Step 5: The three-dimensional structure of bovine rhodopsin

We know from the previous step that the structure of human rhodopsin is yet to be determined. We do have the structure for bovine rhodopsin. Because the sequences for these two proteins are very similar, the 3D structures will be very similar as well. We can therefore use the information we obtain from the bovine protein as a model for the human protein. We will use a program called Yasara to do just that. The program was largely developed in the Netherlands by Elmar Krieger. If Yasara is not already installed on your computer, then look here for an installation guide.
After this practicum you can look at more interesting protein structures. Go to the More in 3D section for details.

Exercise 6:

1. Start Yasara (help). When Yasara is running we can load the rhodopsin structure using the menu at the top left of the screen: File > Load > PDB file. Select the file rhodopsin.pdb and click on OK.
   
   We now see the rhodopsin structure in the so-called Ball representation. In this view individual atoms are displayed as balls. Note: hydrogen atoms are not displayed.
   When you move the mouse pointer to the bottom of the Yasara screen, the sequence bar appears. It contains all amino acids (and other compounds) in the structure. Use the button in the top left corner of the bar (which looks like a blue pushpin) to keep the bar in place.
   
   When you click on an amino acid on the sequence bar the Cα-atom of that amino acid in the structure starts blinking. Holding the  Ctrl-key while clicking moves you to the right position in the structure automatically. You can manipulate the structure with the mouse, feel free to try:
   • Left: Rotate
   • Middle: Move/translate
   • Right: Zoom
   
   The atoms are coloured by element. The text on the left on the screen gives extra information when you select an atom. At first glance, the Ball-model does not look very informative. However, we can still see some interesting features. Look at the colours: there are more red (oxygen) and dark blue (nitrogen) atoms at the ends of the structure, whereas the middle part has more light blue (carbon) atoms. There are a few questions we can answer already:
   • What are the green atoms in the Ball-model?
   • In which of the amino acids do they occur?
   Hint: Besides C, O, N and H, which atom type occurs in proteins? You can refresh your mind if you need to.
   
   
2. A protein structure can be viewed in different representations. When we show the protein chain as a cartoon (use the F6-key) to see how it is folded, we see corkscrew-like structures: the helices (dark blue). They form a channel through the cell membrane. This second representation of the protein structure gives us new information. So let's answer a few more questions:
   • How many helices are there?
   • This protein sits in the cell membrane. Make a schematic drawing of its orientation?
   
   

Step 6: Where are the RP mutations in the 3D structure of rhodopsin?

Lets look at the mutation sites in the protein structure. To do so, we need to load something new into Yasara. Go to File > Load > Complete scene, select the file rhodopsin.sce and click on OK.

The protein chain is now shown as a grey tube and the mutating amino acids are shown in the Ball-representation (blue). Between them is the orange retinal group. It is bound to an amino acid, lysine number 296 (green). A translucent molecular surface is drawn around the retinal group. This represents the actual size of the retinal atoms. You can select individual mutating amino acids with the sequence bar.

Final exercise

Study the mutating amino acids. Can you explain why a person with one or more of these mutations in rhodopsin has problems with his vision?

Answers

You now know that you can figure out a lot about a disease when you have a gene or protein sequence and some information about mutations, even when no 3D structure of the human protein is available. This concludes the RP practicum, so you can now check your answers.

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