Friday 27 May 2016

Introducing our newest aquatic residents to Liverpool Life Science UTC

On Wednesday the 11th of May, Myself and a team of teachers and students (from year 10 - 12 ) from Liverpool Life Sciences UTC went to The Aquarium Liverpool, a shop in our local area  for the afternoon to carefully pick out a number of a different tropical fish that are a range of species and colours. We picked the fish by their natural habitat, as we had set up two types of environments  for two different tanks. The first tank was focused around the African lakes (mainly Malawi) which contain few plants (if any at all), sandy/pebbly lake floor with big boulder rocks for the fish to hide and protect themselves from predators such as birds of prey. Our african lake tank has a pH slightly above neutral (due to the rocks) to represent the pH of the lakes in natural habitat . The second tank theme was Amazonian and these rivers have a lower pH (due to the decaying wood and green plants) to also represent their natural habitat.
After myself and the team had spent the afternoon at the aquarium carefully picking out the fish and deciding on certain names for the fish, we headed back to school to introduce the fish to their new home. Before releasing them from the bags they were transported we allowed them to acclimatize in the bags so that the temperature could even out between the tank and the bag. Putting the fish straight into a new environment can be very stressful for the fish as well as possibly the fish going into shock that could kill them. We slowly introduced them by letting them float on the top of the water in their bags for about 5-10 minutes then slowly allow a small amount of water every 5 minutes to fill the bags allowing the fish to acclimatize to their new environment. Finally we released them into the tanks.
           
Part of the team slowly introducing the new fish into the African Lake Tank

We got these tanks for a few reasons, firstly as a Life Science school we thought the tanks are a good way of introducing students to the different adaptations and behaviours of organisms from different ecosystems. Secondly when owning any type of aquatic tank water checks are crucial to keep the plants and fish healthy, so by doing regular water checks and water changes it teaches us new skills by analysing nutrient levels using chemical test kits, learning which levels are healthy or unhealthy for the fish and how to improve the tank. Thirdly we often have primary school children joining us down in the innovation labs for the afternoon that find it great fun to interact with the fish along with other aquatic model organisms as well as being taught by observing them and learning all about them for example how to care for fish by analysing their water quality and study their behaviour.



We have a range of fish in our two tanks. For example in the Amazon tank we have: Bolivian rams,  they are often described of having the behaviour of being very peaceful and described as being very hardy (meaning it's easy to look after them in aquatic tank environment) but have to be kept at a temperature of 23.3 - 25.6oC. They are found in streams,pools and lagoons and feed on small organisms but every fish in our tank get fed tropical flake food that give them all the nutrients they need to stay healthy. We also have angel fish which are described of having the behaviour traits of being semi- aggressive and are also moderately hardy fish that have to be kept at 23.9 - 27.8oC degrees. In their  natural habitat they are found in swamps or flooded areas that contain their feeding source of small model organisms such as smaller fish and invertebrates. Marbled  hatchetfish behaviour is said to be peaceful but shy  but are moderately hardy that has to be kept at 23.9 - 27.8oC. Thier habitat are throughout the amazon river as they are a very common fish in the Amazon river environment. Lastly we have two types of species tetra,the cardinal tetra and the black neon tetra both described as  peaceful and very hardy that feed on fallen decaying plants and small invertebrates.

Our amazon themed tank


In our African tank we only have two different species but will be shortly adding to their community. At the moment we have a variety of Lake Malawi Cichlids that are mainly found in Lake Malawi in Africa. They are recommended as a starter fish for tropical tanks as they are very hardy, have a great personality of being very active and lively but are very dominant, there is always one main leader in each tank. These Malawi Cichlids often come in bright and natural colours that can add a burst of colour into the tank which can be fun for any fish owner. As these fish are found from Africa the water has to be kept at a high temperature of 26-27oC, in their natural habitat they feed on smaller aquatic organisms. Particularly striking are the two Peacock Cichlids.


Our African Lake themed tank


As any type of aquatic model organism they all need care, some more than others.  For these fish we feed them and take the temperature of each tank every day and record the results on a chart to prevent over or under feeding and maintain good water conditions. We also test the tanks water twice a week and record the results down to keep on eye on the pH, nitrate, nitrite and ammonia in the water as if the levels rise high there is a risk of the fish dying. Finally we do weekly water changes of 25% of each tank to refill the tank with clean water to reduce the risk of rising levels of nitrate, nitrite and ammonia in the water. Once a month we do a larger water change to clean the tank more thoroughly.  


In the future our aim is to increase the range of tanks we have to include a number of different habitats. These tanks are proving a great way of engaging our students and were a huge hit with the primary school students. We hope these tanks will allow students to take part in a variety of projects which educate them and inspire them to find out more about workings of organisms and Biological Systems.

By Y12 Student, Lauren Scott (Manager of aquatic organisms for Greenland BioDesign)

Thursday 26 May 2016

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!