Now that you have the wild type lipoprotein sequence, you can use it to find the mutant lipoprotein sequence that causes familial hypercholesterolemia. Because we know that the mutant lipoprotein equence differs from the wild type sequence by a single amino acid, we can use the wild type sequence to search a database for sequences that are extremely similar (or homologous) to it. In order to carry out the homology search, you are going to use a tool called BLAST.

1. Scroll down the textbox and look for the tool called "BLASTP ¡V Compare a PS to a PS DB". (This is an abbreviation for "Compare a Protein Sequence to a Protein Sequence Database".) Select this tool and ensure that your ILPE lipoprotein sequence has a checkmark in the box next to it ¡V then click on the "Run" button.



2. You will be sent to a screen that will give you many options. These options allow the user to fine-tweak their search. For the purposes of this exercise, we do not need to deal with this. The important step here is to choose the database you want to use for your homology search. Scroll down in the textbox until you come to the "PDBFinder" database. We will use this database again since it is the one that we started with. Highlight this database.



3. Now scroll to the bottom and click on "Submit".



You will be sent to the following screen that contains the results of your BLASTP search:



4. Now, you will need to find the mutant sequence that is responsible for causing familial hypercholesterolemia. But, before you do this, look at the number to the right hand side of the results box. If you look over to the "Score (bits)" column, you will see that the first few sequences have very high Score (bits) values. A Score (bits) value above 200 means that the sequence has high homology with the sequence that you are comparing it to. However, you can be more certain of the extent of the homology between two sequences by looking at the "E Value". This is the number right next to the "Score (bits)" number.

E Value
The E value or "Expect" value is the most intuitive, or instinctive, way to rank the results of a search. The E value estimates the statistical significance of a search result by specifying the number of matches with a given score that could be expected to occur purely by chance in a search of a database of a particular size. For example, an Expect value of 2 would indicate that two matches with that particular score would be expected to occur purely by chance. The expected value changes with the size of the database (in a larger database more chance matches with a given score are expected). Search results with E values much higher than 0.1 are unlikely to reflect true sequence relatives, but sometimes it is useful to examine hits with lower significance (E values between 0.1 and 10) for short regions of sequence similarity. In the absence of longer similarities, these short regions may allow the tentative assignment of biochemical activities to the sequence in question. The significance of any such regions must be assessed on a case-by-case basis. Essentially, the smaller the E-value, the more homologous or similar the sequence is to the original sequence BLASTED. An E-value of zero indicates that no matches would be expected by chance - this would represent a perfect or near perfect match.

Now it is time to decide which sequence is the mutant lipoprotein sequence. The mutation that causes familial hypercholesterolemia is a point mutation in the protein sequence that changes the amino acid arginine in the 158th position to the amino acid cysteine. Find the mutant in these results that matches this description. You can do this by checking the records like we did a bit ago.

The correct sequence record is shown below:



If you will notice, this is a mutant that replaces arginine at position 158 with a cysteine. That is the one we want!!

5. Like before, we want to take a look at this protein structure. Click on the "PDB Structure Explorer" link at the top right of this record. On the window that comes up click on the "View Structure" link on the left side. On the next screen click the link that says "Ribbons (250 x250)." Like before, you can copy the picture that comes up to your Word document. Right click on the picture, copy, then switch to the Word document you still have up. Put the new picture beneath the first one. Make sure the cursor is at the bottom of the Word document, then paste the picture in. Put in a caption for the second picture; make sure to use the word "mutant" in this label.

6. Now, without the captions, could you tell the two proteins apart? They look practically identical -- even though one of them is a mutant, and we know the mutant protein doesn't function correctly! What's going on? Let's go back to the Biology Workbench to see if we can figure this out. If you're using Netscape, you can close the extra Biology Workbench window with the detailed protein records, and just keep open the window with the list of "sequences producing significant alignments." If you're using Explorer, you'll just need to use the "back" button on the browser to get to the sequences screen. We want to import the sequence for the mutant protein.
This is: (1LE2_LIPOPROTEIN|Apolipoprotein ¡V E2_(LDL_receptor_binding_d¡K). Once the sequence is highlighted, click "Import Sequence(s)".



Now, the mutated sequence that causes the disease hypercholesterolemia is stored in the Protein Tools homepage along with the wild-type sequence. We will now further investigate where the mutation occurs in the mutant sequence of the lipoprotein, and try to figure out why the mutant protein doesn't work.