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How to explore a protein

The views expressed are those of the author and are not necessarily those of Scientific American.


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I’m doing a journal club presentation tomorrow, where I take a paper apart in front of my lab through the medium of powerpoint. It’s a nice short little paper but it does bring up some interesting points and also works as a prime example of a very common way that scientists go about exploring how a particular protein works. There are many ways to do this, but this one is quite a common one and if everything works it can generate very nice results.

Stage one: Find your gene

All proteins have a particular gene that codes for them, and as a general rule one gene will code for one protein. In the paper I’m looking at the researchers have a whole host of genes that code for all the enzymes required to make gliotoxin; a poisonous substance made by the fungi Aspergillus fumigatus aka the black mould in the bathroom.

Aspergillus mould, from wikimedia commons.

Stage two: Find out what your gene does

The easiest and quickest way to do this is to compare the gene in question to many other genes. In this paper, the researchers were looking at the gene GliG. By using computer software (i.e programs such as BLAST which are designed to compare genes) they found all the other genes that GliG resembles. It turns out that GliG is related to a lot of other genes that attach to compounds containing sulphur.

Stage three: Knockout your gene

One of the most common ways to explore what a protein does is to remove the gene that makes it and then see what the organism stops doing. Geneticists do this a lot. When you knockout the GliG gene you get no gliotoxin, which is good to know but not particularly specific. Closer examination shows that while you don’t get much gliotoxin, you do get a lot of products which could concievably turn into gliotoxin apart from one vital thing. They are missing the sulphur.

gliotoxin

The chemical structure of gliotoxin. The sulphur atoms are marked in red, and form an important part of the structure.

Stage four: Isolate your protein

So at this stage the researchers have a fairly good idea of what the GliG is all about, but it’s time to start actually looking at the protein in detail. To do this, you have to get hold of a sample and isolate it. The best way is to take the gene for the GliG protein, attach it to a gene for a something you can easily isolate (the equivalent of attaching a ring to the protein and then using a hook to pull it out later) and stick it into a bacteria. The bacteria will (usually!) make shedloads of the protein, and you can then mash up the bacterial cells and pull out your isolated protein.

This isn't exactly how a His-tag for protein isolation works, but it's a pretty good approximation! Separate images from wikimedia.

Stage five: experiment on your isolated protein (in vitro studies)

Once you have the isolated protein, you’re free to explore with it! If you have enough protein you can use various methods to take a look at the molecular structure. You can add things to the protein and see how it reacts, you can test how stable it is at various temperatures, whether it binds to anything, etc. All these techniques deserve blog posts of their own and for biochemists studying protein structure and function they are the main part of the exercise while all the other stages are simply preliminary steps leading to the precious protein.

So what about the GliG protein? By looking at the substrates that it reacted with, and looking at the domains used for binding, they determined that its most probable role is to attach the sulphur onto the precursor of gliotoxin. There are then several more proteins (imaginatively named GliI, GliJ and GliT) that then turn this into the final product; gliotoxin.

Scharf DH, Remme N, Habel A, Chankhamjon P, Scherlach K, Heinekamp T, Hortschansky P, Brakhage AA, & Hertweck C (2011). A dedicated glutathione S-transferase mediates carbon-sulfur bond formation in gliotoxin biosynthesis. Journal of the American Chemical Society, 133 (32), 12322-5 PMID: 21749092

S.E. Gould About the Author: A biochemist with a love of microbiology, the Lab Rat enjoys exploring, reading about and writing about bacteria. Having finally managed to tear herself away from university, she now works for a small company in Cambridge where she turns data into manageable words and awesome graphs. Follow on Twitter @labratting.

The views expressed are those of the author and are not necessarily those of Scientific American.





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