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TUTORIAL (20 - 40 minutes)

In many of the tutorials the beta chain of human hemoglobin has been used as an example. In the following tutorials a very similar protein will be used. Myoglobin is an oxygen binding protein which is very similar to hemoglobin.  However it occurs as a monomer in the muscles, rather than as a tetramer in erythrocytes. 

The muscle tissue of whales carries particularly large amounts of this protein; consequently the muscles are able to absorb large amounts of oxygen.  This is one reason that whales can stay beneath the surface of the ocean without breathing for nearly an hour.  Many people erroneously think that whales are simply holding their breath. In fact, whales may dive to 1000 feet where the pressure on their bodies is so extreme that their lungs actually collapse! Thus, all the oxygen they need is stored in the myoglobin of their muscles.

A key component of a-globin, b-globin and myoglobin is iron, which has a high affinity for oxygen. Because oxidized iron is red (think of rust) myoglobin and muscle is red as well!  The red color of steak in the supermarket is due to myoglobin.  If you ever see a documentary of the Inuit (Eskimos) eating raw whale, notice that the meat is so dark with myoglobin that it is brown.

Since all vertebrates have muscle, they should all have myoglobin as well.  This tutorial  compares the differences in amino acid sequence in the myoglobins of humans and ten other animals.

Click on image to see full-size!	Register for Biology Workbench. Open the Home Page for Biology Workbench V.3.2. Select "set up a free account", as shown by the blue arrow in the thumbnail. Fill in the blanks and then click on the button that reads "Register".  After you have successfully registered, follow the link to the Biology Workbench. Scroll down the page, until you find a series of  buttons, as shown in the thumbnail to the left. Click on the "Protein Tools"  button.
Import the amino acid sequence for human myoglobin. In order to do any kind of BLAST search, a query sequence must be obtained in FASTA format. This is then copied and pasted into Biology Workbench.
BLASTP search for homologs to human myoglobin. Blastp searches in designated protein databases such as PIR or SWISSPROT for matches to a query sequence (either DNA or protein). The user is permitted to select one or more databases to compare. Since human myoglobin was selected, BLASTP will find all of the animals, plants, even bacteria and viruses, that have similar sequences.  The protein sequences are ranked in descending order of bit score, so you can see what is the closest (evolutionarily) to humans. Myoglobin is an oxygen transport protein, and is generally found in muscles - consequently you probably won't see any matches with plants, bacteria or viruses. But, if you search other proteins, like Enolase or Aquaporins, you may find matches with humans.
CLUSTAL W Clustal W  is a multiple alignment algorithm.  It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen.  The results are displayed as sequences of letters.  If proteins are being aligned, each letter represents an abbreviation for a different amino acid.   If DNA is being aligned each letter represents an abbreviation for a different nucleotide.
Boxshade Boxshade presents the results of the alignment to show sequences which are identical, similar or different.  The same proteins from closely related species will be very similar to each other (such as human and chimpanzee myoglobins).  They will have the same amino acids at most positions - these are said to be conserved.  The same proteins from two species which are not closely related will not be similar to each other  (such as human and whale myoglobins).  They will have different amino acids at many positions - the sequence is not conserved at these positions.  In some cases where the same protein is being compared from many species (such as in our present example) the amino acid present at a certain position in the sequence may be the same for most species, but different in a few.  In this case, the amino acid sequence at this position is neither "conserved" or "not conserved".  Instead we speak of a "consensus sequence", which is the amino acid present in most cases.
DRAWTREE and DRAWGRAM The demonstration that proteins are frequently similar in many species begs for an explanation.  These two algorithms use the sequence differences calculated by CLUSTAL W to generate graphics which illustrate phylogenetic trees and genetic distance ( see below for an explanation of these terms), and provide insight into the evolutionary divergence of the species. The biological interpretation of the similarities and differences in the amino acid sequences between species is that these changes represent evolutionary differences.  Two proteins which are very similar to each other (such as human and chimpanzee myoglobins) will have the same amino acids at most positions - these are said to be conserved.  When the amino acid sequences are "highly conserved" the two species are considered to be evolutionarily close to each other.  The extent of conservation is therefore a measure of genetic distance and allows us to retrace the course of evolution which can then be depicted as a phylogenetic tree.

EXERCISE  (20-40 minutes) Open the   Save the web page to your desktop. Launch Microsoft Word. Open the web page from inside the Word application. Save as a Microsoft Word file. Type your answers into the document,  E-mail your answers as a Microsoft Word document, or hand in  a hard copy, as directed by the Instructor.

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