Computational Biology · 2015. 10. 7. · 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

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AP BIOLOGY

2012

Computational Biology

Proteins

Robert S. Goodman

S A R H I G H S C H O O L

2

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.

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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

3

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

http://www.ncbi.nlm.nih.gov/

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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

http://www.ncbi.nlm.nih.gov/protein/203335?report=genbank&log$=prottop&blast_rank=1&RID=







http://www.ncbi.nlm.nih.gov/tools/cobalt/

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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.

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C) Taxonomic Relationship Between The Seven Mammals

Amino Acid Comparison of Catalase in Seven Mammals

Catalase

Comparison

A B C D E F G

Rat Cow Mouse Human Pig Wolf orangutan

1 Rat 0

2 Cow 0

3 Mouse 0

4 Human 0

5 Pig 0

6 Wolf 0

7 Orangutan 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.

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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.

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_________________________________________________________________________ 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 post-

translational 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.

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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 Ala A

2) Arginine Arg R

3) Asparagine Asn N

4) Aspartic Acid Asp D

5) Cysteine Cys C

6) Glutamine Gln Q

7) Glutamic Acid Glu E

8) Glycine Gly G

9) Histidine His H

10) Isoleucine Ile I

11) Leucine Leu L

12) Lysine Lys K

13) Methionine Met M

14) Phenylalanine Phe F

15) Proline Pro P

16) Serine Ser S

17) Threonine Thr T

18) Tryptophan Trp W

19) Tyrosine Tyr Y

20) Valine Val V

http://en.wikipedia.org/wiki/Sickle-cell_disease

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1958/sanger-bio.html

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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

http://www.pnas.org/site/misc/classics1.shtml

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Q-1) Based on that information alone, how would you characterize a peptide bond: single or

double. Explain.

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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.

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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.

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Consider the “R-Groups). Although some are small-such as glycine and alanine who’s R-

groups 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 N-

terminus 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 h-

bond going toward an amino nitrogen is

actually connected to the hydrogen not shown.

(Red=oxygen, gray=carbon, blue=nitrogen)

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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?

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Q-8) What role do these h-bonds play in maintaining the α-helical structure? Explain.

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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-C-

N-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 Figure 8

re-center the image.

Q-9) Where are the (green) R-groups located. Considering their “clunkiness”, why would this

positioning make sense? Explain.

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(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.

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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 32-

36, 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.

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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?

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Using the same procedure that you used when

investigating the length of part of a polypeptide Figure 15

chain in the α-helix, measure the distance between

5 amino acids, specifically the carbonyl oxygens in residues 49-53.

11

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

11


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.



11

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.

11


program:






2. mWith the cursor on the screen, left click and choose various items in the menu or







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

______________________________________________________________________________

______________________________________________________________________________

11

Q-3) Would you expect most of the amino acids facing the outside to be; hydrophobic (nonpolar-

green) 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. Figure 2

Type “select polar” and press Enter. Type

“color yellow” and press Enter. See figure 2.

11

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.

11

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_____





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.

11

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

Atom (Number)

Involved in

IonicBond

Glutamate 18 -COO-

144

Arginine 61 =NH2 +

500

Lysine 200 - NH3 +

1562

Glutamate 240 -COO- 1884

Aspartate 297 -COO- 2343

Arginine 303 =NH2 +

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.

11

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 Figure 6

and oxygens. See 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 h-

bonded amino acid residues are furthest from each other along the chain? ____ and ____.

Which of the highlighted hbonds contribute to secondary structure? ________________

Tertiary structure? _________________

11

************************************************************

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.

11

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


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

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1962/kendrew.html

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1962/perutz

11

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?

11

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.


program:













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.

11

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

11

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.

11

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.

11

“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? ___________________________

11

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.

11

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=______________




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.

11

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.

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

11

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 Figure 9

bound?

________________________________________________________________________

________________________________________________________________________

________________________________________________________________________

________________________________________________________________________

11


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)



http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi

http://www.rcsb.org/pdb/explore/explore.do?structureId=1PYT

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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?

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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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program:










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.

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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?

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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.

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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”.



Type “select 1-74:C” and press “Enter”.



Type “select 79-119:C” 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?

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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?

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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?

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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)

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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.

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_____________________________________________________________________

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?

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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?

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Q-13) What is the charge of the carbonyl oxygens in valine and glycine lining the

channel?

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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.

11

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.

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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.

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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


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

http://www.rockefeller.edu/research/faculty/abstract.php?id=132

http://www.web-books.com/MoBio/Memory/Channel.htm

http://sbkb.org/kb/archives.jsp?pageshow=37

http://adsabs.harvard.edu/cgi-bin/author_form?author=Carrillo-Tripp,+M&fullauthor=Carrillo-Tripp,%20Mauricio&charset=UTF-8&db_key=PHY

http://adsabs.harvard.edu/cgi-bin/author_form?author=Saint-Martin,+H&fullauthor=Saint-Martin,%20Humberto&charset=UTF-8&db_key=PHY

http://adsabs.harvard.edu/cgi-bin/author_form?author=Ortega-Blake,+I&fullauthor=Ortega-Blake,%20Iv%c3%a1n&charset=UTF-8&db_key=PHY

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