Myoglobin as a Probe for Understanding Molecular Evolution
Primary Author: Ajit Chary,
Undergraduate Research Assistant
achary@ncsa.uiuc.edu

Introduction

First of all, what is myoglobin? What's your favorite sport? Let's say basketball. Now you need a lot of strength to play basketball well, right? And what gives you the strength to jump up and dunk the ball like Jordan? That's right, MUSCLES.

Now, how does a muscle get big? Well, you can lift weights, and you can jog, run, jump, and play outside everyday. That's all good. But then you need something to give muscles enough energy to let you jump, lift weights, play, jog, run and dunk. What lets the muscle get big and strong and what lets the muscle live is oxygen, found in the air you breathe.

But how can we breathe in through our noses and get oxygen to our arms, legs, and all the rest of our muscles? The way the oxygen gets there is through the blood. Then, the blood goes through our muscles and gives it the oxygen.

Where exactly where does the oxygen go when this liquidy red stuff gives it to the muscles? And how can the oxygen stay in the muscles so that we can use them and get energy from them? Through a tiny little protein called MYOGLOBIN.

So that means that every single thing that has muscles, meaning all living animals and people have myoglobin right? Therefore is it all the same? I mean does everyone and everything, even your pet dog, cat, or a whale have the same myoglobin as we do? If not, then is it similar to ours? Lets find out.

Before we take a look at the Biology Workbench, we suggest that you either print out a copy of this tutorial or open another web browser window to view this tutorial while playing with the Biology Workbench.

Using the Biology WorkBench

  • First, open the Biology Workbench V.3.2.

  • Now, select set up a free account in the Biology Workbench.

  • Fill in the blanks and then click on the button that reads "Register".

  • After doing so, from now on whenever you enter the biology workbench, it will ask for this login name and password.

  • If you enter your logon and password exactly the way you did when you registered, you should arive at a page that contains a picture of a protein. (It will look something like this):

  • Scroll down past the large protein and click on Protein Tools.

  • Now, you will see many items and a scrollbar inside a box. Click the item NDjinn Multiple Database Search and then hit Run.

    The first item you will see is a gray query box with several parameters. On the right side of the search bar, you will see "Show 10 Hits". Change that (by clicking on the icon) to "Show All Hits".

  • Scroll down and select PIR-- The Protein Information Resource (#'s 1 through 4) and SWISSPROT-- Swissprot Database.

  • In the search bar, at the top of the page, type myoglobin then hit Search.

  • You should now see a page which has the results for the myoglobin search, and several (207) "records" within a scrollbox. Look within this box, and toward the top of the list you should see pir1:myhu - myoglobin - human. Select that and towards the end of the page (not the box), and click Import Sequence.

  • This next page should have a little box next to PIR:MYHU myoglobin - human . Click it. Then in the box above, find BLASTP - Compare a PS to a PS DB, after clicking that hit Run.

    What BLASTP does is quite interesting. It takes the human that you selected and compares it to that huge protein database (for now let's make it PIR) or another huge database (like SWISSPROT). You are permitted to select one or more databases to compare. Now since you clicked human, BLASTP will find all of the animals, plants, even bacteria and viruses, that are similar or the same as the human protein you selected (in this case myoglobin). It doesn't stop there, it also does a big favor by putting the protein sequences in ORDER for you, so you can see what is the closest (evolutionarily) to humans and what is furthest from humans. Since myoglobin is found in muscles and animals with blood, you won't see any plants or viruses. But, if you use other sorts of proteins like Enolase, or Aquaporins (higher level proteins) you can and probably will find all of that. What animals do YOU think have myoglobin thats the most like the myoglobin that we humans have? Dogs? Cats? Whales? Monkeys and other primates? Birds?

    If you guessed Primates, you were right. Now, which PRIMATE is most closely related to humans? Lets see...

  • After hitting Run, you should now be on a page with a large scrollbox to the left. Under Database Selection: click on the PIR Databases. To select more than one database with a PC or Mac hold down the control key for PC or the Apple command key for a Mac. Keep scrolling down the page (ignoring all of the default parameters) until you come up to Number of hits to display. Now select 100 instead of 1000. That means you'll see the first 100 animals closest to humans in the PIR protein databases. (There aren't 100 myoglobins, but then you can ignore the other molecules like hemoglobin on the page you're going to see next.) Hit Submit.

  • The next page will show you all of the animals (the first 100 out of this database) closest to a man. When you scroll down a little bit, you'll find a big box with all of the animal sequences to be selected. For the purpose of this tutorial, select all of the primates (which are monkeys and apes). Here is the list if you are unsure, and don't forget to scroll down if you can't find a primate on this list:

    chimpanzee
    siamang
    agile gibbon
    mountain gorilla
    Bornean orangutan
    hanuman langur
    red guenon
    olive baboon
    crab-eating macaque
    common marmoset
    douroucouli
    brown capuchin
    common woolly monkey
    common squirrel monkey
    potto
    slow loris
    weasel lemur

    IF YOU HAVE A MAC OR A PC, DO AS DESCRIBED ABOVE TO SELECT MORE THAN ONE ITEM

    DO NOT CLICK ON HUMAN because you already are comparing everything to a human. After you get used to this BLASTP program, choose any animal you want and see how closely related it is to humans. After you clicked these animals, hit Import Sequences.

  • The next page will have the same little boxes next to the names of all of the animals and the sequences so click them all. After doing that, look above at the scrollbox with the programs to choose. Find something called CLUSTALW - Multiple Sequence Alignment and select it. Now hit Run.

    Clustal W is a program that will let you see the sequences and it will align all of the sequences for you so that you can see for yourself how closely related and how far apart all they are. In this case, it will show you how closely related they are. These sequences are shown in letters. These letters represent chains called Amino Acids which all form together to make the Myoglobin protein structure. The letters are compared with other letters, and you will see that the letters are arranged columnwise, and that several of these letters (by column) are the same. Don't really worry about what all these letters mean. At the beginning of the page, hit "Import Alignments."

    After you hit this, you will see a little gray box and to the right of the box, you will read CLUSTAL-W Protein -- and all of the sequences you picked.

    Click this box, and then look at the large scrollbox above it.

    Scroll down the box with the programs within it, until you see

    1. DRAWGRAM- Draw Rooted Phylogenetic Tree from Alignment
    2. DRAWTREE- Draw Unrooted Phylogenetic Tree from Alignment
    and click the DRAWGRAM. Now hit "Run".

    Now, at the begining of this new page hit submit, because all of the default parameters work well.

    After you take a look at the flowchart, hit the Return button in the Biology Workbench. That will take you back to the place where you selected the DRAWGRAM and DRAWTREE programs. Now repeat the procedure with DRAWTREE, view the unrooted tree and hit "Return" once again.

    Now to compare the sequences with another program, you can go to BOXShade - Color-coded Plots of Pre-Aligned Sequences, and on the next page hit Submit.

    Now take a look at the COLORS. These colors have a relationship and compare the sequences in this manner:

    YELLOW

    -This means that this region is not completely conserved, and that there is a general consensus of what amino acid there should be in those spots. "Conserved" means the same amino acid throughtout.

    GREEN

    -This means that this region is completely conserved, meaning that at these regions the amino acid will always be found in this spot of the sequence of no matter what organism.

    LIGHT BLUE

    -This means that the amino acids are variable in these positions of the sequences. Some amino acids will appear more often than others.

    NOT SHADED

    -This means that the amino acids in these areas of the sequences are extremely variable, meaning that there will never be the same amino acids at that spot per organism.

    Now after exiting BOXShade by pressing Return, you can start the wahole process again with a different organism, different BLASTP database, (like SWISSPROT), different set of animal comparisons, and you can play around with even different proteins that you know of.

    This page created and maintained by Kristian N. Engelsen.
    E-mail any questions or comments.