PROTEIN PROTEIN MODELMODELCHALLANGECHALLANGE

Lin WozniewskiLin Wozniewskilwoz@iun.edu

Di l iDisclaimerThis presentation was prepared using draft rules There may be some changes in the final 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 th ffi i l lthe official rules

What the Student Need to Bring

Th P b ildThe Pre-buildSomething to Write WithUp to 5 8.5 X 11” pages with whatever references p p ghey want

hat MSOE will ProvideThe TestTh T t A

hat MSOE will Provide

The Test AnswersThe Pre-build Scoring RubricThe On-Site Build ToobersOn-Site Build Scoring Rubric

WHAT THE EVENT SUPERVISOR NEEDS TOROVIDE

Enough expertise to be able to interpret and g p papply the rubric.Computers for the Students with one of the 2

ti f ll i th t d t t th Joptions for allowing the students access to the J-Mol program

Installation of the J-Mol Program on the computersg pAccess to a CD with the J-Mol Program on it

Rulers or meter sticks

WHAT THE STUDENTS NEED TO DORead the article on the molecule they will be ymodelingVisit the J-Mol website and determine how many

i id i th l l d th i damino acids are in the molecule and their orderLearn what the side chains of the various amino acids are and what they doacids are and what they doDivide the toober or other material up into approximately 2 cm segments to correspond to h b f i id i h ithe 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 Fold the protein according to the 3-d model at the appropriate places

WHAT THE STUDENTS NEED TO DOAdd 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 acids that form the bonds that hold the tertiary structure together & the active site) Learn the difference between acid and basic side chainsLearn the difference between hydrophobic and hydrophilic side chainshydrophilic side chainsLearn about hydrogen bonding and sulfur bondingLearn the difference between primary, secondary,

HE COMPETITIONStudents will bring their pre-built models to the g passigned impoundStudents will sit at a computer and build a

ti f th d l ith th t b id d portion of the model with the toober provided using the J-Mol programStudents will answer questions on a testStudents will answer questions on a testThe test will cover

General amino acid structureProtein primary, secondary, and tertiary structureSpecifics about the particular protein being studied

Amino AcidsThere are 20 amino acids that make up the proteinsAmino Acids are a backbone of a

t i & i t fprotein & consist of:Nitrogen atom with 2 Hydrogen atoms (the Amine side)

Nitrogen

atoms (the Amine side)A carbon atom, on which the side chain is hooked

Amino Group

Another carbon atom with a double bonded oxygen and a hydroxyl group (The Carboxylic hydroxyl group (The Carboxylic Acid side)

ide Chains

ide chains are classified as Hydrophobic – water hating de c a s a e c ass ed as yd op ob c wate at g r Hydrophilic – water loving depending on what is in the ide chainsid Ch i th t h l b d h d t ide Chains that have only carbon and hydrogen atoms re Hydrophobic & tend to be buried inside the protein way from the water when it is folded. They are non-polary y pide chains that have hydroxyl, carboxylic acid, or amine roups are Hydrophilic & are generally on the outside of he protein hen it is folded These can be acidic or basic he protein when it is folded. These can be acidic or basic, r just polar

rimary Structure of a ProteinThe Primary Structure of the Protein is the yactual order of the amino acids in the protein.This is the next thing the students must d t i ft th l b t i id & determine after they learn about amino acids & the side chains

Lys- Glu-Thr-Arg-Arg-Arg-Lys-etc.Lys Glu Thr Arg Arg Arg Lys etc.

econdary Structure of a Proteinecondary Structure of a ProteinThe 2 most common types of secondary structure are the alpha helix and beta pleat Th l h h li ONLY il i ht h d d (if 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 acidamino acid

Alpha HelixBeta Pleat

Alpha Helix

ertiary Structure of a Proteinertiary Structure of a ProteinThe tertiary structure of the protein is the final folding that is the result of the molecular interactions formed by the primary and interactions formed by the primary and secondary structure.This is determined using the J-Mol program.What a finished pre-build might look like

Pi ClPipe Cleaner

oberober12 Gauge Wire

HOW TO USE RESOURCEST i Two main resources:

http://cbm.msoe.edu/includes/jmol/SOJmols/2012PreBuild.html12PreBuild.html

Don’t need to know what molecule you are looking for, correct one will come up.

www.pdb.orgNeed to know which molecule you need (1i3o based on A and B chain)based on A and B chain).

DB DATABASEType 1i3o into PDB look-up window & hit search.yp pClick on the sequence tabClick on A and B chain. Complete sequence is thereClick anywhere on sequence-Jmol will come up and h l l ( li k di l J l b tt )show you molecule (or click on display Jmol button)

Scroll down so that you see E chain again. Jmol window will come with you.yPut pointer over any residue & leave pointer there for a couple of seconds. The amino acid letters will appear.

MOLRight Click and move mouse to move moleculeMain commands

B kbBackboneCartoonRibbonWireframeSpacefill

T d d th b (1 499 k)Type command and then a number (1-499 work)Click on ExecuteTo get rid of that picture type the command and then 0 To get rid of that picture type the command and then 0 Click on ExecuteHold mouse over one spot for a second or two to see pletters & position of amino acid

Mini EventWe will be making a small part of one protein to g p pillustrate how to make the model and the resources available to make the model.W ill b l ki t th i b i f th 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 We will be using the rubrics to score our own model.

Making Zinc FingerMaking 1st Zinc finger of KLF2 of 1zaa proteinAmino acids 4-31 of the proteinHas one α chainHas two β pleatsHas one active site that holds zinc ionHas one active site that holds zinc ionThe α chain is near the carboxylic endThe 2 β pleats take up most of the rest and ends

h i idat the amine sideCoordinates to Zinc with two Histidines side chains and two Cysteine side chainsyCoordinates to DNA with Arginine side chainHas hydrophobic core to stabilize (phenylaninine 21 & leucine 27)& leucine 27)

coring Zinc Finger-Score SheetR b i S l R i lRubric-Sample Regional-

Description Points

ap on N terminal amino acid (Pro4) 1ap on N-terminal amino acid (Pro4) 1

ap on C-terminal amino acid (Gly31) 1

ha-helix (19-31) 2ha helix (19 31) 2

-helix right handed 2

-helix properly formed (phone cord) 1p p y (p )

-helix right length (~3.5 turns) 2

Strand #1 (5-7) 2

Strand #2 (14-16) 2

Strands formed properly (zig-zag) 1Strands form beta Sheet @ N terminus 2

Regional Scoring Continued& C terminus in opposite directions 2odel flat 2nc Ion 2Hi tidi (Hi 25 & 29)( di t Z ) 2Histidines (His25 & 29)(coordinates Zn) 2Cysteines (Cys 7 & 12) (coordinates Zn) 2rginine 18 (attaches to DNA) 2g ( )ydrophobic amino acids face in(Phe16, Leu22) 2NA Attached to DNA 2

i ddi i i 2reative additions appropriate 2reative additions accurate 2X5 card clear 2

ull Rubric SheetWhole Molecule Overview

erminus

Alpha-helix

heet

C-terminusterminus

Rubric Detailsue cap on N-terminal amino acid (Pro4) (1 pt)To receive this point the blue cap should be positioned on 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.

ed 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. pSee picture to the right for correct placement of the red end cap.

pha helix (amino acids 19-31) is located at C-terminus of p ( )otein (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.

Rubric Details ContinuedRubric Details, Continuedpha helix is right-handed (2 pts)Alpha helices are right-handed. Check the alpha helix in the model to

fi h h h li i i h h d d If h l h h li i i hconfirm 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 l ft h d d h li d th d l ld t i th i t a left-handed helix and the model would not receive the points.

pha helix is properly formed (helix resembles a telephone cord) (1 )The helix should be formed in such a way that it resembles a telephone e e s ou d be o ed suc a way t at t ese b es a te ep o e 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

pha helix is appropriate length (13 amino acids; ~3 5 turns) (2 pts)pha 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

i t l 3 6 i id i l th Th f th l th f thi

ubric Details, Continued

d #1 ( i id 5 7) (2 )ta 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)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 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.t t d i f d l (1 t)ta 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

t i ht i t th d l Th h ld t b

Rubric Details, Continuedix is arranged next to beta sheet (protein should be compact h a 2-stranded beta sheet lying next to an alpha helix; helix sheet should not be too far apart) (2 pts)

Please note that there is not much space

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 on to coordinate between the 4 amino acids that bind the ion

between the two secondary structures.

on 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 N terminus (blue cap) and C terminus (red cap) are

N-terminus

N-terminus (blue cap) and C-terminus (red cap) are nted 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

C-terminus

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.

Rubric Details, Continueddel should be flat in that the beta strands and alpha helix are occupying 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 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 ative Additions to model (2 pts each):Zinc ionZinc 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)coordinates Zn)

CysteineTo 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.p yArginine 18 (attaches to DNA)

ArginineTo 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.

Rubric Details, Continued2 Cysteines (Cys 7 and 12) (coordinates Zn) Cysteine

To receive these points the model should have 2 Cysteines at positions #7 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 DNATo 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

Arginine

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)

Phenylalanine

LeucineTo receive these points, the model should have a phenylaninine residue at

iti #16 d l i id t iti #22

Leucineanine

position #16 and a leucine residue at position #22.

Rubric DNA attached to protein

T i h i h d l h ld h DNA 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)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 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 p p p yfocus 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

di h i i h i h to coordinate the zinc ion as these are pertinent to the structure of the protein.

RubricCreative additions are accurate (2 pts)( p )

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

f f 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 resourcesresources.

Students submitted a 3x5 card to explain model (2 pts)

T i h i d h ld h b 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 to the model so that the judge is not left guessing what the model represents.

ResourcesMSOE has a lending library available where g ytoobers can be sent for to use for a few days in the classroom

d / b /lib www.rpc.msoe.edu/cbm/lib. J-Mol Program

http://bioportal.weizmann.ac.il/oca-p pdocs/fgij/index.htm

MSOEhtt // b d / t / /i d ht lhttp://cbm.msoe.edu/stupro/so/index.html

Includes tutorials for the studentsIncludes tutorials on how to score models using rubrics.