Grade Level: 4 (4-8) Time Required: 20 minutes

Keywords: protein, tertiary structure, amino acid, contractile, covalent bond, non-covalent bond, hydrogen bond, disulfide bond, hydrophobic effect, charge-charge interaction, Van der Waals, heme group.

Summary: Students are introduced to proteins, and their diverse functions. Students will investigate the three-dimensional model of hemoglobin, and learn why proteins need to fold correctly. Students will learn about the attractive forces that stabilize protein structure such as covalent and non- covalent forces with emphasis on hydrogen bonding.

Engineering Connection: Scientists mutate different amino acids in a protein to determine their effect on protein folding, transition state stabilization, and substrate binding. Scientist can design inhibitors by studying the three-dimensional structure, the active site of the protein, and the amino acid in the active site that helps to coordinate the substrate.

Related Curriculum Subject Area Biochemistry Activity Can a folded string stay intact?

Educational Standards Virginia Standard: Force, Motion, and Energy 4.2

Learning Objectives  Students will be able to define protein and recognize diverse roles of proteins in biological systems.  Students will also learn that a protein must attain its correct tertiary structure to function properly.  Students will be able to understand that the formation of stable tertiary structure depends upon attractive interactions.  Students will be able to understand that there are strong and weak forces and these forces help protein to fold.

Introduction/Motivation Ask some students what their favorite foods are. As they answer, write the names of the food on the board. Ask students if they have looked at the nutrition facts on food items, and what kind of information can they obtain from it. For example, they might say it gives information about the amount of vitamins, minerals, calories, proteins, and so on. Then ask students which foods contain protein. Students might say meat, butter, milk, nuts, eggs and so on.

Ask students why they need to consume protein in their diet. Some might say that it is good for you, and others might say it gives you energy. Students should be able to respond that proteins are an essential part of their diet. Ask students if they know some of the functions of proteins. Students will know that they are suppose to consume a certain amount of protein in their diet, but they will not know that proteins have different biological functions. Then ask students if they can define protein.

Lesson Background & Concepts for Teachers Proteins are found in all living systems ranging from unicellular to multicellular organisms. Proteins are a major component of bacterial cells, viruses, simple eukaryotes, vertebrates, and mammals. Proteins make up about 50% of the dry weight of the cells. Protein is a biomolecule composed of a long chain of amino acids. Amino acids are the building blocks of proteins.

Proteins are involved in almost every biological process in living organisms. Proteins can vary in size ranging from 100 amino acid residues to about 2000 amino acid residues. Proteins have diverse functions such as transport of oxygen, storage of iron, enzymes that catalyze different reactions, and contractile proteins. Proteins also act as an active component of immunity such as antibodies, and they can also be toxins, and structural protein.

Proteins are linear polymer and are assembled from a combination of amino acids. A specific protein has a unique amino acid sequence and is synthesized with the absolute control of the amino acid sequence. The linear polymer chains of all proteins can fold to assume a specific three-dimensional structure. Protein structure is dictated by the amino acid sequence.

Protein must fold properly to assume its correct three-dimensional structure. A properly folded protein can sustain its function. The three-dimensional structure of a protein is the result of a delicate balance between stabilizing and destabilizing forces. The forces that stabilize protein structures are covalent bonds such as disulfide bonds, and non-covalent bonds such as hydrophobic effect, charge-charge interactions, Van der Waals, and hydrogen bonding. Non- covalent interactions are weak, but collectively they can be strong.

Hemoglobin is composed of four polypeptide chains, and each of these subunits contains a heme group. Each heme group can bind one oxygen molecule, and the complete oxygenation of hemoglobin requires the binding of four oxygen molecules. Hemoglobin is found in the blood within the red blood cells. Hemoglobin binds oxygen in the lungs and becomes saturated. In the peripheral tissues where the concentration of oxygen is low, oxygen is unloaded from the hemoglobin. Myoglobin binds the released oxygen from hemoglobin. Hemoglobin facilitates the transport of oxygen from the lungs to the tissues.

Figure 1: Three-dimensional structure of hemoglobin http://www.rcsb.org/pdb/explore.do?structureId=1HDA Figure 2: Three dimensional structure of one subunit of hemoglobin with hydrogen bonding. Program used: ViewerLite. http://www.rcsb.org/pdb Hemoglobin is also a linear polymer, but it has to assume a specific three-dimensional structure to sustain its function. The forces that stabilize the structure of hemoglobin are covalent and non-covalent interactions. We will focus on hydrogen bonding. The alpha-helices and the interaction of beta sheets are stabilized by hydrogen bonding. Hydrogen bonding occurs between the NH and CO groups. Hydrogen bonding involves a donor and an acceptor atom such as (N—H- - - - - O=C). Other types of hydrogen bonding can also occur between a donor and an acceptor atom within the side chains or main chain and side chain. The distance of the hydrogen bond can vary from 0.26 to 0.34 nm. Hydrogen bonding is a weak force, but collectively they are strong.

Body of Lesson Give students an example of a protein such as hemoglobin. Show the three-dimensional structure of the hemoglobin. Hemoglobin is also a protein found in blood that transports oxygen from the lungs to the tissues. Hemoglobin always maintains this specific three- dimensional (Show students the structure of hemoglobin). Ask students if they can determine the four subunits. Then point out the heme group and tell them that this is where oxygen binds. Show students the structure of hemoglobin with hydrogen bonding and ask them what is holding the structure together. They will say dashed lines or green web like structure. Respond by saying, Yes, these are hydrogen bonds. Ask students what will happen to the structure if they had only one hydrogen bond. Student should be able to respond that the structure will fall apart.

Vocabulary/Definitions protein: Biomolecule composed of one or more polypeptide chains containing amino acid residues linked together tertiary structure: a comprehensive three-dimensional structure of a protein. amino acid: the basic building blocks of proteins contractile: capable of contracting covalent bond: a chemical bond formed when two atoms share a pair of electrons non-covalent bond: Bond in which electrons are not shared by atoms hydrogen bond: Bond occurs when two electronegative groups compete for the same hydrogen atom disulfide bond: covalent bond formed between a pair of cysteines hydrophobic effect: non-polar molecule form aggregates in aqueous solution charge-charge interaction: Interaction between oppositely charged residues Van der Waals: Interaction from the induction of dipoles due to fluctuating charge densities within atoms heme group: A prosthetic group with Fe (iron) coordinated in the porphyrin ring

Activity Can a folded string stay intact? Obtain a string and fold the string several times. Now let go of the string. Does the string maintain its folded structure? No. What does the string need to maintain its folded structure? (Answer: Some kind of force). Here we demonstrated the importance of force. The string represented the linear polymer; the folded string represented the three-dimensional structure of the protein, and the requirement of force to keep it intact.

Lesson Closure Can you define protein and give an example? Answer to this question should be protein is a biomolecule composed of amino acids. An example of protein is hemoglobin. What are some of the functions of protein? (Answer: Transport, storage, catalytic reactions and so on) What are some of the forces that stabilize tertiary structure of a protein? (Answer: Hydrogen bonds, Van der Waals, hydrophobic, covalent, and charge-charge interactions.)

The amino acid sequence of a protein defines the three-dimensional structure of a protein. The amino acid sequence and the three-dimensional structure of a protein determine the chemical and biophysical properties as well as biological activities of a protein.

Scientists study the three-dimensional structure of a protein, the active site, and different interactions between the protein and the substrate to design inhibitors. The refolding studies are also done to investigate major interactions in protein that stabilize its structure.

Assessment Pre-assessment Discussion Questions: Write their answers on the board.  Which food contains proteins? (Answer: egg, butter, milk, meat, nuts)  What is Protein? (Answer: a bimolecule composed of amino acids)  What are some of the functions of proteins? (Answer: transport, storage, active component of immunity, contractile, and so on)

Post-assessment Discussion Questions:  What do you know about different kinds of forces? (Answer: There are many strong and weak forces. Force is a push or pull. There are attractive and repulsive forces in a protein.)  What kinds of forces stabilize the tertiary structure of a protein? (Answer: covalent and non-covalent such as hydrogen bond, van der waals, hydrophobic interactions, and charge-charge interactions.)  What happens to a protein if there was no force to stabilize its structure? (Answer: Its structure will fall apart, and it will not be able to sustain its function.)

Lesson Extension Activity This lesson can be extended by taking about protein denaturation. Protein denaturation is the noncovalent change in the secondary, tertiary, or quaternary structure of a protein. Protein can be denatured by different means such as thermal, changing the pH. You can show the thermal denaturation of the protein by using an example of an egg. Crack a raw egg on a pan, student will look at the white portion of the egg and define some of the characteristics. Then heat the egg, student will look at the cooked egg and write down its characteristics. The protein has denatured so the egg becomes firm and loses its solubility. This is due to the change in noncovalent interactions such as hydrogen bonding, van der waals, charge-charge interactions, and hydrophobic interactions.

References Whitford, David. (2007). Proteins Structure and Functions. England: John Wiley & Sons Ltd.

Mangino, Mike. Protein Denaturation. Science Olympiad Food Chemistry 2006-2007. http://www-fst.ag.ohio-state.edu/olympiad/lectures/Lect11.html - accessed March 13, 2008

Owner: College of Science, George Mason University Contributor: Manisha Shrestha