negatively charged, positively charged, and cysteine... and then flip the sidechain over to - examine the atoms that make up each sidechain to learn how the different functional groups determine the chemical properties of amino acids. Your students will then attach sidechains to a mini-toober and fold it into a 15-amino acid protein, following the chemical properties of the sidechains. As they fold their proteins your students will soon discover that it is easy to find a 3D shape in which all of the hydrophobic sidechains are on the inside of their globular structure. As they continue to fold their protein, applying each additional property, it becomes increasingly difficult to find shapes that simultaneously satisfy all of the properties. Following this engaging, hands-on protein folding activity, your students will have a basic understanding of how the physical and chemical properties of amino acids determine protein structure. They also will be ready to begin a more thorough exploration of molecular structure using the computer visualization tools that are included on the CD accompanying this kit. Introduction - Page 1

2 Student Handout 2 In the first set of activities in this kit, Discovering the Properties of Amino Acids and Folding a 15-Amino Acid Protein, your students explored the primary and tertiary structure of proteins, including the role of the amino acid sidechains in determining the overall shape of a protein. The second set of activities in the kit explores the secondary structure of proteins. Simple tools allow students to explore: - the alpha helix and beta sheet are the two primary elements of secondary structure. - hydrogen bonds in alpha helices and beta sheets stabilize protein structures - the alpha helix and the beta sheet represent different ways to optimize the hydrogen bonding that can occur between the amino nitrogen of one amino acid and the carbonyl oxygen of another - although a single hydrogen bond is weak, multiple hydrogen bonds in the alpha helix and beta sheet contribute significantly to the stability of molecular structures - the alpha helix and beta sheet are features of the backbone atoms Using the mini-toobers and their fingers as bending jigs your students will form a beta sheet and stabilize it with hydrogen bond connectors. Then they will form an alpha helix and stabilize it with hydrogen bond connectors. Then with a couple of folds, you students will make a Zinc Finger and learn about its function. The computer visualization tools on the CD (included with the kit) will allow your students to begin a more thorough exploration of the Zinc Finger. National Standards National Content Standard A: Science as Inquiry Scientific explanations must adhere to criteria such as: - a proposed explanation must be logically consistent; - it must abide by the rules of evidence; it must be open to questions and possible modification; - it must be based on historical and current scientific knowledge. National Content Standard C: Life Science Standards Students should understand: - the chemical basis of life; - Inside the cell is a concentrated mixture of thousands of different molecules; - Most cell functions involve chemical reactions; - Smaller precursor molecules (subunits) can be used to assemble larger molecules with biological activity, including proteins. Introduction - Page 2

3 Amino Acid Starter Kit Contents 1 Magnetic Chemical Properties Circle 1 Laminated Amino Acid Sidechain List 21 Magnetic Amino Acid Sidechains (1 each of the 20 Amino Acids plus 1 additional cysteine) 1 4 Mini-Toober 15 Metal Mini-Toober Clips 120 CPK Coloring Dots 6 Hydrogen Bond Connectors 1 CD with Teacher Notes, Student Handouts and Jmol Visualization Tools Please contact us if any part are missing or damaged. CD Contents Introduction, National Standards, and Contents Teacher Notes Student Handout 1 Student Handout 1 Key Student Handout 2 Student Handout 2 Key Toober Variations Toober Variations Key Read Me First (for Jmol and PDF information) Amino Acids Jmol* Basic Principles of Chemistry that Drive Protein Folding Part 1 Jmol* Basic Principles of Chemistry that Drive Protein Folding Part 2 Jmol* Zinc Finger Jmol* Word Files (PC)* Photo Files* Center for BioMolecular Modeling Tertiary and Quaternary Protein Structure, Lesson 6 Please note that most of the files are PDFs for universal viewing and printing. (A link is provide to download Adobe Acrobat Reader (free software) for those who don t have it installed.) We also provide Word (PC) and photo files for teachers who want to develop their own student handouts or worksheets. (If you have trouble opening the Jmol files, please consult with your school IT staff.) * Jmol files developed by John Regner CAUTION: This product should be kept out of the reach of children under the age of 3, because the molecules or their pieces may present a choking hazard to small children. This is a science education product, not a toy. It is not intended for children under 8 years old. Introduction - Page 3

4 Amino Acids - Building Blocks of Proteins Amino Acids are small molecules used by cells to make proteins. There are 20 Amino Acids and each one consists of two parts a Backbone and a Sidechain. The backbone is the same in all 20 Amino Acids and the sidechain is different in each one. Each sidechain consists of a unique combination of atoms which determine its 3D shape and its chemical properties. When different amino acids join together to make a protein, the unique properties of each amino acid determine how the protein folds into its final 3D shape. The shape of the protein makes it possible to perform a specific function in our cells. The activities described in this handout primarily focus on amino acid sidechains. They will help you understand how the unique properties of each sidechain contribute to the structure and function of a protein. 1. First, look at the components in your Amino Acid Starter Kit. Make sure you have: - Chemical Properties Circle - Amino Acid Chart - Mini-Toober (foam-covered wire) with one Blue End Cap and one Red End Cap - 21 Foam Amino Acid Sidechain Models with Magnets - 15 U-shaped Metal Clips - Red, Blue, Yellow, Green and White Dots - Hydrogen Bond Connectors Photo shows the Three-Group Amino Acid Starter Kit. All Rights Reserved. U.S. Patents 6,471,520B1; 5,498,190; 5,916,006; other U.S. and International Patents Pending. Student Handout 1 Key - Page 1

5 Chemical Properties Circle & Amino Acid Chart 2. Place each foam amino acid sidechain on the magnetic Chemical Properties Circle according to its chemical properties. The sidechains are colored to reflect their chemical properties according to the following coloring scheme: Hydrophobic Sidechains are Hydrophilic Sidechains are Acidic Sidechains are Basic Sidechains are Cysteine Sidechains are You will need to consult the Amino Acid Sidechain List in your kit (photo above) to find the name of each sidechain, so you can position it correctly on the circle. 3. After each sidechain has been correctly positioned on the circle, you should add the colored dots to the gray side of each sidechain to color code the atoms that make up each sidechain. Use the amino acid sidechain list in your kit. Scientists established this CPK Coloring Scheme to make it easier to identify specific atoms in models of molecular structures. Carbon is Oxygen is Nitrogen is Hydrogen is Sulfur is CPK Coloring Scheme 4. After placing the correct dot(s) on each sidechain, return each one to its place on the circle. Position the circle so you can easily read the names. (See the circle photos above.) Student Handout 1 key - Page 2

6 Chemical Properties Circle (continued) Turn all of the sidechains so that the colored side (yellow, white, red, blue) of each one is facing you as you read the names of the sidechains. (Photo on left) Now rotate the circle so that the names of the sidechains are upside down and the gray side of each sidechain is facing you. (Photo on right) Examine the sidechains and their positions on the circle. Describe Your Observations - Do you see similarities or patterns in the sidechains? Yes Explain what you observed: Hydrophobic amino acids are composed primarily of gray carbons. Acidic amino acids have oxygen-containing sidechains. Basic amino acids have nitrogen-containing sidechains and Hydrophilic amino acids have a mixture of oxygen and nitrogen. - Hydrophobic sidechains are composed primarily of carbon atoms. - Acidic sidechains contain two oxygen atoms. This is called a carboxylic acid functional group. - Basic sidechains contain nitrogen atoms. This is called an amino functional group. - Hydrophilic sidechains have various combinations of oxygen, nitrogen and sulfur. - An exception to the above observation is: Tryptophan - a hydrophobic amino acid that contains a nitrogen atom. - Optional Activity - Amino Acids Jmol (See CD or Amino Acid Starter Kit resources on website) Student Handout 1 Key - Page 3

7 Folding a 15-Amino Acid Protein Once you have explored the chemical properties and atomic composition of each sidechain, you are ready to predict how proteins spontaneously fold up into their 3D shapes. Predicting What Causes Proteins to Fold into their 3D shapes - From your experience with oil and water, which sidechains might position themselves on the interior of a protein, where they are shielded from water? The hydrophobic amino acids - Tryptophan, Leucine, Isoleucine, Valine, Proline, Alanine and Glycine. - From your experience with magnets or electricity, which sidechains might be attracted to each other? The basic amino acids (+ charge) and the acidic amino acids (- charge). - Would the final shape of a protein be a high energy state or a low energy state for all of the atoms in the structure? A low energy state. Why? _ A low energy state is more stable than a high energy state. Student Handout 1 Key - Page 4

8 Folding a 15-Amino Acid Protein (continued) Unwind the 4-foot Mini-Toober (foam covered wire) that is in your kit. Notice the blue and red end caps on the ends of your mini-toober. The blue end cap represents the N-terminus (the beginning) of the protein, and the red end cap represents the C-terminus (the end) of the protein. Select 15 metal u-shaped clips from your kit. (A spare clip is usually shipped with each kit.) You will also need a ruler. Beginning at the N-terminus of your mini-toober, measure about three inches from the end of your mini-toober and slide the first clip into place there. (See photo below.) Place the rest of the clips three inches apart on your mini-toober until all 15 are attached to the minitoober. Find the part of the drawing that represents the backbone section of the amino Acid. What do you think the clips represent? The alpha-carbon, because this is the atom that the sidechains are bonded to. Student Handout 1 Key - Page 5

9 Folding a 15-Amino Acid Protein (continued) 3. Select methionine from the chemical properties circle and place it on the clip closest to the blue end cap. Choose any other sidechains from the chemical properties circle as long as you have the right number of each color, as indicated in the chart to the right. Mix the Sidechains together and place them (in any order you choose) on your mini-toober. The sequence of Amino Acid Sidechains that you determined when placing them on the mini-toober is called the Primary Structure of your protein. As a general rule the final shape of a protein is determined by its primary structure (the sequence of its Amino Acids). 4. Now you can begin to fold your 15-amino acid protein according to the chemical properties of its sidechains. Remember all of these chemical properties affect the protein at the same time! - Start by folding your protein so that all of the hydrophobic sidechains are buried on the inside of your protein, where they will be hidden from polar water molecules. - Next, fold your protein so the acidic and basic (charged) sidechains are on the outside surface of the protein and pair one negative sidechain with one positive sidechain so that they come within one inch of each other and neutralize each other. This positive-negative pairing helps stabilize your protein. - As you continue to fold your protein to apply each new property listed below, you will probably find that some of the sidechains you previously positioned are no longer in place. For example, when you paired a negatively charged sidechain with a positively charged one, some of the hydrophobic sidechains probably move to the outer surface of your protein. Continue to fold until the hydrophobic ones are buried on the inside again. Find a shape in which all the properties apply. Student Handout 1 Key - Page 6

10 Folding a 15-Amino Acid Protein (continued) - Continue to fold you protein making sure that your polar sidechains are also on the outside surface of your protein where they can hydrogen bond with water. - Last, fold your protein so that the two cysteine sidechains are positioned opposite each other on the inside of the protein where they can form a covalent disulfide bond that helps stabilize your protein. The final shape of your protein when it is folded is called the Tertiary Structure. - Why should Methionine be next to the Blue End Cap? Because all proteins begin with Methionine - and protein synthesis begins at the N-terminal end of a protein. - What happened as you continued to fold your protein and applied each new chemical property to your protein? It became more compact and more complicated. - Were you able to fold your protein, so that all of the chemical properties were in effect at the same time? Yes. (Note to teachers: some students may answer No.) - If not, do you have any ideas why you weren t able to fold your protein in a way that allowed all of the chemical properties to be in effect simultaneously? Some sequences simply do not allow for a single shape that simultaneously satisfies all the principles of chemistry that drive protein folding. - Did your protein look like the proteins other students folded? No. Explain. Because everyone had a different sequence of amino acids. - How many different proteins, 15 amino acid long, could you make given an unlimited number of each of the 20 amino acids? = 3.28 x Student Handout 1 Key - Page 7

11 15-Amino Acid Protein Questions (continued) -Most real proteins are actually in the range of 300 amino acids long. How many different possible proteins, 300 amino acids in length, could exist? = 2 x How many different proteins are found in the human body? (Another way to ask this question is How many different genes are there in the human genome?) Approximately 50,000 or 5 x Assuming that all human proteins are 300 amino acids long, what fraction of the total number of possible different proteins is found in the human body? 2 x = miniscule! - Why do you think there are fewer actual proteins than possible ones? Because only a relatively small number of amino acid sequences can adopt a stable shape that simultaneously satisfies all of the principles of chemistry. Student Handout 1 Key - Page 8

12 15-Amino Acid Protein Questions (continued) This is the Primary Structure of your protein. In the space below, sketch the Tertiary Structure of your protein. The next student handout provides folding activities and information that will help you understand the Secondary Structure of proteins. - Optional Activity - Basic Priciples of Chemistry that Drive Protein Folding Part 1 Jmol Basic Priciples of Chemistry that Drive Protein Folding Part 2 Jmol (See CD or Amino Acid Starter Kit resources on website) Student Handout 1 Key - Page 9

13 Secondary Structure In the previous protein folding activity, you created a hypothetical 15-amino acid protein and learned that basic principles of chemistry determine how each protein spontaneously folds into its 3-dimensional shape. You learned that the sequence of amino acids in a protein (from N-terminus to C-terminus) is its Primary Structure. The final folded, 3-dimensional shape of your protein is its Tertiary Structure. In this second protein folding activity, you will now learn about the Secondary Structure of proteins. Secondary Structure consists of alpha helices and/or beta sheets. Proteins can be thought of as a series of alpha helices and beta sheets, joined by loops of less regular protein structure. An alpha helix is a compact right-handed helix, with 3.6 amino acids per turn of the helix. The amino acid sidechains are bonded to the alpha carbon of each amino acid and radiate outward from the helix. The alpha helix is stabilized by hydrogen bonds weak bonds between the amino nitrogen of one amino acid (x), and the carbonyl oxygen of another amino acid (x+4) located four residues further along the chain. A beta sheet is an extended, zig-zag structure in which individual strands are positioned parallel or anti-parallel to each other to form flat sheets in proteins. Since the amino acid sidechains are bonded to the alpha carbons of each amino acid, they are alternately orientated above and below the plane of the sheet. The beta-sheet is stabilized by hydrogen bonds between the amino nitrogen of one amino acid and the carbonyl oxygen of another amino acid in an adjacent beta strand. Student Handout 2 Key - Page 1

14 Folding a Toober Model of the Zinc Finger In this activity, you will fold a model of the first of three zinc fingers of the Zif268 protein. The primary structure of this zinc finger is: The sidechains of the seven amino acids circled in the above sequence will be included in the model you fold. 1. Primary Structure: Map the positions of the seven amino acids on the mini-toober. Since the toober is 48 inches long, and the zinc finger is 28 amino acids long, each amino acid occupies 1.7 inches of toober. Using a ruler, measure the distances shown below, and add a labeled metal clip to the toober at each position. Label the clip with an appropriately colored dot. 2. Secondary Structure: Fold the toober into its secondary structure. The first 13 amino acids (the first 22 inches from the N-terminus) should be folded into a 2-stranded beta sheet. This can be made by creating a zig-zag structure that is bent in the middle as shown in the photos below. You can also add the plastic hydrogen bonds to your model as shown in the far right photo. Student Handout 2 Key - Page 2

15 Folding a Toober Model of the Zinc Finger (continued) The last 14 amino acids of the zinc finger exist as a compact, right-handed alpha helix. This can be made by wrapping the mini-toober around your finger to create four 360 degree turns as shown in the photos below. Add the hydrogen bond connectors as shown in the far right photo. Your mini-toober should now look similar to the one shown below. 3. Tertiary Structure: Fold the beta-sheet and alpha-helix into the final tertiary structure of the zinc finger. Add the seven amino acid sidechains to the clips on the toober. In its final tertiary structure, these seven sidechains will be positioned such that: - The two cysteine and two histidine sidechains will be oriented so that they can simultaneously bind to a single zinc atom (not included in this kit) in the center of the structure. - The two hydrophobic amino acid sidechains phenylalanine (Phe, F) and leucine (Leu, L) will be orientated toward the inside of the structure. - The positively-charged arginine sidechain will be exposed at the top of the alpha helix, where it is available to bind to the negatively-charged phosphate backbone of DNA. Student Handout 2 Key - Page 3

16 Folding a Toober Model of the Zinc Finger (continued) As a folding guide, you can either use the photo shown below or the interactive Jmol image of a zinc finger on the CD included in the Amino Acid Starter Kit. Note: As you fold your toober, you may need to rotate the sidechains around the mini-toober to make them adopt the desired final shape. The zinc (not included with the kit) binds simultaneously to the two histidines and two cysteines. Student Handout 2 Key - Page 4

17 Folding a Toober Model of the Zinc Finger (Questions) 1. Both alpha helices and beta sheets are stabilized by hydrogen bonds. - Which atoms share the hydrogen in these weak bonds? The nitrogen of an amino group and the oxygen of a carbonyl group. - Are these backbone atoms or sidechain atoms? Backbone atoms. 2. Describe the secondary structural elements that comprise a zinc finger: A 2-stranded beta sheet and a short alpha helix. 3. How is a zinc atom involved in the stabilization of the zinc finger motif? The zinc atom is simultaneously bound by the 2 cysteine and the 2 histidine sidechains. 4. Zinc fingers often bind to DNA. How might the arginine sidechain (positively-charged) shown on your model be involved in DNA binding? DNA has a negatively-charged phosphate backbone. Therefore, the positivelycharged arginine of the zinc finger can bind to DNA via an electrostatic interaction. - Optional Activity - Zinc Finger Jmol (See CD or Amino Acid Starter Kit resources on website) Student Handout 2 Key - Page 5