Part 2: Analyzing mutations

In the second part of this practical you will look at the structural context of some mutations involved in a condition known as 'favism'.

Favism results from mutations in Glucose-6-Phosphate Dehydrogenase, an enzyme which generates NADPH which in turn reduces glutathione. Glutathione is used to maintain the correct oxido-reductive state of the cell. If glutathione production is reduced, cells suffer under oxidative stress resulting from certain food stuffs (including broad beans - or 'fava beans') and a number of drugs including anti-malarials.

First we will visit the resources that you will need and then we will go on to analyze the mutations.

OMIM and SwissProt

AIM: To become familiar with the OMIM and SwissProt resources

First we will visit Online Mendelian Inheritance in Man (OMIM). This is a comprehensive resource containing information on Mendelianly inherited genetic diseases. Where known, a sample of mutation data related to the disease are provided. We will visit this resource to identify mutations known to be involved in G6PD deficiency.

Go to OMIM: http://omim.org/
Enter G6PD into the search box and click 'Search'
Click on the first result (*305900)

The resulting page will give you extensive information about the disease.

Scroll down until you reach the section headed ALLELIC VARIANTS (XX Selected Examples):, or click Allelic Variants in the menu on the left.

Here you will see a long list of mutations known in G6PD and linked to disease. Each is headed with a line like
.0001 G6PD A+
followed by a line detailing the mutation
G6PD, ASN126ASP ......
and text describing the mutation. The important part for these purposes is the residue number (in this case 126).

You are going to look at a small selection of these in the context of the protein structure.

On the right hand side of the page is a list of External Links. Under this click Protein and then click UniProt.

A new window (or tab) will open containing the UniProtKB/SwissProt entry for the protein.

In the menu on the left of the UniProt page, click Disease & Variants.

As you will see, UniProt also provides a list of mutations in G6PD.

In the menu on the left of the page, click Structure.

Just below the 3D image of the protein, you will see that there are a number of structures of G6PD available in the PDB.

One of these (2BH9) has only one chain (labelled 'A') while the others have multiple chains (copies of the same protein) in the crystal structure. For simplicity, we will use 2BH9 for our analysis.

Go to the PDB web site at http://www.rcsb.org/ and download 2BH9 (go to the PDB web pages and search for the code - click the 'Download Files' link on the right and select 'PDB Format' as the format for the download. Right click the link so you can save the file. Ensure you download in PDB format, not a compressed file or a file in an alternative format.