Hydropathy Plot of Protein Sequences

1. Now that we know how the secondary structure of the wild type prion protein differs from that of the mutant prion protein that causes CJD, let's look to see what the differences are at the amino acid level. Amino acids are what constitute a protein. Amino acids can either be hydrophilic (that is, soluble in water and other polar solutions), or hydrophobic (not soluble in water). These properties of amino acids have a significant effect on how a protein chain folds up.

2. We are going to use a tool called GREASE to determine the distribution of hydrophobic and hydrophilic amino acids in each of the prion protein sequences. This will give us a better idea of why the proteins are folded the way that they are. Let's begin:

3. First, make sure that your mutant and wildtype sequences have checkmarks in the boxes next to them. Scroll down the menu and highlight "GREASE ˇV Kyte-Doolittle Hydropathy Profile". Click "Run".

5. The default screen will appear. Just click "Submit".

6. The results of the plot will appear. Look for the code numbers of the wildtype and mutant sequences to identify which plot represents which sequence.



7. First, let's understand what we are seeing here. The plot is divided into two sections, an upper half and a lower half. If you look at the very top left corner of the graph, you will see "CH3". This represents a methyl group and it is there to let you know that the upper half of the plot represents hydrophobic, or nonpolar, amino acids. All of the amino acids in a protein sequence that are nonpolar will be found in the top-half of the plot. On the other hand, if you look at the very bottom left corner, you will see "H2O". This is of course water and it is there to let you know that the bottom half of the plot represents hydrophilic, or polar, amino acids (which are soluble in water or don't mind contact with water). All of the amino acids in a protein sequence that are polar will be found in this bottom-half of the plot. How far the lines of the actual plot extend into the bottom or the top halves depend on just how many amino acids are polar or nonpolar in a particular region.

8. Now, if you look all the way to the right hand side of the plot, you will notice a bunch of uppercase letters (some stacked one on top of the other). These letters represent the twenty amino acids and where they can be found according to their hydrophilic or hydrophobic properties.

9. Whew! Now that we understand what we are looking at, we can take a look at the wildtype plot and see that there are both hydrophobic/nonpolar and hydrophilic/polar regions within the wild type protein. However, if you scroll down the page a bit to see the mutant prion proteinˇK



10. Watch out hereˇXthis is the mutant sequence that we entered into the GREASE tool, but the mutant graph that you see above corresponds to only a region of the wildtype graph. Remember that the mutant sequence is much shorter than the wildtype sequence (refer to the GOR4 section to see the lengths of the wildtype and mutant protein sequences and to the CLUSTALW section to see the alignment of the two sequences). If the "Edit" tool is used to substitute the mutant prion protein sequence for the corresponding region in the wild type prion sequence, it is easy to see how the mutation affects the hydrophilic properties of the prion sequence (don't worry about actually doing the editing, just view the plots below to understand how the protein sequences are different):

Wildtype


Mutant


(In order to get these graphs to correspond to one another, the "edit" tool was used to copy and paste the extra sequences of the wild type prion on either side of the mutant prion to make the lengths of the prion proteins the same). Now that we are comparing protein sequences of the same length, we can see that the mutant sequence and the wildtype sequence differ in the first hydrophilic regions. The mutant region is much longer, reflecting the increased number of octapeptide repeats (and beta-sheets).

Conclusion
The prion mutations studied here leads to a higher percentage of beta-sheets in the resulting protein. The secondary structure of the wildtype protein contains approximately 40% alpha helices. However, the mutant prion protein contains a higher percentage of beta sheets, which is thought to change the overall structure of the protein and contribute to its seemingly indestructibility. In other words, this change in structure causes mutant prions to be very difficult to degrade. Heat, acid, and protease enzymes can normally denature proteins; however, the mutant prion protein can withstand all of these, thus aiding it in its transmission.