1. PROTEIN MODEL CHALLANGE Lin Wozniewski lwoz@iun.edu

2. Disclaimer This presentation was prepared using draft rules. There may be some changes in the final copy of the rules. The rules which will be in your Coaches Manual and Student Manuals will be the official rules

3. What the Student Need to Bring The Pre-build Something to Write With Up to 5 8.5 X 11” pages with whatever references they want The Test The Test Answers The Pre-build Scoring Rubric The On-Site Build Toobers On-Site Build Scoring Rubric What MSOE will Provide

4. W HAT THE EVENT SUPERVISOR NEEDS TO PROVIDE Enough expertise to be able to interpret and apply the rubric. Computers for the Students with one of the 2 options for allowing the students access to the J- Mol program Installation of the J-Mol Program on the computers Access to a CD with the J-Mol Program on it Rulers or meter sticks

5. W HAT THE STUDENTS NEED TO DO Read the article on the molecule they will be modeling Visit the J-Mol website and determine how many amino acids are in the molecule and their order Learn what the side chains of the various amino acids are and what they do Divide the toober or other material up into approximately 2 cm segments to correspond to the number of amino acids in the protein Label the amino acids on the chain in order. Fold the protein according to the 3-d model at the appropriate places

6. W HAT THE S TUDENTS N EED TO DO Add creative items to emulate the side chains and form appropriate intramolecular bonds (side chains are ONLY to be attached to those amino acids that form the bonds that hold the tertiary structure together & the active site) Learn the difference between acid and basic side chains Learn the difference between hydrophobic and hydrophilic side chains Learn about hydrogen bonding and sulfur bonding Learn the difference between primary, secondary, and tertiary protein structure

7. T HE C OMPETITION Students will bring their pre-built models to the assigned impound Students will sit at a computer and build a portion of the model with the toober provided using the J-Mol program Students will answer questions on a test The test will cover General amino acid structure Protein primary, secondary, and tertiary structure Specifics about the particular protein being studied

8. Amino Acids There are 20 amino acids that make up the proteins Amino Acids are a backbone of a protein & consist of: Nitrogen atom with 2 Hydrogen atoms (the Amine side) A carbon atom, on which the side chain is hooked Another carbon atom with a double bonded oxygen and a hydroxyl group (The Carboxylic Acid side) Nitrogen Amino Group

9. Side Chains Side chains are classified as Hydrophobic – water hating or Hydrophilic – water loving depending on what is in the side chains Side Chains that have only carbon and hydrogen atoms are Hydrophobic & tend to be buried inside the protein away from the water when it is folded. They are non-polar Side chains that have hydroxyl, carboxylic acid, or amine groups are Hydrophilic & are generally on the outside of the protein when it is folded. These can be acidic or basic, or just polar

10. Primary Structure of a Protein The Primary Structure of the Protein is the actual order of the amino acids in the protein. This is the next thing the students must determine after they learn about amino acids & the side chains Lys- Glu-Thr-Arg-Arg-Arg-Lys-etc.

11. Secondary Structure of a Protein The 2 most common types of secondary structure are the alpha helix and beta pleat The alpha helix ONLY coils right-handed (if you are going up the stairs, your right hand rests on the outside banister going up) The beta pleat should bend back and forth in a zigzag pattern of about 20  at each start of a new amino acid Beta Pleat Alpha Helix

12. Tertiary Structure of a Protein The tertiary structure of the protein is the final folding that is the result of the molecular interactions formed by the primary and secondary structure. This is determined using the J-Mol program. What a finished pre-build might look like Toober Pipe Cleaner 12 Gauge Wire

13. Mini Event We will be making a small part of one protein to illustrate how to make the model and the resources available to make the model. We will be looking at the scoring rubrics for the model and how to use them. We will be using the rubrics to score our own model.

14. Making Zinc Finger Amino acids 4-31 of the KLF2 protein Has one  chain Has two  pleats Has one active site that holds zinc ion The  chain is near the carboxylic end The 2  pleats take up most of the rest and ends at the amine side Coordinates to Zinc with two Histidines side chains and two Cysteine side chains Coordinates to DNA with Arginine side chain Has hydrophobic core to stabilize ( phenylaninine 16 & leucine 22)

15. Scoring Zinc Finger-Score Sheet Rubric-Sample Regional- DescriptionPoints Blue cap on N-terminal amino acid (Pro4)1 Red cap on C-terminal amino acid (Gly31)1 1 Alpha-helix (19-31)2 Alpha-helix right handed2 Alpha-helix properly formed (phone cord)1 Alpha-helix right length (~3.5 turns)2 Beta Strand #1 (5-7)2 Beta Strand #2 (14-16)2 Beta Strands formed properly (zig-zag)1 Beta Strands form beta Sheet @ N terminus2 Beta Sheet & Alpha helix not far apart2

16. Regional Scoring Continued N & C terminus in opposite directions2 Model flat2 Zinc Ion2 2 Histidines (His25 & 29)(coordinates Zn)2 2 Cysteines (Cys 7 & 12) (coordinates Zn)2 Arginine 18 (attaches to DNA)2 Hydrophobic amino acids face in(Phe16, Leu22)2 DNA Attached to DNA2 Creative additions appropriate2 Creative additions accurate2 3X5 card clear2

17. Full Rubric Sheet Whole Molecule Overview C- terminus N-terminus Alpha- helix Beta-sheet

18. Rubric Details Blue cap on N-terminal amino acid (Pro4) (1 pt) To receive this point, the blue cap should be positioned on the first amino acid. This should be next to the beta- strand. See picture to the right for correct placement of the blue end cap. Red cap on C-terminal amino acid (Gly31) (1 pt) To receive this point, the red cap should be positioned on the last amino acid. This should be next to the alpha-helix. See picture to the right for correct placement of the red end cap. Alpha helix (amino acids 19-31) is located at C-terminus of protein (2 pts) There should be an alpha helix located at the C-terminus of the protein. See figure to right. On the model and in the figure, the alpha helix is colored magenta.

19. Rubric Details, Continued Alpha helix is right-handed (2 pts) Alpha helices are right-handed. Check the alpha helix in the model to confirm that the helix is right-handed. If the alpha helix is right- handed, the model is awarded two points. To determine if the helix is right-handed, find one of the ends of the helix and imagine that the helix is a spiral staircase. Pretend that you are climbing that staircase and the helix is the hand-rail, which is always on the ourside edge of the staircase. If you would put your right hand on the toober as you go up the staircase, you have a right- handed helix. If you would put your left hand on the toober, you have a left-handed helix and the model would not receive the points. Alpha helix is properly formed (helix resembles a telephone cord) (1 pt) The helix should be formed in such a way that it resembles a telephone cord stretched out slightly. The helix should not be compacted down so that there is not any space between the turns. It should also not be so stretched out that there is a lot of space between the turns Alpha helix is appropriate length (13 amino acids; ~3.5 turns) (2 pts) The helix is 13 amino acids, and each turn in the helix is approximately 3.6 amino acids in length. Therefore, the length of this helix should be ~3.5 turns.

20. Rubric Details, Continued Beta strand #1 (amino acids 5-7) (2 pts) To receive these points, the model should have a beta strand from amino acids 5-7 (3 amino acids in length). The first beta strand should be located near the blue end cap. The model and the figure to the right have the beta strands colored yellow.. Beta strand #2 (amino acids 14-16) (2 pts) To receive these points, the model should have a beta strand from amino acids 14-16 (3 amino acids in length). The second beta strand is located 6 amino acids (12 cm) away from the first. The model and the figure to the right have the beta strands colored yellow. Beta strand is formed properly (1 pt) To receive this point, the model should have properly formed beta strands. The model can have the beta strands in a zig-zag shape (a bend every 2 cm) or it could have them be represented as straight regions to the model. There should not be any helical or coiled portions in this area

21. Rubric Details, Continued Helix is arranged next to beta sheet (protein should be compact with a 2-stranded beta sheet lying next to an alpha helix; helix and sheet should not be too far apart) (2 pts) To receive these points, the beta sheet and alpha helix should be located close to one another. There should only be enough space between the two secondary structure to allow for a zinc ion to coordinate between the 4 amino acids that bind the ion. In other words, there should not be much space between the alpha helix and the beta sheet 12. N-terminus (blue cap) and C-terminus (red cap) are pointed in opposite directions (2 pts) To receive these points, the N-terminus and C-terminus of the protein should be facing away from each other. If you hold the model so that the beta sheet is facing the left and the helix is on the right (like the picture shown to the right), then the N- terminus should be pointing upward and the C-terminus is pointing downward. Please note that there is not much space between the two secondary structures. N- terminus C- terminus

22. Rubric Details, Continued Model should be flat in that the beta strands and alpha helix are occupying the same plane (2 pts) To receive these points, the alpha helix and beta sheet should be in the same plane (please see figure to the right). The model should be “flat” in that neither the helix nor the sheet protrudes upward or downward form the main axis. You should be able to look through the beta sheet and see the alpha helix Creative Additions to model (2 pts each): Zinc ion To receive these points, the model should have a zinc ion located between the alpha helix and beta sheet closer to the C-terminus than the N-terminus. Please model and figure to the right (zinc ion is colored dark red). 2 Histidines (His 25 and 29) (coordinates Zn) 2 Cysteines (Cys 7 and 12) (coordinates Zn) Cysteine To receive these points, the model should have 2 Cysteines at positions #7 and #12. If zinc ion is present, then these Cysteines should be connected to the zinc ion. Arginine 18 (attaches to DNA) Arginine To receive these points, the model should have an Arginine at position 18. If DNA is present on model, this amino acid should interact with the DNA. To receive these points, the model should have 2 Histidines at positions #25 and #29. If zinc ion is present, then these Histidines should be connected to the zinc ion

23. Rubric Details, Continued 2 Cysteines (Cys 7 and 12) (coordinates Zn) Cysteine To receive these points, the model should have 2 Cysteines at positions #7 and #12. If zinc ion is present, then these Cysteines should be connected to the zinc ion. Arginine 18 (attaches to DNA To receive these points, the model should have an Arginine at position 18. If DNA is present on model, this amino acid should interact with the DNA. Hydrophobic amino acids facing inward to create hydrophobic core stabilizing protein (Phe16, Leu22) Leucine To receive these points, the model should have a phenylaninine residue at position #16 and a leucine residue at position #22. Arginin e Leucine Phenylal anine

24. Rubric DNA attached to protein To receive these points, the model should have DNA bound to the zinc finger. Zinc finger should be in the major groove of the DNA. Creative additions are appropriate (2 pts) To receive these points, the creative additions need to be relevant to telling the functional story of the protein. These points should not be awarded to models that have displayed all of the amino acids. The focus of adding the amino acids should be on the ones that play a role in the function of the protein. Additionally, this point should not be awarded if amino acids outside of those that play a specific functional role in the protein are displayed. The focus of this protein is binding to DNA to induce transcription. Therefore, it is relevant to highlight these amino acids. It is also relevant to highlight the amino acids that are involved in the stability of the protein and to coordinate the zinc ion as these are pertinent to the structure of the protein.

25. Rubric Creative additions are accurate (2 pts) To receive these points, the creative additions need to be accurate – in other words, the model cannot have the amino acids or other additions in inappropriate places. The additions need to reflect the scientific information that has been provided in Goodsell’s Molecule of the Month, the PDB file or alternative resources. Students submitted a 3x5 card to explain model (2 pts) To receive these points, a 3x5 card should have been submitted along with the model, describing the model in terms of what additional features have been added to the model so that the judge is not left guessing what the model represents.

26. Resources MSOE has a lending library available where toobers can be sent for to use for a few days in the classroom www.rpc.msoe.edu/cbm/lib. www.rpc.msoe.edu/cbm/lib J-Mol Program http://bioportal.weizmann.ac.il/oca- docs/fgij/index.htm http://bioportal.weizmann.ac.il/oca- docs/fgij/index.htm MSOE http://cbm.msoe.edu/stupro/so/index.html Includes tutorials for the students Includes tutorials on how to score models using rubrics.