AP BIOLOGY

Computational Biology Proteins Robert S. Goodman

2012

11

SAR HIGH SCHOOL

Computational Biology: Proteins  Robert S. Goodman, 2012 All Rights Reserved. No Part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage system without written permission from the author, Robert S. Goodman. ******************************************************************************

TABLE OF CONTENTS

1

Primary Structure of Proteins

3-10

2

Secondary Structure of Proteins

11-22

3

Tertiary and Quaternary Structure of Proteins

23-31

4

Carboxypeptidase A-An Example of Substrate Binding and Catalysis

32-42

5

The Mystery of the Potassium Channel

43-53

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The Primary Structure of Proteins Goals:   

To examine the primary structure of proteins To see how sequence comparisons give us insight into disease and evolutionary relationships To examine the relationship between an active and inactive proteins (enzymes).

Background: In 1951 Frederick Sanger was the first to work out the amino acid sequence of a protein-bovine insulin. He was awarded the Nobel Prize in Chemistry in 1958 for that accomplishment. In 1980 he was awarded a second Nobel Prize in Chemistry for discovering a method for sequencing DNA. He is one of only four two time Nobelists and the only two time Nobelist in chemistry. Sanger was able to cut apart the insulin using different techniques: for example using the enzyme trypsin in one case or strong acid in another case. Each time he was able to separate the small peptides and work out their sequences. But to string together those peptides required some clever analysis. Here is what he did. Suppose that you did not know the order of the alphabet. In one experiment you cut it up using the imaginary enzyme “alphabetase” and you get these pieces: ghijkl

pqrstuvw abcdef

mno

xyz

But you do not know what order the fragments are in. However, using another imaginary enzyme, “letterase” to cut up the alphabet you get these fragments: vwxyz

ijklmnopq

abc

defgh

rstu

Now you reason the following… 1) It seems like nothing ever comes before “a”…so maybe “a” is first. 2) And nothing ever comes after “z”…so maybe “z” is last. 3) So take the long fragment with “a” which goes like this: “abcdef” and from the “defgh” fragment obtained with the second method we know that “gh” comes next. We then look at the fragment “ghijkl” obtained using the first method and we know that “ijkl” comes have “gh”. 4) Keep going with that same “overlap” analysis of fragments and you will figure out the whole alphabet: abcdefghijklmnopqrstuvwxyz !!!! Of course it wasn’t quite this simple, but this analogy give you some idea of Sanger’s approach.

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Using Sanger’s method, the amino acid sequences of many proteins were worked out by researchers around the world. The sequence of amino acids in a protein is just one important aspect of protein structure known as the “primary structure”. In later activities you will learn about the secondary, tertiary and quaternary structure as well. Although the primary structure plays a major role in ultimately determining the higher levels of structure (secondary, tertiary and quaternary), we are becoming more aware that there are other factors which affect the ultimate shape of a protein. Those factors would include the role of chaperonins, post translational modifications of the primary structure as well as the action of signal molecules that activate or inactive various proteins. The primary structure of proteins gave scientists a method of comparing variations in various organisms which provided clues to understanding disease as well as evolutionary relationships. This activity will focus on the primary structure of various proteins. Procedure: A) One protein, an enzyme that you may study in the laboratory later in the course is catalase. This enzyme is found in virtually all aerobic organisms. It catalyzes the decomposition of hydrogen peroxide (H2O2), a byproduct of oxidative reactions. Hydrogen peroxide can be quite toxic, but this enzyme breaks it down before it can build up to dangerous concentrations. The reaction is summarized below: 2H2O2 → 2H20 + O2 . Let us start out by using the “National Center for Biotechnology Information” website to ascertain the sequence of amino acids in this protein. The URL for the web site is: http://www.ncbi.nlm.nih.gov/ Go to this site and click Protein (see yellow arrow in figure 1).

Figure 1

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In the pull down menu (see yellow arrows in figure 2) select protein and in the “search” box type “Catalase Homo sapiens”. Click on “Search”.

Figure 2 There are many different versions of this enzyme listed. Select the first one and click on the term FASTA (see yellow arrow in figure 3). It is pronounced “Fast A”. This will give you the amino acid sequence of catalase.

Figure 3 Each letter stands for an amino acid. See the abbreviation code at the end of this activity. Here are the 527 amino acids in catalase. Considering the effort that it took the early protein researchers such as Sanger’s, it is amazing that we now have this information at our fingertips…and for thousands of other proteins as well. MADSRDPASDQMQHWKEQRAAQKADVLTTGAGNPVGDKLNVITVGPRGPLLVQDVVFTDEMAHFDRERIP ERVVHAKGAGAFGYFEVTHDITKYSKAKVFEHIGKKTPIAVRFSTVAGESGSADTVRDPRGFAVKFYTED GNWDLVGNNTPIFFIRDPILFPSFIHSQKRNPQTHLKDPDMVWDFWSLRPESLHQVSFLFSDRGIPDGHR HMNGYGSHTFKLVNANGEAVYCKFHYKTDQGIKNLSVEDAARLSQEDPDYGIRDLFNAIATGKYPSWTFY IQVMTFNQAETFPFNPFDLTKVWPHKDYPLIPVGKLVLNRNPVNYFAEVEQIAFDPSNMPPGIEASPDKM LQGRLFAYPDTHRHRLGPNYLHIPVNCPYRARVANYQRDGPMCMQDNQGGAPNYYPNSFGAPEQQPSALE HSIQYSGEVRRFNTANDDNVTQVRAFYVNVLNEEQRKRLCENIAGHLKDAQIFIQKKAVKNFTEVHPDYG SHIQALLDKYNAEKPKNAIHTFVQSGSHLAAREKANL

Q-1) What are some of the ways that such information might be useful? Explain _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________

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B) Comparing Catalase in Different Mammals. Each protein in the NCBI files has a different code called an “Accession Number”. The accession number for Homo sapiens catalase is given on the web page. It is NP_001743.1. Here are the accession numbers for the enzyme catalase in seven different mammals:

Accession Number

Description

AAB42378.1

Rat

NP_001030463.1

Cow

NP_033934.2

Mouse

NP_001743.1

Human

NP_999466.2

Pig

NP_001002984.1

Wolf

NP_001124739.1

Orangatan

We are going to compare the amino acid sequences of the catalase in these mammals using a computer based NCBI alignment tool called “COBALT”. Go to the following web page: http://www.ncbi.nlm.nih.gov/tools/cobalt/. Type in the accession numbers for the catalase which was derived from seven different mammals. Be sure to include the “underscore” _ in all of the animals’ accession numbers except the rat. In the Job Title box type in “Mammalian Catalase Compared” and then click on the “Align” Box. (see figure 4)

Figure 4

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If you scroll down, you will see the amino acid sequences for the seven mammals. It is given 80 amino acids at a time. Look over these sequences carefully. Q- 2) Do the sequences seem more “alike” or more “different”? Explain giving examples. _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ C) Taxonomic Relationship Between The Seven Mammals Amino Acid Comparison of Catalase in Seven Mammals Catalase Comparison 1 Rat 2 Cow 3 Mouse 4 Human 5 Pig 6 Wolf 7 Orangutan

A Rat 0

B Cow

C Mouse

D Human

E Pig

F Wolf

G orangutan

0 0 0 0 0 0

Indicate the number of amino acids differences in the enzyme “Catalase” between each two mammals in the chart. There are 21 boxes that need to be filled in. Depending on your class size you will be asked to fill in a number of the boxes (ie, A2, A3, B3, etc). Your teacher will share this chart as a google document giving you editing privileges. Once you and your classmates have finished filling in the chart, be sure to include it in the write up of this activity. It may be easier for you to do this by aligning only two organisms at a time using “Cobalt”. For example, if you are assigned B4, then you might want to just use the accession numbers for Catalase in the human and cow. Once you get the aligned amino acid sequences of these two mammals, simply count the number of amino acid differences in the compared sequences. Q-3) Construct a “horizontal” phylogenetic tree based on your results showing each of the seven mammals. Compare yours with your neighbor’s. Draw it below:

Your Phylogenetic Tree (Pig, Mouse, Rat, Human, Wolf, Orangatan, Cow)

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↑

Go to the top of the web page and and click “Phylogenetic Tree” to give you the evolutionary relationship based on catalase structure (see red arrow in figure 5).

Figure 5

Q- 4) How does YOUR phylogenetic tree compare with the COBALT generated version? Explain. _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ D) Sickle Cell Anemia: The Primary Structure of Proteins and Disease One of the more dramatic examples of the significance of primary structure involved the disease sickle cell anemia. This is a disease affecting red blood cells or erythrocytes. Specifically, there is a problem in the amino acid sequence of hemoglobin (Hb) in those afflicted with this genetic disease. Each hemoglobin protein is made of four polypeptides, 2 alpha globin chains and 2 beta globin chains. The problem is with the beta chains. The accession numbers for normal human beta globin is AAA16334.1 and the accession number for sickle cell beta globin is AAN11320.1. Using the NCBI Cobalt Alignment Tool, determine what the fault is with the sickle beta globin. It may surprise you to see how one single error can have dire consequences! Q- 5) Describe the error in the primary sequence of SSA beta globin. Be as specific as possible. _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________

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E) The Activation of Pepsinogen into Pepsin The enzyme pepsin is secreted by the gastric glands in your stomach. It is secreted as “pepsinogen”, which is an inactive form of the enzyme. Q-6) Why is it beneficial to secrete this enzyme in the inactive form? Explain. _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ The accession numbers for pepsinogen and pepsin are “3PSG_A” and “5PEP_A” respectively. Using the NCBI Cobalt Alignment Tool, determine the difference between these two forms of the enzyme (inactive and active). Before to type the “underscore” in the accession numbers. (“3PSG_A” and “5PEP_A”) Q-7) Explain how pepsinogen and pepsin are different. Explain what is meant by posttranslational modification and explain how it is relevant in the case of pepsinogen and pepsin. ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ____________________________________________________

Here are 3 dimensional representations of the two enzymes. Such images are really the subject of the next few activities on the secondary, tertiary and quaternary structure of proteins. The primary sequence of the inactive and active form of the enzyme, along with these images will serve as a good bridge to the forthcoming activities. (see figure 6)

Pepsinogen (inactive enzyme)

Pepsin (active enzyme) Figure 6

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Q-8) Summarize what you have learned about the primary structure of proteins. _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ Further Investigations 1) Choose a group of organisms from a taxonomic clade that you are interested in investigating. Choose a protein that is found in all of the organisms in that clade (there are many candidates: ie, enzymes in the glycolysis family) and use the alignment tool, followed by the “Phylogenetic Tree” option to create an evolutionary tree of the clade that you are investigating. 2) Investigate the relationship between catalase and the organelle called a “peroxisome”. References 1) Wikipedia article on Sickle Cell Anemia: http://en.wikipedia.org/wiki/Sickle-cell_disease 2) Nobel Prize Website on Frederick Sanger: http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1958/sanger-bio.html

Appendix: Amino Acid Abbreviations 1) Alanine 2) Arginine 3) Asparagine 4) Aspartic Acid 5) Cysteine 6) Glutamine 7) Glutamic Acid 8) Glycine 9) Histidine 10) Isoleucine

Ala Arg Asn Asp Cys Gln Glu Gly His Ile

A R N D C Q E G H I

11) Leucine 12) Lysine 13) Methionine 14) Phenylalanine 15) Proline 16) Serine 17) Threonine 18) Tryptophan 19) Tyrosine 20) Valine

Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

L K M F P S T W Y V

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The Secondary Structure of Proteins Goals:  To learn how to use computer modeling to examine the secondary structure of proteins.  To examine the intricate structure of the peptide bond and the nuanced relationship to protein folding.  To consider the factors that both prevent and cause polypeptides to bend. Background: In 1951 Linus Pauling and Robert Corey worked out the two models for the initial folding of a polypeptide. The two conformations were named the alpha helix and the beta pleated sheet. Christen Brownlee writes in “Classics of the Scientific Literature: The Protein Papers (http://www.pnas.org/site/misc/classics1.shtml), “Grasping the structure of these molecules would give scientists a head start on understanding how proteins function in the body. Pauling and Corey's research, now over a half-century old, guides today's biotechnology revolution and the search for hundreds of disease cures--drugs that may someday conquer Alzheimer's disease, cystic fibrosis, Mad Cow disease, and many forms of cancer.” Indeed their findings have had so much impact on our understanding of proteins. We are going to use data from experiments on protein structure as well as computer modeling to get a handle on Pauling and Corey’s models that show different secondary structures in polypeptides. Procedure: A) Consequences of the “peptide bond” joining amino acids. As discussed in the activity on the “Primary Structure of Proteins”, polypeptides are made by joining together a “string” of amino acids. The diagram to the right (Figure 1) show the dehydration reaction by which two amino acids are joined together to form a dipeptide made of two amino acid residues and a water molecule. Note the box showing the C-N peptide bond. It is interesting to note that most carbon-nitrogen single bonds measure 1.49 Ȧ. Most C-N double bonds measure 1.27 A. The peptide bond measures 1.32 A.

Figure 1-Forming a Dipeptide

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Q-1) Based on that information alone, how would you characterize a peptide bond: single or double. Explain. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ What actually happens is a resonance stabilization reaction in which the peptide bond “swings” back and forth between double and single. The same is true for the bond connecting the carbonyl carbon and carbonyl oxygen. (See figure 2). This results in the carbonyl oxygen being slightly negative and the amino hydrogen of the next amino acid residue is slightly positive.

Figure 2 Q-2) How might the slightly negatively charged carbonyl oxygen interact with the slightly positively charged amino hydrogen of two amino acids that are some distance from each other on a polypeptide chain? Explain. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Q-3) Based on your knowledge of organic (carbon) chemistry, what can be said about the rotation on both sides of a single bond? Double bond? If the peptide bond has “double bond” properties, what can be said about rotation on both sides of a peptide bond? Explain. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________

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Examine figure 3 which shows four amino acids peptide bonded together. The double bond nature of the peptide bond may “discourage” rotation on either side of the peptide bond, but it does NOT preclude rotation on both sides of the alpha carbon (α carbon) in each amino acid residue.

Figure 3 Q-4) Examining the tetrapeptide above, note that starting with the amino nitrogen, it goes (left to right) N-C-C-N-C-C-N-C-C and so on. To be more precise, its amino nitrogen, α carbon, carbonyl carbon, amino nigrogen, α carbon, carbonyl carbon and so on. Where is the molecule free to rotate? Be specific and explain. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Consider the “R-Groups). Although some are small-such as glycine and alanine who’s Rgroups are H and CH3 respectively, some of the R-groups are larger and a bit “clunky”. So, as we look into the folding of a protein, we need to consider: 1) where the polypeptide can twist or fold; 2) where it cannot do so; 3) what to do with these “clunky” R-groups; and 4) what factors will cause the polypeptide to fold. B) Using Jmol to Investigate the Alpha Helix. We are now going to investigate the secondary structure of an enzyme called carboxypeptidase. In exercise # 4 we will revisit carboxypeptidase as we investigate how this enzyme can bind to its substrate and catalyze a reaction. But, our emphasis here will be to investigate the alpha helical and then the beta pleated sheet regions of this enzyme. To do so, we will use the computer program called Jmol.

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There are three ways that we will manipulate and measure our model using the Jmol program: 1. Type directions onto the Jmol Script Console in the window to the left. All jmol instructions that you need to type on the Jmol Script Console will be in bold and within quotation marks. Type in the instructons just after the $ sign. Leave out the quotation marks when you type your directions on the Jmol Script Console. It is important to apply the correct syntax in doing so. 2. With the cursor on the screen, left click and choose various items in the menu or submenus. Each step will be followed with a “>” sign and will be in bold. For example, style>scheme>ball and stick. 3. Use the menus, submenus and shortcuts in the toolbar. Some of the instructions are cumbersome to type out and so for those we will use the script editor. In such cases, you will find it easiest to copy and paste instructions from this document onto the script editor box and then select “run” to enact the instructions. Open your Jmol program and drag the Enzyme/Substrate (5CPA PDB) file onto the blackened screen. A version of the protein will appear on the screen. (see Figure 4) This protein is made of 307 amino acid residues. The image shows many surrounding water molecules (the red dots) and the single polypeptide, some of which is in the “cartoon” scheme (the pink and orange regions) and some of it is in the “trace” scheme (the white regions). First we are specifically interested in investigating the pink regions. Move the cursor across the image. Figure 4 Note that you can rotate the image in different directions. Q- 5) How would you describe the pink regions of this molecule? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________

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If you move the cursor onto the image, information about the amino acid that your cursor is sitting above. We are going to isolate one spiral region of the protein, specifically that α helical region encompassed by amino acids 14-27. Type “Display 14-27” and press Enter. Enlarge the α helix by typing “zoom 150” and pressing Enter. Drag the cursor across the molecule such that it is turned in an “upright” position. Then press both ctrl and alt to move the helix into the center of the screen. See figure 5.

Figure 5 Convert the molecule to the “ball and sticks” format by right clicking and choosing style>scheme>Ball and Stick. See figure 6. As we continue to analyze the alpha helix, it would be helpful if everyone conducting this activity positioned the helix in the same way. Recall that biochemists number the amino acid residues in a polypeptide in order, going from the Nterminus to the C-terminus. Place the cursor on one of the Figure 6 atoms at the bottom of the screen. We want the lower numbered N terminus (residue 14) to be at the bottom of the screen. If “[Thr]14…” appears, then you are ok. But if “[Ala]27…” appears, then the molecules is upside down and you need to reposition it. See figure 7 showing the right position. Your molecule may look a bit different if it is twisted more than the way it is shown in figure 7. In order to see the position of the hydrogen bonds, type “Select 14-27” and press Enter. Then type “Calculate hbonds” and press Enter.

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Ok, it is time to begin analyzing this structure. Note the hydrogen bonds. Q-6) Which atoms (element name) are joined by the hydrogen bonds. In identifying the elements, be as specific as possible. Keep in mind that hydrogens are not shown, so an hbond going toward an amino nitrogen is actually connected to the hydrogen not shown. (Red=oxygen, gray=carbon, blue=nitrogen) ____________________________________ ____________________________________ ____________________________________ ____________________________________ Note the number of the amino acid residues joined by the h-bonds. To do so, move the cursor onto the oxygen or amino nitrogen and note the number of the residue. You need to be a bit careful in doing so because the dotted red/blue line representing the h-bond my go behind some of the atoms that you are observing leading you to name the wrong residue. Be sure that you can see the entire h-bond.

Figure 7

Q-7) What is the difference in residue numbers of the h-bonded amino acid residues? _____________________________________________________________________________ Q-8) What role do these h-bonds play in maintaining the α-helical structure? Explain. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Type “select sidechains” and press Enter. Type “Color green” and press Enter. See figure 8. This molecule is called the α helix. The designation “α” is simply due to the fact that Pauling and Corey discovered it first, hence “α” for first and then later they discovered the β conformation, hence “β” for second. But let us see why they called the “α helix” a helix. So to do this, let’s examine the backbone of the molecule, ignoring the green side chains which we will discuss shortly.

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The backbone of the molecule is made of nitrogens and carbons. Starting with amino acid residue 14 at or near the bottom of your figure, follow the backbone. You will need to drag the molecule to the left or right each time an atom in the backbone is obscured. Note the backbone goes….N-C-C-N-C-CN-C-C….and so on. Although you might feel like you are zig zagging a bit, if you follow the backbone you will see that it is like going up a spiral staircase, a staircase the goes up in a counter-clockwise direction. To view the molecule from all directions, type “move 0 360 0 0 0 0 0 0 10” and press Enter. The molecule will turn 360 degrees in 10 seconds. (Some of the zeroes in this instruction hold the place of instructions that can be used to move the molecule in other ways including rotating the molecule along the Y or Z axis.) You can repeat these instructions if desired. Now drag the bottom of the molecule up so that it rotates in a way such that the center of the helix is facing you. You may have to press ctrl and alt and re-center the image.

Figure 8

Q-9) Where are the (green) R-groups located. Considering their “clunkiness”, why would this positioning make sense? Explain. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ (Bonus) : Try to figure out about how many amino acids residues there are per turn of the helix. This is a bit difficult, because you will need to reposition the molecule somewhat as you go up the backbone. It is NOT a whole number as some suspected when the model was first described. See how close you can come. Once you have the number (again, it will not be a whole number, so you will have to make an estimate), check with your instructor. Here is another bit of advice. Get the sidechains out of your way by typing “select 14-27” and press Enter and then type “restrict backbone” and press Enter. This will remove everything except what you restricted….in this case the backbone of the helix. See figure 9. Figure 9

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Here is another trick you may try. Starting with residue 14, color each residue differently…you need not go too far with this, but try coloring residues 14-20 differently. Just type “Select 14” and press Enter and then type “color tan” and press Enter. Repeat with each residue up to 20 using a different color each time. Then type “Select oxygen” and press Enter. Type “color red” and press Enter. Type “Select nitrogen” and press Enter. Type “color blue” and press Enter. So now the α carbon and carbonyl carbon for each residue in the backbone has different color carbons, but the blue nitrogen and red carbonyl oxygen serve as markers so that you can see when you have completed one turn of the helix. How many residues per turn of the helix? Check your results with your instructor. See figure 10. END OF BONUS Measuring the length of the polypeptide in the α-helix: Figure 10 Left click the “ruler” image in the toolbar above at the top of the display window. See figure 11. Move the cursor above the carbonyl oxygens until you find the oxygen in residue 19. Left click while the cursor is over that oxygen. Then, move the cursor over the carbonyl oxygen in residue 23 and left click. A dotted line will be shown connecting those oxygens and the Figure 11 distance between them will be measured. Record that distance between these 5 amino acids. You will carry out a similar measurement when examining the β-conformation for comparison purposes later in this activity. Q-10) Summarize what you have learned about the structure of the α-helix being as complete and specific as possible. There are a lot of good answers one could give….make it super. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________

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C. Using Jmol to Investigate the Beta Conformation (Pleated Sheat) Drag the Enzyme/Substrate (5CPA PDB) file onto the screen. If the image from part B of this activity is still on the screen, the 5CPA PDB file will replace it. This time we are not interested in the pink α-helical regions of the molecule, but rather the yellowish orange β-pleated sheet (or β-conformation). It is referred to as “β”…the second letter in the Greek alphabet…because it’s structure was worked out second by Pauling and Corey. We will only display a part of the molecule which shows this structure. Rotate the molecule by dragging the cursor across it in such a way that maximizes the view of regions of the polypeptide in the β-conformation. Now we are going to remove from view much of the polypeptide which is NOT in the β-conformation. Type “Display 32-36, 49-53” and press Enter. The cartoon display of a region of the polypeptide is shown. The arrows indicate the direction of the chains going from the N-terminus toward the C-terminus. Type “zoom 200” to increase it’s size 200%. Press Ctrl and Alt while you drag the molecule to the center of the window. See figure 12. Figure 12 Q-11) What does it mean that these two sections of the polypeptide are antiparallel? Explain. ________________________________ ________________________________ ________________________________ ________________________________ Now, to better visualize the details of the structure will put this region of the polypeptide in the “Ball and Stick” scheme. Convert the molecule to the “ball and sticks” format by typing “Select 3236, 49-53” and pressing Enter and then right clicking and choosing style>scheme>Ball and Stick. See Figure 13.

Figure 13

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In order to see the position of the hydrogen bonds, type “Calculate hbonds” and press Enter. In order to highlight the sidechains so that they can be seen as distinct from the backbone of the chains, type “select sidechains” and press Enter. Type “color green” and press Enter. See figure 14. Viewing this structure from different angles will give us insights about it’ structure. The two sections of the polypeptide go from residues 32 to 36 and from residues 49-53. Q-12) What joins the two sections of the polypeptide? More specifically, note what atoms are connected. Where a nitrogen is involved, it is actually the hydrogen attached to the nitrogen (not shown), that is involved in the bonds. Note the alternating nature of the bonds; going from nitrogen Figure 14 of one section to the oxygen of the other section and the next bond going from the oxygen of one section to the nitrogen of the other section. ________________________________________ Turn the molecule such that the backbones of the two sections of the polypeptide are as aligned as possible. To facilitate view this, one section of the polypeptide will be removed from view. Type “display 49-53” and press Enter. See figure 15. Q-13) In what directions are the green sidechains pointing? ________________________________________ Using the same procedure that you used when investigating the length of part of a polypeptide chain in the α-helix, measure the distance between 5 amino acids, specifically the carbonyl oxygens in residues 49-53.

Figure 15

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Q-14) Summarize what you have learned about the structure of the β-conformation being as complete and specific as possible. There are a lot of good answers one could give….again, make it super. ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Q-15) Make a comparison between the α-helix and the β-conformation. Consider the following:

Characteristic

α-helix

β-conformation

General Shape

Position of the H bonds

Position of the R-groups

Length of Section of Polypeptide Per 5 Amino Acid Residues Other Features

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Further Investigations 1) Determine what percentage of the protein which is in the α-helix and what percentage is in the β-conformation. 2) What factors determine whether or not a section of a polypeptide is in the α-helix or in the β-conformation.

References 1) PNAS at 100: Classics of Scientific Literature. The Protein Papers by Christen Brownlee. http://www.pnas.org/site/misc/classics1.shtml

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The Tertiary and Quaternary Structure of Proteins Goals:  

To examine the factors that cause a protein to fold into its three dimensional tertiary structure For those proteins constructed of more than one polypeptide, to examine how those polypeptides are joined together to make a functional protein

Background: In 1962 John Kendrew and Max Perutz were awarded Nobel Prizes for their work on the structure of proteins. Using X-ray analysis, they were able to work out the three dimensional tertiary structure of the proteins myoglobin and hemoglobin. In our prior activity we examined the factors which cause the initial folding of a polypeptide into either an alpha helix or a beta conformation. The next level of folding is described as the tertiary structure of the protein. We will consider four factors that cause this level of folding: 1)Hydrophobic and hydrophilic interactions; 2)Disulfide bonds between cysteines; 3)Ionic interactions between fully charged amino acids; and 4)Hydrogen Bonds. We will then briefly examine the quaternary structure of hemoglobin. Procedure: A) Hydrophobic and Hydrophilic Interactions Assume for a moment that hydrophobic and hydrophilic interactions between amino acids and their surrounding (both internal and external to the rest of the protein) were the only factors that affect protein folding: Q-1) How would the way the protein folds be different if it were moved from a watery (polar) environment to an oily (nonpolar) environment (or in the opposite direction)? Explain. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ We are going to use the program Jmol to analyze several different proteins as we try to understand the basis of protein folding. The box below has some general instructions for using this powerful program and there is further information in the appendix at the end of the book which may be helpful.

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There are three ways that we will manipulate and measure our model using the Jmol program: 1. Type directions onto the Jmol Script Console in the window to the left. All jmol instructions that you need to type on the Jmol Script Console will be in bold and within quotation marks. Type in the instructons just after the $ sign. Leave out the quotation marks when you type your directions on the Jmol Script Console. It is important to apply the correct syntax in doing so. 2. mWith the cursor on the screen, left click and choose various items in the menu or submenus. Each step will be followed with a “>” sign and will be in bold. For example, style>scheme>ball and stick. 3. Use the menus, submenus and shortcuts in the toolbar. Some of the instructions are cumbersome to type out and so for those we will use the script editor. In such cases, you will find it easiest to copy and paste instructions from this document onto the script editor box and then select “run” to enact the instructions. Open your Jmol program and drag the Enzyme pepsin (5PEP PDB) file onto the blackened screen. A version of the protein will appear on the screen. In the Jmol script console, type “cpk” and press Enter. Type “zoom 90” and press Enter. Type “hide hoh” and press Enter. The image of pepsin now fits the screen and is in a space filling scheme. Type “select hydrophobic” and press Enter. Type “color green” and press Enter. Type “select polar” and press Enter. Type “color yellow” and press Enter. See figure 1. Pepsin does its work in the watery, acidic solution of your stomach. Q-2) Does pepsin sit in a polar or nonpolar solution when it is in your stomach? Explain. ____________________________________ ____________________________________ Figure 1 ______________________________________________________________________________ ______________________________________________________________________________

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Q-3) Would you expect most of the amino acids facing the outside to be; hydrophobic (nonpolargreen) or hydrophilic (polar or charged-yellow)? Does the diagram support your hypothesis? Explain. Consider the following information when you answer this question. Although only 45% of the atoms are associated with polar residues, it appears that over 50% of the visible residues are yellow. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Q-4) How would the answer to Q-3 change if this protein were sitting in a nonpolar environment? Explain. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ The protein potassium channel is just such a protein. It traverses the plasma membrane. Most of the protein channel is embedded in the nonpolar region of the lipid bilayer. Q-5) Given that information, where would you expect the hydrophobic (nonpolar) or hydrophilic (polar or charged) residues to be located? Explain. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Drag the potassium channel (1BL8 PDB) file onto the blackened Jmol screen. A version of the protein will appear on the screen. In the Jmol script console, type “cpk” and press Enter. Type “zoom 90” and press Enter. The image of potassium now fits the screen and is in a space filling scheme. It is a teepee shaped protein complex. Turn the channel such that the narrow end is facing you the observer. You should be able to see straight into the channel and the purple potassium ions should be visible. Type “select hydrophobic” and press Enter. Type “color green” and press Enter. Type “select polar” and press Enter. Type “color yellow” and press Enter. See figure 2.

Figure 2

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Unlike the last protein, pepsin which sat in a polar environment, this one is sitting in a mostly nonpolar environment, although, each end of the protein is facing either the watery inside of the cell or the watery outside of the cell. Q-6) Is this coloration consistent with your expectations? Explain why or why not. Also, to see a “slice” of the protein right down the middle, type “slab on” and press Enter and type “slab 50” and press Enter. Are the colors what you predict in the middle of such a molecule? Explain. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Q-7) Summarize what you have learned about hydrophobic and hydrophilic intereactions and how they vary with the environment that the protein is sitting in. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ B) Covalent Disulfide Bonds Between Cysteines Drag the enzyme amylase (1PPI PDB) file onto the blackened Jmol screen. A version of the protein will appear on the screen. Type “hide hoh” and press Enter. That instruction will hide the water molecules. To convert them to a ball and stick format, right click and then choose style>scheme>ball and stick (see figure 3).

Figure 3 Type “select sulfur” and press Enter. Type “cpk 200” and press Enter. Type “ssbonds on” and press Enter. Type ssbonds 100 and press Enter. This series of events selects sulfur atoms, enlarges them, turns on the disulfide bonds and thickens them so they are easily visualized.

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Out of the many amino acid residues in amylase, only 12 are cysteines. Let us determine how many of them are involved in forming disulfide bonds. Type “display [cys]” and press Enter. Only those 12 cysteines are displayed and all of the other amino acid residues are hidden. Q-8) How many of those 12 form disulfide bonds. You may need to rotate the molecule by dragging the cursor across the screen in order to answer this question. ___________________________________________________________________________ There are 496 amino acid residues in this particular amylase derived from wild pig. Five pair of cysteine residues are linked by disulfide bonds. Move the cursor onto each sulfur and record the number of the residue within the polypeptide chain. Cys _____linked to Cys_____ Cys _____linked to Cys_____ Cys _____linked to Cys_____ Cys _____linked to Cys_____ Cys _____linked to Cys_____ Q-9) Which linkages likely result in the sharpest folds in the polypeptide? Which linkages are between cysteines that are most distant? ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ In order to visualize the relatively sharp turn caused by the linkage of cysteine 378 and 384 we will look at a few amino acid residues before, between and after those two cysteines. Type “display 370-390” and press Enter. Then type “select 370-377, 379-383, 385-390” and press Enter. Right click and then choose style>scheme>trace. Then type “color green” and press Enter. See figure 4. Type “zoom 250” and move the model to the center by pressing Ctrl and Alt and dragging the model to the center.

Figure 4

Disulfide bonds between nearby and distant amino acid residues play an important role in protein folding. Let us now look at another factor which plays a role in folding.

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C) Ionic Bonds Between Oppositely Charged R-Groups Q-10) Which amino acids have positively charged R-groups? Negatively charged R-groups? ___________________________________________________________________________ ___________________________________________________________________________ Q-11) What type of bond forms between fully charged atoms? _________________________ We are going to examine the role of this type of interaction between amino acid residues in determining the 3-dimensional shape of a polypeptide. These bonds may form between oppositely charged R-groups that are some distance from each other on the polypeptide chain. Drag the enzyme amylase (1PPI PDB) file onto the blackened Jmol screen. A version of the protein will appear on the screen. Type “hide hoh” and press Enter. Type “display 294-306, 15-64, 197-243” and press Enter. With the cursor on the screen, right click Style>Scheme>Trace. Type “select all” and press Enter. Type “color green” and press Enter. The following pairs of amino acid residues contain oppositely charged R-groups that are close enough together to form bonds even though they are somewhat distant on the polypeptide chain: 18 and 61; 200 and 240; 297 and 303. This chart summarizes the properties of those amino acids. There are many other charged amino acid residues in amylase…we are just examining a few examples here.

Amino Acid Amino Acid Number Charged Group Glutamate Arginine Lysine Glutamate Aspartate Arginine

18 61 200 240 297 303

-COO=NH2 + - NH3 + -COO-COO=NH2 +

Atom (Number) Involved in IonicBond 144 500 1562 1884 2343 2396

Type “select 18, 61, 200, 240, 297, 303” and press Enter. With the cursor on the screen, right click Style>Scheme>Ball and Stick. To show the ionic bonds that form…   

Type “select atomno=144, atomno=500 and press Enter. Type “connect single” and press Enter. Type “color yellow” and press Enter. Type “select atomno=1562, atomno=1884 and press Enter. Type “connect single” and press Enter. Type “color yellow” and press Enter. Type “select atomno=2343, atomno=2396 and press Enter. Type “connect single” and press Enter. Type “color yellow” and press Enter.

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Note how the ionic bonds are yet another factor causing the polypeptide to fold. There are many other ionic interactions in this molecule. Rotate the part of the molecule visible on the screen so that you can see the ionically bonded amino acid residues and the folds in the adjacent polypeptide regions. See figure figure 5.

Figure 5 D) Hydrogen Bonds Between Slightly Oppositely Charged R-Groups, Amino Acyl Groups and Carbonyl Oxygens We examined the role of hydrogen bonds in determining the secondary structure of a polypeptide. Hydrogen bonds also play a role in tertiary structure. Though weak, they are numerous and thus significant factors in causing a polypeptide to fold. Drag the enzyme amylase (1PPI PDB) file onto the blackened Jmol screen. A version of the protein will appear on the screen. Type “hide hoh” and press Enter. Type “display 342-346, 355-370, 381-383” and press Enter. With the cursor on the screen, right click Style>Scheme>Ball and Sticks. In order to visualize the H-bonds, type “calculate Hbonds” and press Enter. Type “color hbonds yellow” and press Enter. Type ”select sidechains” and press Enter. Type “color green” and press Enter. At this point you can see the selected amino acids with their sidechains in green and the back bone in gray (carbon), blue (nitrogen) and red (oxygen). Note the yellow hydrogen bonds between several of the nitrogens and oxygens. See figure 6.

Figure 6

ii

Q-12) How many h-bonds can you find (you may need to rotate the molecule a bit to see some of them) in this region of the protein? ___________________Which 2 h-bonded amino acid residues are closest to each other along the chain? ____ and ____. Which 2 hbonded amino acid residues are furthest from each other along the chain? ____ and ____. Which of the highlighted hbonds contribute to secondary structure? ________________ Tertiary structure? _________________

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************************************************************ One of the biggest challenges in biochemistry is to develop a way of predicting tertiary structure of a protein, who’s primary structure is known. Based on what you have learned about primary, secondary and tertiary structure, why would making such a prediction be so difficult…even with the use of the most sophisticated computer programs? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ E) The Quaternary Structure of Proteins: Hemaglobin Many proteins are composed of a single polypeptide chain. For those proteins, analyzing the primary, secondary and tertiary structure is adequate when fully elucidating its three dimensional structure. For other proteins made of more than one polypeptide chain, a fourth level of structure must be investigated. Hemaglobin is such a protein. Hemaglobin transports oxygen in our blood, specifically in our red blood cells or erythrocytes. It is made of four polypeptide chains, 2 alpha globins and 2 beta globins. Earlier in this activity we compared the sequences of normal and sickle cell beta globin. All of the factors which affect the tertiary structure of a protein, may also affect the quaternary structure. Now we are going to take a brief look at hemoglobin. Drag the hemaglobin (1A3N PDB) file onto the blackened screen. A version of the protein will appear on the screen. Type “hide hoh” and press Enter. Convert the molecule into a “ball and sticks” format by typing “select all” and pressing Enter. With the cursor on the screen, right click Style>Scheme>Ball and Sticks. In order to better visualize the distinct polypeptide chains, type “select *A” and press Enter. Then type “color green” and press Enter. Repeat for the other chains, B, C, and D coloring them yellow, magenta and orange respectively. Each of the four polypeptides has a “heme” ligand attached to it. To highlight the ligands, type “select [hem]” and press Enter. Then, type “color white” and press Enter.

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The heme ligands are all attached to histines. To highlight this, type “select [his]87:A, [his]92:B, [his]87:C, [his]92:D” and press Enter. See Figure 7 below.

Figure 7

Further Investigations 1) Choose a protein that you are curious about. Do a google search of its functions. Then find its primary structure (FASTA sequence) and then find a PDB file of that protein. Examine it fully looking at how much of its structure is in the alpha helix, beta pleated sheet and what factors are causing it to fold. Look at which regions are hydrophobic and hydrophilic, where are there charged amino acids or cysteines. Locate disulfide linkages….in other words, do a full study of your protein’s structure and function. References 1) Biography: John Cowdery Kendrew http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1962/kendrew.html 2) Biography: Max Perutz http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1962/perutz

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Carboxypeptidase A-An Example of Substrate Binding and Catalysis Learning About Proteins Through Computer Modeling Goals:  

To learn how computer modeling can help us understand how an enzyme binds to it’s substrate. To learn about the mechanisms of catalysis according to the induced fit model of enzyme action.

Background: Enzymes are remarkable! They speed up chemical reactions, sometimes up to 10 billion times faster than if the enzyme were absent. Ezymes commonly react with about 10,000 substrates per second and that number may go up to 500,000 in some cases. Today we will use the program Jmol to get some insights on how enzymes bind to their substrates and then catalyze a reaction. The enzyme that we are studying today is carboxypeptidase, a pancreatic exopeptidase that removes amino acids, one at a time, from the C-terminus of a polypeptide. The carboxypeptidase A that we are studying today is a digestive enzyme, normally secreted in an inactive form and then activated once inside the intestines. Carboxypeptidases act in other ways. For example, many proteins, such as insulin are modified by the removal of amino acids after they are initially translated. This is referred to as post translational modification and may be carried out by a type of carboxypeptidase. Considering this brief introduction to enzyme action, you are now ready to employ computational techniques to explore a protein. Q-1) Based on that background information, what questions do YOU have about enzyme action? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ The questions that you will answer are: How do enzymes selectively bind to substrates and how do they perform their catalytic role in cells?

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Procedure: In this activity you will be using computer modeling of proteins to learn about enzyme action. To do so, you will use the Jmol Program. There are three ways that we will manipulate and measure our model using the Jmol program: 1. Type directions onto the Jmol Script Console in the window to the left. All jmol instructions that you need to type on the Jmol Script Console will be in bold and within quotation marks. Type in the instructons just after the $ sign. Leave out the quotation marks when you type your directions on the Jmol Script Console. It is important to apply the correct syntax in doing so. 2. With the cursor on the screen, left click and choose various items in the menu or submenus. Each step will be followed with a “>” sign and will be in bold. For example, style>scheme>ball and stick. 3. Use the menus, submenus and shortcuts in the toolbar. Some of the instructions are cumbersome to type out and so for those we will use the script editor. In such cases, you will find it easiest to copy and paste instructions from this document onto the script editor box and then select “run” to enact the instructions. A) Open your Jmol program and drag the Enzyme/Substrate (3CPA PDB) file onto the blackened screen. A “cartoon” version of the enzyme will appear on the screen. Convert it to a ball and stick version by moving the cursor onto the screen, right click, and then choosing style>scheme>ball and stick. See figure 1.We want to discover how this molecule works by manipulating the model. You will see that this program is a powerful tool for studying proteins. Figure 1 B) Start out by moving your cursor onto the black display screen. Note that you can rotate the molecule in different directions by dragging different parts of it. This diagram is quite overwhelming, so we need to manipulate it in ways which will be instructive.

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Q-2) Based on what you know about enzymes, what part of the molecule would you like to focus on? Explain ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ This particular PDB file also includes an artificial substrate, a dipeptide made of glycine at the N-terminus peptide bonded to a tyrosine at the C-terminus. It is sitting in the active site. Q-3) Know that the substrate is sitting in the active site, theoretically, how might you use the Jmol program to locate the active site? (Hint: Historically, scientists have used dyes and radioactive substances to do essentially the same thing.) Explain. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ C) OK, so let us use the program to find the substrate…and therefore the active site. The substrate of made of two amino acids which are residues 501 and 502. Type “select 501, 502”. Now press “Enter” or “Return”. Then type “color green” and then press “Enter”. Type “cpk 250” and press “Enter”. See figure 2A. Q-4) Can you tell where the substrate is? Can you tell where the active site is? Explain. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ Using your cursor, drag across the screen to rotate the protein in different directions. Note, that there are times when the substrate is totally visible and there are times when it is partially or almost completely blocked by other amino acid residues in the carboxypeptidase A. See Figure 2A-C.

Figure 2

A

B

C

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Q-5) How does such manipulation allow you to learn where the opening to the active site is? Explain. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ If you move the cursor to amino acids around the substrate, the name of those amino acid residues and the position of them in the polypeptide chain will appear. Our concern is with the amino acids that play a role in binding (residues ) and those that play a role in the catalytic event (residues and Zinc). Accordingly, we will display those amino acids, the substrate (501 and 502), Zn and hide the rest of the amino acid residues. Type“Display[Tyr]248,[Glu]270,[Tyr]198,[Phe]279,[Arg]145,[His]69,[Glu]72, [His]196, [Arg]71,501, 502,Zn”. Now press “Enter” or “return”. (Note: you could copy and paste the instructions above or you could just type the residue numbers without the names.) D) The 9 amino acids of the active site (remember, there are a total of 307 amino acids in the enzyme) and substrate are now the only visible part of the structure. The hydropobic side chain of the tyrosine residue of the substrate fits into a similarly hydrophobic (nonpolar) pocket in the enzyme, but that is not shown here. You can rotate this structure in different directions by “dragging” it up or down or back and forth sideways. You can also enlarge it any percentage. Figure 3 Type “zoom 150” to enlarge it 150%. Center the molecule by holding down ctrl and alt and dragging the molecule to the center of the screen. See figure 3.

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E) In the next part of the activity we are going to make changes in the appearance of the active site and either color various structures or label them to allow for easy reference. First we are going to change the scheme of the amino acids comprising the active site. Type“select[Tyr]248,[Glu] 270,[Tyr]198,[Phe]279, [Arg]145,[His]69,[Glu]72, [His]196, [Arg]” and press Enter. Then, with the cursor on the screen, right click Style>Scheme>Wireframe (see figure 4).

Figure 4

Type “Select 501, 502” and press Enter. Then with the cursor on the screen, right click Style>Scheme>Ball and Stick. Then type “Select Zn” and press Enter. Type “Color green” and press Enter. Type “cpk 150” and press Enter. At this point, the amino acids in the active site are in the wire frame scheme, the dipeptide substrate is in the ball and stick scheme and the Zn is colored green. (See figure 5). We need to make some other features easily identifiable. First we will color the two atoms directly involved in the peptide bond within the substrate. Remember, that this bond is severed during the hydrolytic reaction catalyzed by this enzyme. Figure 5 Type “Select [GLY]501:A.C #2441” and press Enter. Then type “Color orange” and press Enter. Type “select [TYR]502:A.N #2443” and press Enter. Then type “Color white” and press Enter. Finally, in order to make it easier to refer to the different residues, we are going to name each one. To do this, first go to the File Menu and open the Script Editor.

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“Cut and paste” or “Type” the following into the “Script Editor” and then select “Run” in the script editor window. (Each of the atoms named is the alpha carbon in the amino acid residue. %n=name of the residue and %r= residue number in the polyeptide) select atomno=1958 label %n%r select atomno=2137 label %n%r select atomno=1160 label %n%r select atomno=1552 label %n%r select atomno=578 label %n%r select atomno=551 label %n%r select atomno=1568 label%n%r select atomno=2211 label%n%r select atomno=568 label%n%r

Figure 6

As stated in the introduction, carboxypeptidase A “chops off” the terminal amino acid at the C-terminus of a polypeptide or even a dipeptide such as the one in this active site. We are now going to carefully dissect the image before us by answering a series of questions. For some of the questions, hints will be given for answering the question. First let’s look at the substrate, a dipeptide. The Substrate

F) We are now going to use our model to learn about the substrate: glycyltyrosine. Q-6) What are the two amino acids which make up the dipeptide? (Hint: move the cursor of one of the atoms in the dipeptide and the amino acid residue will be named with a three letter abbreviation.) ___________________________

_____________________________

Q-7) Which amino acid is at the N-terminus? ___________________________ C-terminus? ___________________________

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Q-8) What does the orange atom represent? (Be as specific as possible.) __________________________________________________________ What does the white atom represent? (Be as specific as possible.) __________________________________________________________ Q-9) When this substrate is hydrolyzed, what molecule NOT SHOWN in the image is involved in the reaction? Answer this question by completing the following sentence: Glycyltyrosine +

? → Gycine + Tyrosine

?=_______________________

Q-10) What charge is associated with: the carbonyl oxygen of the peptide bond?

_________________________

the NH hydrogen of the peptide bond?

_________________________

the zinc ion?

_________________________

the carboxyl group of the substrate?

_________________________

the amino group of the substrate?

_________________________

the amino group in the sidechain of Arginine 145?_____________________ the carboxyl group of glu 270?

_________________________

the OH group in the sidechain of tyrosine 248? _______________________ The Active Site: Binding to the Substrate G) When an enzyme and substrate come together there must be both a “spactial fit” and a “bonding fit”. There are several sites where the enzyme carboxypeptidase bonds to it’s substrate, in this case glycyltyrosine. We are going to identify those binding sites and place a bond between the relevant atoms. Look back at your responses in question 10. While viewing your responses and the model and while rotating the model so that you can see it a various angles, try to identify places where bonds might form. This is more or less like a “matching question” on an exam….but you need to look at the spatial orientation of the Zn, the residues in the active site and the substrate in order to ascertain your answers. There are four places where a bond forms. By placing the cursor on the atom you can see the 4 digit number of the atom. (NOTE:, hydrogens are not shown in the model, so for any NH groups, find the number of the nitrogen.

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Q-11) Fill in the answers below and then check with your instructor before you proceed. (atomno refers to the number of the atom in the model): atomno=_____________bonds to atomno=______________ atomno=_____________bonds to atomno=______________ atomno=_____________bonds to atomno=______________ atomno=_____________bonds to atomno=______________ Ok, now let us form those bonds on our model. For each of the bonds, Type “select atomno=?, atomno=?” and substitute the ? with the number that you determined in Q-11. The press “Enter”. Then type “connect single” and press “Enter”. Type “color bond purple” and press Enter. Repeat these three steps for each of the four bonds. The enzyme is now bound to the substrate forming an enzyme-substrate complex. According to Koshlands induced fit model, there are several changes in the enzyme’s structure which are induced by the binding event. Figure 7 shows the enzyme bound to the substrate. Your model may appear different depending on its particular orientation. If you “play” with the orientation, rotating it in different x,y,z direction, you should be able to see your model in a position which is similar to the one shown in figure 7. This is optional.

Figure 7

Note that the bond between the substrates amino group and glu 270 actually occurs through an intervening water molecule. The Active Site: The Catalytic Event The joining of the enzyme to the substrate causes a conformational change in the enzyme (induced fit) which cannot be easily shown with this program. These changes include the movement of groups in glu 270, arg 145 and tyr 248. The actual catalytic event which occurs in the active site is quite complex and there are different models which have been proposed to explain the event.

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According to one model (see figure 8) an OH- group from the intervening water molecule between glu 270 and the carbonyl oxygen “attacks” the carbonyl carbon. The Zn ion which was pointed toward the carbonyl oxygen facilitates this action. Tyr 248 donates a proton (H+) to the NH group of the susceptible peptide bond.

Figure 8 (http://openlearn.open.ac.uk/file.php/4238/!via/oucontent/course/482/s377book1chapter3_f046hi.jpg)

Examining the Active Site in Context with the Whole Enzyme H) We are now going to display the rest of the enzyme so that we may see the active site and substrate as part of the whole enzyme. Type “Display all” and press Enter. Type “select [Tyr]248,[Glu]270,[Tyr]198,[Phe]279,[Arg]145,[His]69,[Glu]72,[His]196,[Arg]71” and press Enter. Type “labels off” and press Enter. Type or “cut and paste” the following: Select 1-68, 70, 73-144, 146-195, 197, 199-247, 249-269, 271-278, 280-307 and press Enter. Left click Style>Scheme>Trace. Type “color white” and press Enter. Type “zoom 75” and press Enter.

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You can now see the active site and substrate in contex with the rest of the molecule. Rotate the structure along the x, y or z axes so that you can see the whole active site as it sits in the rest of the protein. Note in figure 9 that when positioned as you see it here, the active site is all bunched up in the lower right hand corner of the enzyme…all contained within the red square. Perhaps most remarkable is that out of 307 amino acids, only a small number are directly involved in binding the substrate and in the catalytic events leading from reactants to products. Q-12) That being the case, what is the role(s) might the other amino acids in this enzyme play? How might your answer differ is the enzyme was membrane bound?

Figure 9

________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________

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Further Investigations 1) Examine the binding and catalytic event of another enzyme. 2) Examine the binding of an inhibitor to the allosteric site of an enzyme. 3) Examine the activation (or inactivation) of an enzyme acted upon by a kinase in a signal pathway. References 1) http://openlearn.open.ac.uk/file.php/4238/!via/oucontent/course/482/s377book1chapt er3_f046hi.jpg (Source: Figure 8) 2) http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi (NCBI: Carboxypeptidase A) 3) http://www.rcsb.org/pdb/explore/explore.do?structureId=1PYT (RCSB Protein Data Bank)

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The Mystery of the Potassium Channel Learning About Proteins Through Computer Modeling Goals:  

To learn how computer modeling can help us understand the relationship between protein structure and function. To learn about the selectivity filter in ion transport protein potassium channels.

Potassium channels play an important role in many organisms from bacteria to humans. In humans, those roles include involvement in heart muscle contraction and nerve cell impulse transmission. In 2003 Dr. Rod MacKinnon, of Rockefeller University, won the Nobel Prize for his research on ion channels. Here is some background information about the potassium channel (KCSA) from Streptomyces lividans that we will be analyzing today: It is made of four identical polypeptide chains, each containing 119 amino acids. Much of the structure plays a significant role in allowing the protein to sit in a phospholipid bilayer. It is more or less shaped like an upside down teepee with the wider end facing the hydrophilic outside of the cell and the narrow end facing the hydrophilic inside of the cell. Most of the protein sits in the hydrophobic region of the membrane amidst the “tails” of the phospholipids. However, the part of the molecule that we will focus in on is the “selectivity filter” (see Figure 1), the part of the protein channel which determines which ions may pass through the membrane…in this case potassium.

The selectivity filter is an amazing feature of the potassium channel. It allows approximately one million K+ ions to pass through it per second and yet it only allows 1 Na+ ion “sneak through

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for every 10,000 K+ ions that enter the cell. That would be like a ticket collector at a football stadium allowing one person without a ticket to enter through the gate for every ten thousand fans entering through the gate which have tickets. It is interesting to note that Na+ ions are smaller than K+ ions. Both ions have the same +1 charge. It seems like Na+ ions DO have the “tickets”, yet they cannot get into the “stadium” (read cell). Q-1) Based on that background information, what questions do YOU have about the potassium channel? ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ The question that you will answer is: How does the selectivity filter work such that it allows potassium ions through, yet it prevents the smaller, equally charged sodium ion from entering the cell? Procedure I) Open your Jmol program and drag the Potassium Channel (1Bl8 PDB) file onto the blackened screen. A mostly magenta computer model of the potassium channel will appear on the screen. See figure 2.

Figure 2

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There are three ways that we will manipulate and measure our model using the Jmol program: 4. Type directions onto the Jmol Script Console in the window to the left. All jmol instructions that you need to type on the Jmol Script Console will be in bold and within quotation marks. Type in the instructons just after the $ sign. Leave out the quotation marks when you type your directions on the Jmol Script Console. It is important to apply the correct syntax in doing so. 5. With the cursor on the screen, left click and choose various items in the menu or submenus. Each step will be followed with a “>” sign and will be in bold. For example, style>scheme>ball and stick. 6. Use the menus, submenus and shortcuts in the toolbar. We want to relive the discovery of how this molecule works by manipulating the model. You will see that this is a powerful and useful program. There is an appendix at the end of the lab which provides a summary of the “Command” instructions used to manipulate this model. J) Start out by moving your cursor onto the black display screen. Note that you can rotate the molecule in different directions by dragging different parts of it. Position the molecule so that you can see straight through the selectivity channel. You will see “potassium” ions right in the center of the channel. They are colored purple. Q-2) Are you viewing the channel as if you are inside the cell or from the outside of the cell? How can you tell? You will probably have to manipulate the image to answer this question. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Q-3) When viewing this model from the side (as if you were looking at the protein positioned in the lipid bilayer), is it possible to see the channel? Why or why not? Explain. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________

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K) Lets check out this protein a bit more. There are four identical polypeptides in this protein. Q-4) What would you like to do to this model so that we can visualize or highlight each polypeptide separately? _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ L) Type “select *A”. Now press “Enter” or “return”. Then type “color pink” (or whatever color you would like to choose: see the appendix for choices) and then press “Enter”. Repeat this step for chains B, C and D using a different color for each chain. Be sure to type the asterisk (*). Q-5) It is still a bit difficult to get a handle on how the selectivity filter is structured and how it works. In order to get a handle on how the selectivity filter works, what would you like to do with this model (be creative)? Explain why. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________

M) In order to see in detail what is going on along the length of the selectivity filter, we must “slice” through this protein. Since the protein is made of four identical polypeptides, by removing two of them, we can, in effect, slice down through the middle of the protein. Type “display *A, *C,” (which in effect causes polypeptide chains B and D to disappear) and then press “Enter”. Let us color the remaining polypeptides one color. Type “Select*A,*C” and press “Enter”. Then type “Color magenta”. To color the potassium white, type “Select K” and press “Enter”. Type “Color white” and press “Enter”. (see figure 3). Note that if you position the remaining Figure 3 polypeptides, you can see the channel. You can also see the white potassium ions in the channel. Let us explore how the selectivity filter works.

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N) We need to take a closer look at the channel and simplify the appearance of the rest of the polypeptides. That will help us to get a handle on how the selectivity filter works. Each of the polypeptides is made of 119 amino acids. We will be able to identify them and analyze them better in a ball and stick format. Type “select all” and press “Enter”. To convert them to a ball and stick format, left click and then choose style>scheme>ball and stick (see figure 4).

Figure 4 O) The structure looks a bit too complex (see figure 5) to really analyze. Lets simplify the whole molecule except for the actual channel or selectivity filter. Each of the 119 amino acids are numbered. In order to see which amino acids are in the channel move the cursor along atoms in the amino acids in the channel to identify their numbers. If you freeze the cursor on an atom, the amino acids that it is part of will be identified in a little box. You will see the three letter abbreviation of the amino acid, its number in the chain and the name of the polypeptide chain that it is in [ie, Ser69A]. This is a bit trickier than it may sound. Some of the atoms appear to be adjacent to the channel, Figure 5 but when you drag the molecule to rotate it you may discover that while it appeared to be in the channel, actual, it was not.

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P) We are going to simplify the structure of both chains except for amino acids 75-78. Type “select 1-74:A” and press “Enter”. Left click style>scheme>Trace. Type “Color green” and press “Enter”. Type “select 79-119:A” and press “Enter”. Left click style>scheme>Trace. Type “Color green” and press “Enter”. Type “select 1-74:C” and press “Enter”. Left click style>scheme>Trace. Type “Color green” and press “Enter”. Type “select 79-119:C” and press “Enter”. Left click style>scheme>Trace. Type “Color green” and press “Enter”. See Figure 6. Figure 6 Q) In order to understand how the selectivity filter works, we need to center the filter on the screen and enlarge it…even if it means blocking out much of the rest of the molecule. Before doing so, let us take a good look at the parts of the polypeptides which are NOT part of the channel. Q-6) What roles do those parts serve with respect to the lipid bilayer and the selectivity filter? ____________________________________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ R) To move the molecule along the X or Y axis, press +right click and drag. Drag the molecule such that the channel is centered. To enlarge the channel or to zoom, press Shift + left click and drag vertically (↕). You may need to center the model a few times as you enlarge it. Lets make the potassium ions almost invisible by coloring them black.

Figure 7

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Type “Select K” and press “Enter”. Type “Color black” and press “Enter”. See Figure 7. S) OK, now we are in a position to analyze the selectivity filter. Note that carbon is shown in gray, nitrogen in blue and oxygen in red. Q-7) What atoms are facing the inside of the channel? _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Q-8) Look at the model of the selectivity filter. You may have to rotate it a bit to answer the following question. How can you tell if an oxygen is a carbonyl oxygen or an oxygen that is part of the R-group of the amino acid? Explain. What is the charge of these oxygens? _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ T) Measure the length of the channel (in angstroms Å) in the following way: Click the ruler symbol in the toolbar. A small “Measurements” screen opens up. Double click the Thr 75 of chain A and then double click the Tyr 78 of chain A. The distance will appear on the diagram and also in the “Measurements” box. Do the same for Thr 75 of chain C and Tyr 78 of chain C. Q-9) What is the length of the channel in angstroms (Å) _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Q-10) Measure the width of the channel by measuring the distance between [Thr]75:A and [Thr]75:C. Do the same between [Tyr]78:A and [Tyr]78:C. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________

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U) Potassium ions do not travel by themselves when they are outside or inside a cell. Q-10) What molecules surround potassium ions in the intra- or intercellular fluid? Explain the basis for bonding between potassium and the molecules which accompany it. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ V) Note that the channel is lined with charged oxygens. In addition, your model is only showing two of the four polypeptides. When all four are present, the negatively charged oxygens form a cage around the potassium. Consider the potassium or sodium going through this channel with it’s “water partners”…that is hydrated potassium or sodium ions. Remember that water is a dipole having a oppositely charged regions. Q-11) What is the charge of a potassium ion? What is the charge of the oxygen in water in the first hydration shell around the potassium? _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Q-12) What is the charge of a sodium ion? What is the charge of the oxygen in water in the first hydration shell around the sodium? _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Q-13) What is the charge of the carbonyl oxygens in valine and glycine lining the channel? ____________________________________________________________________ ____________________________________________________________________ ____________________________________________________________________ ____________________________________________________________________ W) In order for a potassium ion or sodium ion to fit through the channel, water molecules will have to be stripped off of the respective ions. Based on the structure of the channel and based on the structure of the hydrated ions, we need to explore which ion is more likely to be “dehydrated” and thus more likely to go through the channel.

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The diameter of dehydrated Na+ and K+ ions are 1.96Å and 2.66Å, respectively. Q-15) Are the negatively charged oxygen atoms in water closer to the positively charged sodium nucleus or the positively charged potassium nucleus (see figure 8)? Explain. Based on your answer, which ion requires less dehydration energy to remove the hydration shell, potassium or sodium? Explain. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________

Figure 8 -

X) The O s in the selectivity filter are the same distance from K+ as the O-s in the first hydration shell of potassium (see Figure 9). The O-s in the first hydration shell surrounding Na+ is closer in and thus not in the right position to be displaced by the O-s lining the selectivity filter.

Figure 9

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Q-16) Based on that information, which ion is more likely to become dehydrated as it passes through the selectivity pore? Explain. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Y) And finally, here is a look into a cell through a potassium channel (see Figure 10).

Summary-The potassium channel has a selectivity filter which allows larger, positively charged potassium to enter at a rate which is approximately 10,000 times higher than the rate of entry of the smaller, positively charged sodium ion. Based on what you learned through this activity, explain how the selectivity pore operates to selectively allow the higher rate of potassium ion entry than sodium ion entry. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________

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Further Reading/Vido Clip 1) Rod MacKinnons lecture at the Nobel Prize Ceremony. POTASSIUM CHANNELS AND THE ATOMIC BASIS OF SELECTIVE ION CONDUCTION http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2003/mackinnon-lecture.pdf 2) Rod MacKinnon’s biography on the Rockefeller University Web Site. http://www.rockefeller.edu/research/faculty/abstract.php?id=132 3) Char-Chang Shieh, Michael Coghlan, James P. Sullivan AND Murali Gopalakrishnan. Potassium Channels: Molecular Defects, Diseases, and Therapeutic Opportunities. Pharmacol Rev 52:557–593, 2000 4) YouTube Video showing mechanism of action of the potassium channel. http://www.youtube.com/watch?v=UqxzSrjzJ70&feature=related Further Investigations 1) The selectivity channel actually has more than one binding site at the potassium ions move through the selectivity filter. Discuss what causes the ions to be “forced” out on one side as they enter the other side. 2) Learn about voltage and ligand gated channels as they specifically apply to potassium, calcium and sodium channels. 3) What are the physiological effects of various defects in potassium channels (see further reading #3). References 1) Web Books: Memory-Ion Channels http://www.web-books.com/MoBio/Memory/Channel.htm 2) PSI Nature. Structural Biology/Knowledgebase TrkH Potassium Ion Transporter http://sbkb.org/kb/archives.jsp?pageshow=37 3) Carrillo-Tripp, Mauricio; Saint-Martin, Humberto; Ortega-Blake, Iván. A comparative study of the hydration of Na+ and K+ with refined polarizable model potentials. Journal of Chemical Physics, Volume 118, Issue 15, pp. 7062-7073 (2003). 4) Mancinelli, R. A. Botti, F. Bruni, M. A. Ricci and A. K. Soper. Hydration of Sodium, Potassium, and Chloride Ions in Solution and the Concept of Structure Maker/Breaker. Journal of Physical Chemistry. B 2007, 111, 13570-13577 5) Yu. Noskov and Benoît Roux. Importance of Hydration and Dynamics on the Selectivity of the KcsA and NaK Channels. The Journal of General Physiology. January 16, 2007

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