In order to compare two protein sequences side-by-side, they must be aligned one on top of the other. This is the purpose of the CLUSTALW tool. The alignment process takes place by comparing the two sequences and finding common regions within them. The Biology Workbench then uses an algorithm to compute the most likely position in which the two sequences line up. Therefore, alignment is a key step for you to determine where a mutation is located. The two sequences are aligned one on top of the other and a color-coding system is used to differentiate highly conserved regions and semi-conserved regions (in royal blue and green, respectively) from the non-conserved regions. On to the alignment...

1. Scroll down in the text box menu and highlight "CLUSTALW ¡V Multiple Sequence Alignment". Click on the two lipoprotein sequences, 1LPE and 1LE2. Once there are checkmarks in the boxes next to the sequences, click on the "Run" button.



Another screen will appear in which the alignment parameters can be altered ¡V we are going to use the default settings so just click on the "Submit" button. You will now be taken to a screen that will show you the aligned sequences.



2. Scroll down until you see the alignment. If you will notice, the wild type sequence is located on the bottom row and the mutant sequence is located on the top row. The letters that you see each represent an amino acid in the protein sequence. Almost all of the amino acids in both protein sequences match up perfectly¡K You can see this by the royal blue color (see consensus key at the top). However, there is a single position in which the two protein sequences do not match, and are color-coded black instead of royal blue. This is the site of the mutation. This is where the arginine amino acid was replaced by a cysteine amino acid in the mutant sequence. So you can easily see here that the mutation is near the end (or "C-terminus" in biochemistry jargon) of the protein sequence.