Step 2: Protein databases

Nearly all known protein sequences are deposited in databases that are freely accessible and searchable through the Internet. We will try to find the human rhodopsin sequence in the Uniprot protein sequence database. The database search system MRS is the right tool for the job.

Exercise 2:

  1. Open the MRS website. The homepage looks like this:

    MRS home, click for a larger version
  2. We must select Uniprot KB in the dark blue search bar because we are looking for a protein. Uniprot is a collection of several databases, together describing millions of proteins.
  3. Type rhodopsin in the search bar and click on search. We now get hits for all Uniprot entries that contain the word "rhodopsin". These are far too many hits to use.
  4. Change rhodopsin to de:rhodopsin and search again. This time, we only find the entries that contain the word "rhodopsin" in a short description of the entry (de: stands for description). Even though we have much fewer hits than before, there are still too many.
  5. We have to be even more specific. After all, we are only looking for human proteins. Change de:rhodopsin to de:rhodopsin os:human (os: stands for organism species) and search again. Now, we are only considering the proteins that have the word "rhodopsin" in their description and belong to humans.
  6. Only a few hits remain. Based on their description we pick opsd_human. If you click on that word you are forwarded to a page with a comprehensive description of the protein with many links to other databases and scientific publications about the protein. On the bottom of the page we find what we are looking for: the human rhodopsin sequence. Beware: This is the sequence one finds in healthy persons.

Step 3: Mutations in human rhodopsin

Mutations occur when one or more bases of the DNA change (or are inserted/removed). This changes the codon with that base, which results in the codon coding of a different amino acid.

One of the mutations Dr. Bashir found is Lysine 296 to Glutamate (Lys296Glu or K296E). This means that healthy people have Lysine at position 296 of rhodopsin whereas RP patients have Glutamate. Let's find out more about the mutations found by Dr. Bashir.

Exercise 3:

  1. Look up the codons for Lysine and Glutamate in the codon table.
  2. Which base in the rhodopsin gene was mutated to cause the amino acid mutation K296E (Lysine becomes Glutamate)?
  3. A few other mutations in rhodopsin that cause RP are:
    • Ser186Pro
    • Cys187Tyr
    • Met207Arg
    • Ala292Glu
    Translate these amino acid mutations from the 3-letter code to the 1-letter code. Hint: use the amino acid drawings.
  4. Locate the five mutation sites mentioned above in the rhodopsin sequence you found in Step 2. You can also use the copy of the rhodopsin sequence on this site.

Step 4: Is the three-dimensional structure of human rhodopsin known?

To explain the effect of the mutations thoroughly, we need to know what the structure of human rhodopsin looks like. For this we will use a databank with three-dimensional protein structures: the PDB. This databank contains all scientifically described protein structures (over 50000).

You can search the PDB with the program BLAST. This tool enables you to compare a protein sequence with all the sequences in a databases like the PDB and Uniprot. We will use the BLAST option in MRS.

Below you see which steps to take to use BLAST and how to interpret the results.

Exercise 4:

  1. Go to the MRS website.
  2. Select BLAST in the menu bar on the left to go to the BLAST form.

    BLAST, click for a larger version

    Here is an example BLAST screen with human rhodopsin sequence in the query box. The first line must always start with a > followed by a name for your query (this is the so-called FastA format). We chose the name rhodopsin.
  3. Copy the sequence from Step 2 and paste it in the BLAST query box. You can also use the copy of the rhodopsin sequence on this site.
    • Select the PDB as the databank
    • Make sure that the Filter Query Sequence box is cleared.
    Then click Run BLAST (top right).

You are forwarded to the next screen that keep reloading while your BLAST query is running. This might take a while when many people are using BLAST. After all, you are searching a database with large numbers of protein structures. On the next page we will look at the results.

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