Cysteine for June and Half Term. Molecule of the Month

There can be fewer amino acids that have received as much attention from protein chemists and enzymologists than cysteine:  one of two proteogenic, sulphur containing amino acids (the other being methionine). Even Michaelangelo thought so: he celebrated it in his famous work in Rome! Well, joking aside, the connection between the hand of God and Man on the ceiling of the Sistine Chapel (sorry!) could represent one of the most well studied interactions in Biochemistry, the disulphide bond (or bridge). I shall return to this shortly after a brief description of the cysteine molecule. 

The structure shown left illustrates the essential features of the "free" amino acid, cysteine (abbreviated to Cys or C) with its characteristic carboxylate and amino groups attached to the central, alpha carbon. Its systematic name is 2-Amino-3-sulfhydrylpropanoic acid (note the US spelling, which is now just as common in the UK, but seven years at a grammar school means I am unable to substitute "f" for "ph" without trembling). As you will be aware, the carboxylate and amino groups are consumed in the formation of a peptide bond, leaving the sulphydryl group the main distinguishing feature of a Cys residue in a polypeptide chain. At neutral pH there is a slight tendency for the SH to dissociate, creating the nucleophilic thiolate anion (S-). The pKa of cysteine is just above neutral at pH8.2 (serine is 9.2, approximately), a feature deployed in some active sites including the cysteine protease, papain and the Cytosine-specific DNA methyltransferases (which I have discussed before). Cysteine is synthesised in vivo through the union of the related amino acid serine and methionine, followed by a number of enzymatic interconversions (see here for details (this link will taKe you to a free pdf with an excellent summary of amino acid biosynthesis: Cys is at the foot of the document). Cys is a non-essential amino acid, but alternatively, perhaps when dietary methionine is unavailable, Cys may be provided by a number of the constituents of our gut microbiota?

Free cysteine may be oxidised to form the dipeptide cystine, through formation of an S-S bond: conversely, disulphides can be reduced by compounds such as beta mercaptoethanol or dithiothreitol, to generate free cysteine. An oxidation  reaction often occurs, leading to the formation of inter- and intra-molecular disulphide bonds in some polypeptide chains (as mentioned earlier). This provides me with the opportunity to clarify the role of this interaction in protein folding and stabilisation: an area of frequent confusion amongst undergraduate Biochemists. To begin with disulphide bond formation is not a feature of most proteins, therefore it is illogical to assume that it is a generic feature of all protein structures. (I find this the best way to remember this point). The peptide bond, hydrophobic interactions, hydrogen bonds etc are present in ALL proteins: disulphide bonds are not. Next, disulphide bonds are common in extracellular proteins, a phenomenon that was critical in facilitating the work of Porter and Edelman, who were jointly awarded the 1972 Nobel Prize for elucidating the molecular arrangement of polypeptide chains in antibodies (see below). Finally, disulphide bonds are found in many of the "historically revered" proteases and their precursors, again, perhaps "skewing" the perception of their importance in protein structure formation? 

The iconic antibody structure (left) provides a nice example of how disulphides play a role in protein stabilisation. IgG (for example) can be considered as a tetramer comprising two heavy chains and two light chains. The 4 black lines represent disulphide bonds. The whole structure is generated by hundreds of weak interactions (hydrogen bonds, hydrophobic interactions etc.), on top of the covalent bonds that of course link the amino acid residues. [Protein folding is coming soon, but see here for a taster!]. The disulphide bonds serve to stabilise the folded oligomer. This is seen most elegantly when antibodies in a reasonably pure form are separated on a denaturing polyacrylamide gel in the presence or absence of beta mercaptoethanol. In its presence, the disulphide bonds are reduced and 2 chains are detected. In the absence of the reducing agent, a single molecular species is observed. [Can you work this out?]. Think of disulphides as buttons on a shirt. The shirt fits your body without the buttons fastened: it just says put better with the buttons fastened!

Cys can coordinate metal ions like Zinc. One of the great surprises (to me at least) of the 1980s, was the discovery of a very common protein motif involved in the recognition of nucleic acids: the Zinc Finger. [Without spending too much time discussing the details, the original motif is now known to come in a number of structural varieties, but I will only discuss the generic type here]. It was known that DNA recognition by bacterial repressors was often mediated via an alpha helical motif, called the helix turn helix motif. The crystal structure of repressors, such as the lambda cro repressor, provided a focus for this work. However, as DNA sequencing became more popular, protein sequences began to emerge that pointed to repeated Cys residues in several proteins from eukaryotes, known to play a role in gene regulation. Structural studies then revealed that a cluster of 4 Cys (as I said there are variants, such as 2 Cys-2 His) residues coordinate a zinc ion and in doing so, provide a platform for an alpha helix-beta sheet unit, that can recognise DNA (and in some cases RNA) in a sequence-specific manner. I remember being quite shocked when the publication of the Drosophila Genome in the late 1990s, revealed an abundance of putative Zn-finger transcription factors. It seemed at the time that prokaryotes preferred the helix turn helix motif for gene regulation, while eukaryotes utilised the Zn-finger. In the near 20 years that have passed, it has become clear that DNA recognition motifs are more diverse and this oversimplification hasn't (as is often in Molecular Biology!) stood the test of time. However, the importance of Cys residues in facilitating DNA recognition by proteins remains robust: it has in fact become the basis of designing novel sequence-specific gene regulators. 

The final function of Cys residues in protein function in this Post is given by FNR: the  master regulator of anaerobic gene expression in E.coli. My former colleague, John Guest and my current colleague Jeff Green have devoted much of their careers (John latterly) in pursuit of an understanding of the mechanism of action of this gene regulator, shown schematically on the right. As you can see, the DNA binding region contains a helix turn helix motif, but I want to draw your attention to the (yellow) [4Fe–4S]²⁺cluster which comprises 4 Cys residues, and is exquisitely oxygen sensitive. Since the job of this protein is to modulate expression of sets of genes in response to changes in oxygen levels, this mechanism is ideal. You can read more about the molecular details here, in this nice open access review. The iron sulphur cluster is found in a number of (perhaps) better know proteins which are critical in electron transfer processes such as oxidative phosphorylation. There is a nice wiki page here, where you can learn more about the properties and diversity of these fascinating structures.
 

I hope I have given you a taste for the beauty and versatility of amino acids, and in particular cysteine. I will leave you with the amazing discovery in the early 1970s by Thressa Stadtman that a small number of critical redox enzymes have selenium in place of sulphur in the form of the amino acid selenocysteine. With a substantially lower pKa (less than 6), this enhances the nucleophilicity of the side chain. What an elegant evolutionary adaptation to overcoming the limitations of Nature's 20 proteogenic amino acids! Who says Bioinorganic chemistry isn't fun!

I have highlighted text in red that I think may require a definition and a glossary will follow, thanks to a suggestion from my partner in crime at the Widnes Sci Bar, Bob Roach. I am just working out how to insert tables effectively, but I thought it best to post in time for the weekend!

Introducing Ant Man, the Queen and the Colony

We came up with the idea of using ants to conduct behavioral experiments at Liverpool Life Sciences UTC. We chose a species called Messor barbarus which is a species that eats seeds and farms aphids. These ants need to be kept at around 25OC, with good ventilation and little disturbance, conditions found in the model organism room in the innovation labs.

On April 12th the ants arrived and we placed them into their new home, it was at this point that we had found that the queen had laid a large amount of eggs whilst she was in transit from Spain to the UK, this created a very strong foundation for a new colony. At first no ants left the tube that they had arrived in but over the coming days they soon ventured out into the pipette tip box that we are using to contain them (so that when they are big enough they can move into the formicarium). The hive exploded with newly hatched ants some were just minor worker ants, some were major and some were the soldier ants. The main way to tell the difference is the size, this can be measured using image analysis software such as Image J.

The pipette tip box in the bottom right contains the ants, so far only the minor workers have ventured outside and into the formicarium. We use a red light to observe the ants without disturbing them, this is because the ants vision is shifted away from the red end of the spectrum towards the UV wavelengths.

A few days ago the queen became very inactive and the ants swarmed her, they covered her, we believed that she was dying due to the fact that there was no activity at all, in retrospect we probably should have noticed how much sugar solution was being used up in just one day. Usually the ants only need to have a new eppendorf of sugar solution once every 3 days but at this moment in time they were using up one every day. As turns out the queen had been lay large amounts of eggs at least 10 in her three days of inactivity, from that we guessed that the queen was being fed and looked after by the other ants.

In the coming weeks we have planned a few experiment including one based on testing the effect of concentration of sugar solution on habitat choice. To do this we needed to make a choice chamber for the ants. When the hive is big enough we will start feeding them mealworms, after some time we will give them meal worms infected with fungi to see how their behaviour changes. What this space for details of how our ant colony and experiments progress.

By Jack McKenzie (Ant Man), Erin Ruddock and Mazuba Maambo

Bugs, Bites and Parasites - A Totally Tropical Tour!

On Friday afternoon, I, along with Dr Dyer and various Year 10 and 12 students, visited the Liverpool School of Tropical Medicine, which, for the uninitiated, is an institution that works on improving healthcare in developing countries by working on cures for such diseases as Malaria, Schistosomiasis, and Lymphatic Filariasis. I mention these three illnesses in particular because these are the three groups we were split up into, and also the three illnesses that made up the majority of our tour.

The tour started (after a brief health and safety talk, of course) with my group, which was called malaria, being taken into one of the rooms where the mosquitoes are housed. One of the scientists there then showed us the mosquito’s life cycle from the 1st, 3rd, and 4th instars (Larval stage) through to the pupal stage, and finally adulthood, at which point they must be housed in an inescapable environment in order for them not to bite anyone, compared to all previous forms of the mosquito that must be housed in water.  

We then moved on to the parasitology department, where we were shown pictures of sufferers of lymphatic filariasis, a disease affecting the lymphatic system (the system that is responsible for the removal of toxins, waste, and other unwanted materials via the production of lymph, a liquid containing infection-fighting white blood cells). The cause of Lymphatic Filariasis is small worms of the roundworm family, known as filarial parasites, being transferred from mosquitoes into humans through the skin, these worms then create thousands of larvae that enter the bloodstream. This causes ‘Lymphoedema’ (A swelling of limbs and other body parts with Lymph) which can lead to ‘Elephantiasis’, a permanent deformity making the limbs look like Elephants’ and requiring amputation. So all in all it isn’t a very pleasant disease.

On the walk between different areas of the facility we were shown smaller labs that were responsible for blood testing, and testing on more infectious diseases,  and some larger labs that were responsible for less possibly infectious diseases and parasitology.

The next illness we saw was Schistosomiasis, which is an illness caused by a genus of parasitic flatworms called Schistosomes, which infect freshwater snails who then go on to release the Schistosome larvae which penetrate the skin during contact with infested water. This can cause intestinal/abdominal pain, blood in urine, and liver enlargement, among other things.

Finally we were taken to the oldest part of LSTM in which we were able to talk to researchers and PhD students, which was particularly interesting to me as, personally, pharmacology is a field I’m very interested in going into.

By Isaac Basque-Rice

Year 10 student at Liverpool Life Sciences UTC