In this section, you will learn how to search databases for a protein sequence. As mentioned above, you will be working with apolipoprotein E to study this form of hypercholesterolemia.

Using the Ndjinn Tool: to Search Sequence Databases

1. Notice the scrollable textbox at the top of the page¡K this box contains a variety of tools, some of which you will now explore. Highlight "Ndjinn ¡V Multiple Database Search " and click "Run ".



2. The next screen list the different databases that you can search. In the search box at the top of the page, type: "lipoprotein ". This tells the search engine what to look for. Also, notice the box to the right of the input box. This simply allows you to decide how many sequences you want to display. For this exercise, you want to see all of the sequences that are found, so select "All ".



Scroll down the page. Below the input box, you will see a list of many different databases, all containing a variety of sequence information. The databases are separated into two distinct groups: the first group contains sequences from many different organisms (for example, "GBBCT " contains a large number of sequences from many different bacteria), whereas the second group contains the entire genome of specific organisms (for example, "Mthe " contains the entire genome sequence of the bacterium Methanobacterium thermoautotrophicum).

3. Click on the box that is next to the PDBFinder Database. This database contains protein sequences that have a crystal structure formulated so that a 3-D picture of the protein can be visualized. A little later we will look at some of these pictures.



4. Scroll back up to the top of the Screen and click on "Search ". You will then be sent to a page that contains the results of your search. At the time this tutorial was written, the search engine found 53 matches for "lipoprotein ". If you get more than 53 results, do not panic. Inconsistencies in the number of search results can occur because new sequences are being added to the databases on a daily basis.



From the descriptions of the search results, we need to determine which one is the wild type sequence for the lipoprotein receptor protein. In order to find the correct sequence, we will need to check the records of the sequences to find out which sequence is wild type. To do this, highlight the 53 sequences with your mouse.



Once all of the sequences are highlighted, click on the button below that says "Show Records ". This tool will give a detailed description of each sequence that is highlighted.




5. If you are using Netscape, a new window will appear; in Windows Explorer the browser will move to a new page. Scroll down this page until you can locate the wild type sequence for Apolipoprotein E. Specifically, we want the "LDL receptor-binding domain " of the protein. If you have correctly identified the wild type protein, you should have picked the following:



This file (1LPE) is the wild type, or normal, protein sequence for lipoproteins found in people that do not have hypercholesterolemia. This is the protein that we want to work with. We need to do two things: look at a model of the protein structure, and second, download the amino acid sequence of the protein into our Biology Workbench session. First, let's look at at structural model of this protein. Click on the link to "PDB Structure Explorer " in the upper-right hand of the entry for our protein.



6. This will bring up a new window with a page from the Protein Databank web site. Click on the link to "View Structure " on the left side of this page:

7. You will come to a page with several options for viewing the protein crystal structure. We will look at a still image of the protein. Scroll down and click on the link that reads "Ribbons (250 x250) ":

This will take you to a ribbon diagram of the protein. In this diagram you can't see individual atoms or amino acids, but you can see the overall shape of the protein molecule. Note the "corkscrews " that make up most of the protein chain. These represent alpha helices, a common kind of protein structure.

8. So that later on you can compare this picture side-by-side with a picture of the mutant protein, copy this picture into an application like Word. To do this, RIGHT click on the picture itself, and then click on the "Copy " command that comes up in the command box:


9. Next, open Word, and use the command bar go to Edit --> Paste (or press "CTRL " and "v " simultaneously):



10. The picture should appear on your Word document as shown below. Be sure to label your picture so you will remember what it is and not confuse it with the next picture.


Now that we've got the picture of the protein, next we want to import the sequence of this protein from the Protein Databank into the Biology Workbench. Keep the Word document open so we can look at it again later, and go back to the Biology Workbench screen with the Ndjinn search results. (Note: If you are using Netscape, you can close the pop-up Biology Workbench window with the full records. If you are using Windows Explorer, you will need to hit the "back " button on the browser to get back to the search results page).

11. Highlight the correct sequence: "pdbfinder:1lpe ¡V lipoprotein ". Now click on the button at the bottom that says "Import Sequence(s) ". This will bring the wild type protein sequence for the lipoprotein (the sequence we just highlighted) into the protein tools homepage for further investigation.