Exploring protein interactions: yeast two hybrid systems

>Proteins are one of the key molecules inside cells; involved in signalling, intracellular transport, metabolism and gene control. They rarely work alone, most of the proteins in the cell are part of large complex networks consisting of many interacting proteins. Various techniques exist in order to find these interactions, and one of the most common is the use of yeast two hybrid systems.

Yeast is apparently quite a nice organism to work with (I've never worked with it myself, I must say, apart from a few practicals in second year, where I almost set my lab-partners hand on fire). The genome is fully annotated, the organism is well characterised, and yeast grows and responds quite fast, so experiments shouldn't take too long.

The yeast-two hybrid system is based around molecules called transcription factors, which are normally used by the cell to active gene expression. In yeast, there are several transcription factors which consist of two separate molecules, which need to be in close proximity in order for a gene to be expressed. If you attach an experimental protein to one half of the transcription factor, and another experimental protein (that you think interacts with the first) to the other half you can test for interactions. If the two proteins do interact then the transcription factors will be brought close together and the gene downstream of them (which acts as a reporter gene) will be expressed:The diagram above (taken from the reference below) shows this process. The blue jigsaw-shaped 'X' and 'Y' proteins are the experimental proteins, being tested for interactions and the yellow shapes are the two parts of the transcription factor. The big white cloud is the polymerase, which begins the process of turning the DNA into protein. The reporter gene can be set to code for a vital compound such as histidine; stick the whole system into a histidine deficient mutant and you have a marker system to see if the proteins interact. If they do, the histidine is produced and the cells can grow, if they don't, the cells die.

One of the most useful things about this technique is that it can be automated, and used to scan whole libraries of proteins to see if they interact. By using a matrix, each protein X (the 'bait' protein) can be given a defined position and then systematically exposed to a number of different protein Y (the 'prey' protein). If you're taking Y from a clonal library, and have a sufficiently intelligent robot, the whole procedure can be carried out with minimal human input.

The yeast two-hybrid system has been invaluable for determining many important protein-protein interactions however there are some problems with it. Firstly, this is a yeast two component system, and most protein complexes consist of many interacting proteins, certainly more than two! Secondly, this whole system relies on two soluble proteins interacting in the nucleus (where the DNA is) and so doesn't work for membrane bound protein interactions.

In view of this, several modifications have been made to the original methodology to make it more useful for trapping a wider range of protein interactions. It's been expanded into the three component system, which identifies proteins that interact with (or inhibit) both the the bait and the prey. Using another natural yeast system (the G protein system) has allowed transmembrane proteins to be identified as well:
In this system protein Y contains a binding site for a subunit of the G protein, while X is a membrane-spanning protein. G proteins are membrane bound proteins that activate transcription factors inside the nucleus. If the two proteins interact then the G protein subunit bound to the Y is sequestered away from the rest of the complex, and the G protein signal cannot be transmitted. There is therefore no signal to the transcription factors, meaning reporter genes (in this case genes for pigmentation rather than for death) will be turned off.

Even with these modifications there still are problems with the yeast two hybrid system. There is very little quantitative analysis involved to see how strongly the protein is binding, and the discovery of new interactions will always be limited by the choice of proteins to screen. However one of the largest advantages of yeast two hybrid systems is that it carries out protein binding analysis inside the cell, in proper cellular conditions, unlike affinity binding which, while it can identify much larger protein complexes, involves taking proteins out of the cell and handling them in vitro.

Yeast two hybrid systems are therefore still one of the main practical methods used for determining protein interactions. The field of systems biology is a fascinating one, and while attempting to catalogue the whole spectrum of cellular interactions may seem like a daunting task ('interactomics' for the funding people) working towards it will only reveal more and more useful information about the complex and fascinating networks of proteins within the cell.

[btw: The reference below is a great source of information about the many different modifications made to the basic yeast two hybrid system, thoroughly recommended for anyone interested in protein interactions or systems biology]

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Brückner A, Polge C, Lentze N, Auerbach D, & Schlattner U (2009). Yeast two-hybrid, a powerful tool for systems biology. International journal of molecular sciences, 10 (6), 2763-88 PMID: 19582228

Half plant, half predator, all weird.

I was planning on brushing up my knowledge of chloroplasts today, as next week I'm starting a plantsci course for my options lectures, but I got sidetracked by Captain Skellet alerting me to Hatena. I've heard of several organisms containing proto-plasmids; symbiotic chloroplasts which haven't completely been endosymbiosed, but Hatena was a new one so I went to look it up. And I'm very glad I did, because it's pretty amazing.

Hatena, taken from the reference. Green blob is the symbiont. Scale bar is 10um

Quick background: chloroplasts are little membrane enclosed vesicles in plants which carry out photosynthesis. Current theory for how they developed is that they were once free-living bacterial type organisms (cyanobacteria) which were engulfed by a larger cell and over time lost their own identity and became little photosynthesising factories inside the larger cell. (I've got another post on it here for anyone particularly interested in the subject.)

Hatena arenicola doesn't have a chloroplast, but it does have a symbiotic relationship with another organism; nephroselmis. The nephroselmis is always found in the same place in the Hatena, and carries out photosynthesis to provide energy for both of them. Unlike regular chloroplasts, nephroselmis has it's own proper nucleus and even it's own mitochondria although most of the internal cellular organisation and any kind of motile apparatus (such as flagella) has been lost.

The weirdest thing about these two organisms though, is their replication cycles. When Hatena replicates, the nephroselmis doesn't, and as a result only one of the offspring gets the photosynthesising symbiont. The other organisms remains colourless and develops a complex feeding apparatus at the apex of the cell, presumably as it can no longer rely on the symbiont for food. This wierd 'half plant, half predator' lifecycle is shown below. (Picture taken from the reference, scale bar 10um):


That's just weird. Seriously odd. The Hatena is able to move seemingly freely between being a predator consuming other cells for food, and being a plant-like organism, once it settles down with it's symbiotic partner. The grey non-symbiont organisms can be induced to take up free-moving nephroselmis and (in the words of the paper) "tentitavely" maintain a symbiotic relationship with them.

The paper suggests that Hatena cycles between these two modes of living, depending on circumstance. Thus the 'predator' grey cell shown above will continue eating fellow cells until it consumes a nephroselmis, at which point it degrades its complex feeding apparatus, accepts energy from the symbiont until it's ready to divide. One of the daughter cells will then go through the whole cycle again while the other remains as a non-predating plant. The authors freely admit that there is little evidence for much of these stages, but it seems a reasonable way to explain what is going on.

As this is clearly a very early stage in symbiotic capture it has important implications for the endosymbiotic theory of chloroplast evolution. Along with various other 'intermediate' symbionts (such as Karenia mikimotoi and Lepidodinium viride) the Hatena helps to show how chloroplasts might have first formed in the cellular ancestor of plants. Hatena and its symbiont have already acquired an intimate structural association, only the coordination of their cell cycles would be required to turn the nephroselmis into an internally replicating plastid.

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OKAMOTO, N., & INOUYE, I. (2006). Hatena arenicola gen. et sp. nov., a Katablepharid Undergoing Probable Plastid Acquisition Protist, 157 (4), 401-419 DOI: 10.1016/j.protis.2006.05.011

Cycling Cells - The circadian rhythm

Circadian rhythm is the cyclic control of cellular processes over a period of roughly twenty-four hours. There are many processes within the body that are held under circadian control; the need to eat and sleep, blood pressure and some hormone production to name a few. Circadian control is an important development in evolution, as it allows behaviour to adapt to appropriate times in the day. Humans have not adapted to function particularly well at night, so using that time for sleeping means they can be more alert during the day and in the hour or so before you wake up (on weekdays at least), the body gets busy increasing the blood-pressure, preparing you to need the bathroom and, slightly bizarrely, increasing testosterone levels.

I wasn't planning on doing a post on circadian rhythms, but Alejandro mentioned it in one of the comments, so it was in the back of my head when I was looking for a paper to review this week. And it's about time I started getting back into more general molecular biochemistry (less than five months left till exams!) rather than concentrating exclusively on bacteria.

From a cellular point of view circadian rhythms are controlled by careful feedback loops between interacting proteins. In this paper, the main positively regulating proteins they were looking at were proteins named CLOCK and BMAL1, which bind to promoter sequences of circadian rhythm genes and switch them on. They also turn on the negative regulators PER and CRY which, when they get to high enough levels, bind to CLOCK and BMAL1 and stop them from functioning, turning the circadian rhythm genes off again.

Many clock proteins undergo post-translational modifications in order to give a further level of control. In particular they can be phosphorylated (a phosphate group is added onto the protein) by proteins known as protein kinases. The main work of the paper I decided to look at (reference below) was identifying GSK3β, a protein kinase which phosphorylates BMAL1. Like most kinases, GSK3β is not just involved in circadian rhythm control, but takes part in many other cellular functions such as control of glucose homeostasis, cell fate determination, and cell survival. It's not surprising, therefore, that is can be involved in a number of pathological conditions, including diabetes, Alzheimer's disease, cancer and bipolar disorder.

The first study done for the paper was to show that GSK3β will bind to BMAL1 and phosphorylate it. This was done by adding pure samples of the proteins together, along with radiolabelled ATP as a source of phosphate. The result is shown below (all diagrams taken from the reference at the bottom) wiith GST used as a control to check what the GSK3β does when there's nothing for it to phosphorylate (it phosphorylates itself):


As only radiolabelled material shows up on the autoradiogram, this shows that the GSK3β has transferred the radiolabelled phosphate onto the BMAL1.

For further proof that the GSK3β was carrying out the phosphorylation under physiologically relevant conditions, cell lines were used which contained no GSK3β (-/- mutants). Comparing the levels of BMAL1 in these cells with wild type cells showed higher levels of BMAL1 in the mutant strain. This is expected as the addition of the phosphate is thought to lead to the addition of another group, ubiquitin. Ubiquitin is (as far as I am aware) an almost universal signal for 'Degrade This Protein'.

The next stage was to look for the actual sites of phosphorylation; the places on the BMAL1 where the GSK3β sticks the additional phosphate group. As with most kinases, GSK3β recognises a specific pattern of protein residues (T/SXXXS/T for anyone who's interested) and fifteen of these sequences were found in BMAL1. The exact residues were found by point mutation; certain amino-acids were changed and the resulting change in phosphorylation measured:
The wild type protein is shown on the left and the mutant, with the significant T residue converted to an alanine(A) on the left (the change is notated as T21A; the T in position 21 has been changed to an A). This change in one amino acid has decreased the phosphorylation by 40%, although it would be nice to see some actual values rather than relative ones.

The final test to confirm that GSK3β is involved in circadian rhythm control of BMAL1 was to show some actual cyclic behaviour of the protein. For this two cell lines were used, the wild type and the GSK3β -/- mutant. α-tubulin was used as a control; this protein is expressed at constant levels over time and therefore shows that the decreasing and increasing levels of BMAL1 is actually due to changing levels in the cell, rather than just a smaller cell sample containing fewer proteins overall:
That's quite a beautiful gel; in the wild type cells the BMAL1 cycles nicely over the time period (they don't run the experiment for terribly long, but BMAL1 cycling has been proved adequately elsewhere, and the wild type cells are more of a control than the actual experiment). When you knock out the GSK3β, however, the cycling pretty much stops. The paper is careful to point out that is doesn't completely stop, some evidence of differing levels is still seen, but this is to be expected. It's very rare that important cellular processes in mammals are placed entirely under the control of one protein, and there are likely to be other pathways involved in the circadian control of BMAL1.

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Sahar, S., Zocchi, L., Kinoshita, C., Borrelli, E., & Sassone-Corsi, P. (2010). Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008561

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For those interested in such things, I now have a twitter. Like the blog, it will be strictly sciency, rather than personal and will be updated far more often.

Wonderful Life - book review

This is my first attempt at a proper scientific book review, so any feedback would be much obliged. Thanks!

The Burgess Shale is a collection of fossils of soft-bodied invertebrates that was formed soon after the Cambrian Explosion, an apparently rapid appearance of a many groups of complex animals. Initially discovered in 1909 by Charles Walcott, the fossils were later examined by three researchers in Cambridge; Charles Whittington and two graduate students, Derek Briggs and Simon Conway Morris. What began as an exercise to catalogue a group of ancient arthropods turned into the discovery of several new phyla of organisms, and ultimately changed the way evolution and selection were viewed. “Wonderful Life” by Stephen Jay Gould is a documentation of this process, exploring and explaining this new view of evolutionary change through Deep Time.

Although Gould was not personally involved in the cataloguing of the Burgess Shale organisms, he was in close communication with the people who were and his clear enthusiasm for the subject shows through. In the preface Gould sets out three main aims; to chronicle the intellectual drama that *was* the Burgess Shale examination, to explore the implications that this change in the perceived workings of evolution brought about, and finally to briefly look at *why* the discovery of the Burgess Shale seems to have passed so unnoticed by the general public, and even non-paleontological scientists.

The book is divided into five sections. The first sets up the central theme that the book is out to destroy; the iconographic idea of the March of Progress, that evolution is a kind of ‘onwards and upwards’ affair, with each generation leading to greater complexity. This was the idea that the discovery of new creatures in the Burgess Shale, creatures that belonged to no known phylum (and could therefore not be simpler and less developed forms of current animals) began to destroy. The second section covers background information considered necessary for an understanding of the Burgess Shale, a quick course on the Paleontological timeline and arthropod anatomy. Being the rather badly specialized microbiologist that I am, I read through these dutifully and promptly forgot them, so can safely say that it’s perfectly possible to enjoy the book without a great depth of scientific understanding.

The third section was for me the most exciting, as it was the actual description of the Burgess Shale creatures, written in order of their discovery (by Wittington, Briggs and Conway Morris). As they slowly discovered more new creatures, they began to realize that these were animals that had never been seen before, that had been wiped out by some extinction event. Furthermore, there was no particular evolutionary *reason* for certain animals to have been saved, the ‘March of Progress’ was beginning to look more like a lottery of chance. The last part of this section discusses the implications of this point of view, that humanity is not a strived-for evolutionary point of perfection, but simply a small twig on a tree of life which has had several branches snapped off altogether at different points in time.

The fourth section leaves the Burgess Shale (in a rather anticlimactic and in my opinion a slightly disappointing shift) to discuss Walcott, the man who found the fossils in the first place. Being a busy man, who in later life was caught up with various family tragedies, Walcott never properly had time to examine his fossils, and in the few papers he did write about them, he ‘shoehorned’ every fossil into modern phyla. Although I couldn't find the discussion of the life of an Edwardian scientist anywhere near as exciting as the beautiful fossil-creatures of the Shale, this section allowed a detailed examination of the shift in the way evolution was viewed as a theory, and why the original iconography of the March of Progress was so seductive and successful. In the fifth section Gould takes a brief but fascinating look at how things might have changed had life had a chance to play out a second time, if different branches of the tree of life had been cut off at different stages in history.

Overall I really enjoyed the book; I was especially pleased as this is (embarrassingly) the first book by Stephen Jay Gould I’ve ever read. The writing style is easily accessible, even to people with a very sketchy view of Deep History and the importance of arthropods, and is, a little surprisingly, highly immersive. The creatures of the Burgess Shale are so beautiful and wonderful that they stand up perfectly well on their own, and Gould lets them do so, his writing concentrating on exploring the philosophies surrounding their discovery rather than over-elaborate descriptions. The many accompanying pictures, most of which are the original drawings made by Wittington taken straight from the fossil samples, help to provide a wonderful visual image of the amazing and sometimes quite frankly weird creatures that populated the Burgess Shale.

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For those interested in such things, I now have a twitter. Like the blog, it will be strictly sciency, rather than personal and will be updated far more often.

Obligatory January The First Post

I like organising myself, which may come as a surprise to people who know me. When it comes to revision time I like making revision plans, when it comes to holidays I like planning what I'm going to do each day, and when it comes to New Year I tend to go a bit crazy with the resolutions.

What I am not very good at is actually doing things. I'm good at starting things, just not sticking with them. The main problem with all my wonderful plans is not so much that they aren't good plans, but that I never follow them through. I'm surprised I'm still blogging to be honest; over one and a half years now when most of my new-exciting-idea-things barely make it to the six month mark.

So this year, instead of making a whole page of resolutions and then breaking them all by February, I'm going to make just six, and really try and stick with them. Three things I won't do, and three things I will.

Things I will not do next year:

1. Waste time on the Internet. I'm not talking about the fifteen minutes morning webcomic-check, or an hour spent browsing on scienceblogs or research-blogging. I'm talking about four hours looking up TV-tropes kind of time wasting.
2. Spend longer than fifteen minutes in the shower. My current average is thirty minutes. My getting-up routine consists mostly of shower. This is not good.
3. Buy non-fairtrade chocolate. This one is so hard not to break. It's amazing how you can be half way through a Twix before you realise you're eating forbidden chocolate.

Things I will do next year:

1. An hours work every evening (at least), unless I am out of the house. Taking an evening to go out with friends (or just the one special friend :D ) is fine. Spending the whole evening lying on the sofa breaking the other resolution-number-one is not.
2. Go to Yoga at least once every two weeks, ideally every week. I tried to do this last term, but things kept getting in the way. This term I should be able to keep it up more regularly.
3. Blog regularly. Ideally two posts a week, dropping back to one post a week in times of Stress (due to exams, job-hunting, etc.)
   

My holiday is officially over now, as is the Hiatus. I have so many research-blogging posts to catch up with it is unreal.

Hiatus

I was hoping to avoid this by maybe writing some future-posts to appear during the holidays, but time just ran out on me. So I'll be going on holiday-induced hiatus for the next two weeks, while I enjoy some time with my family and turn off the science brain for a bit (and work my way through the most AMAZING CHRISTMAS PRESENT EVER aka the complete works of Shakespeare =D )

I've got plans for when I get back though! Starting from January I'm going to try and keep to the whole twice-a-week-blogging I managed last term, which will include, among other things:

• Book reviews. I've been catching up on all the science books that I never quite got around to reading, and I'm planing on writing proper reviews for them. Expect some Stephen Gould, and Schrodinger's 'What is life' among others. (I might even attempt the Stephen Hawking).
  
• The molecular clock. I've been wanting to write about this for a while. Hopefully I'll manage to get my thoughts and references together manage something half-way decent.
• Plants. The course I've chosen for next term is primarily about plant and chloroplast evolution and diversity. As I'll have to read around this subject I'll probably be dissecting some of the papers in my blog, so there will probably be quite a heavy plantsci slant to the papers coming up.
• More bacteria. As always, I'll be reading papers concerning my project and my major interests, so those will feature as usual.
• Shakespeare quotes. Oh yes.

I've had a great time blogging this term. Hopefully I can keep it up next year!

Lab Rat would like to wish everyone who is having a holiday a Happy-Holiday!

Plastic from bacteria

I'm on holiday at the moment, so today's post is another section from my long essay last year, about the potential uses of biorefineries. It was written for a more scientific-based audience so might be a little harder to decipher than my usual posts.

Bioplastics

Bioplastics are polyesters that accumulate intracellularly in microorganisms in storage granules. They are usually built up from hydroxyl-acyl CoA derivatives through a range of different pathways in different microorganisms. As they are both biodegradable and biocompatible they have found numerous applications within medical and surgical fields, as well as having a greater environmental advantage over petroleum based plastics. The main disadvantages of bioplastics for commercial use are their high production and recovery costs.

The most widely produced bioplastics are poly(3-hydroxybutyrate) and poly(hydroxyalkanoic acid), referred to as PHB and PHA respectively. These both contain different β-oxidation intermediates as monomers, which are enzymatically polymerised through a condensation reaction. The structure of PHA is shown below (the 'n' indicates that the section show below is repeated multiple times):The first bioplastic to be described was PHB, found in Bacillus megaterium in 1926 by Lemoigne. It is stored in polymer form in granules within the cell.In order to decrease the recovery costs of the PHB granules, several attempts have been made to produce the secreted monomers, for polymerization outside the bacterial system. This has been achieved by expressing recombinant genes in E. coli

There are a large number of PHA polymers, ninety-one of which have been fully characterised. They are produced by both Gram negative and Gram positive bacteria via at least five different metabolic pathways. The main enzyme involved in polymer formation is PHA synthase (of the α/β hydrolase family), which polymerizes the monomers by connecting the coenzyme A thioesters of one monomer to the hydroxyl groups at positions 3, 4, 5 or 6 of the acyl moiety of the second monomer. There are four classes of PHA synthase, which are distinguished by their primary structures, substrate specificity and subunit composition. PHA synthases are found on the surface of the PHA storage granules, along with other proteins, and phospholipids.

(The structure of a PHA granule is shown above, image taken from Rehm 2003)

Engineering of recombinant bacteria that are capable of producing bioplastics requires both the transfer of a functional PHA synthase enzyme (there is no evidence as yet to suggest that any post-translational modifications of the enzyme are important for its function), and the engineering of suitable substrates that provide the enzyme with suitable substrates and sufficient concentrations. While the enzyme has been successfully transferred into model organisms such as E. coli, S. cerevisiae and even some transgenic plants, the provision of substrates is a more difficult problem as it involves dealing with large numbers of interlinked metabolic pathways. Metabolic flux analysis, carried out in transgenic E. coli, has substantially increased the carbon flux towards the production of PHB without detriment to the health of the bacteria, however this form of analysis has not yet been carried out on more complex PHA polymers.

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Madison LL, & Huisman GW (1999). Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiology and molecular biology reviews : MMBR, 63 (1), 21-53 PMID: 10066830

Steinbuchel, A., & Valentin, H. (1995). Diversity of bacterial polyhydroxyalkanoic acids FEMS Microbiology Letters, 128 (3), 219-228 DOI: 10.1111/j.1574-6968.1995.tb07528.x

Rehm BH (2003). Polyester synthases: natural catalysts for plastics. The Biochemical journal, 376 (Pt 1), 15-33 PMID: 12954080

Bacterial evolution: negative to positive?

The most commonly used distinction in bacterial populations is that of Gram negative and Gram positive bacteria. They are named after the method used to distinguish them: the Gram stain (developed by Hans Christian Gram). Gram positive bacteria have larger peptidoglycan cell walls, and therefore retain the crystal violet stain, whereas Gram negative bacteria have two membranes with a thin peptidoglycan wall between them, do not retain the crystal violet stain, and pick up the safranin counter-stain. The practical upshot of this is that you end up squinting at small blotched shapes under the microscope, trying to work out whether they look more pink or purple:

Gram +ve on the left, Gram -ve on the right.

As well as hinting that Mr Gram was one of those people who knows what shade 'fuchsia' is, the Gram stain is also one of the most important ways of telling what kind of bacteria you're dealing with. Despite being seemingly arbitrary, the composition of the cell wall plays a major role in determining behaviour. Gram negative bacteria (small cell wall, two cell membranes, see the picture below) tend to be motile, opportunistic, and able to colonise a wider range of environments. Gram positives on the other hand (big cell wall) are not so motile, but tend to have a huge range of excretory proteins to make up for this; almost all known antibiotics come from Gram positive bacteria.

Again, Gram +ve on the left, -ve on the right. Image from soil microbiology webpage

One thing that I've never really considered before is which one of them evolved from which. I haven't done much taxonomy, and the only time I really covered bacteria (unrelated to lab work) was in my Pathology course, which didn't seem too concerned about where different types of bacteria had come from, only what they were currently up to. The few times I did vaguely think about this though, I would have gone for the positive to negative direction. After all, surely you start with one cell membrane, and move on to two.

I recently came across a paper that came to the complete opposite conclusion, and therefore was too interesting not to read. The thing about bacterial taxonomy is that a lot of the major changes to morphology took place in Deep Time, and bacteria leave precious few fossils. Bacteria (and archaea...) had somewhere in the region of over one billion years to evolve before eukaryotic-things even started to be considered. That's a lot of time to try and sort out. To put that into context, one billion years ago from now things were just about starting to think about going multicellular. No dinosaurs, no plants even; the most complex form of life was something resembling a sofa cushion.

So how to sort out what was going on in that billion years or so? There are four main ways of going about it:

• Paleontological evidence. Bacteria don't form a huge number of fossils, but they can occasionally leave some physical evidence of their presence. For example, bacteria that eat iron will leave behind little fossilised iron cases; those that eat rocks can leave microscopic drilling holes. These provide temporal evidence for changes in structure and metabolism.
• Transition analysis. This is used to polarise major changes by turning them into a simple before-or-after question, and uses comparative, developmental, and selective arguments for determining answers. For example: did legs or wings develop first? Or, in bacterial cases: Which came first, Gram negative or Gram positive?
• Congruence testing. This searches for similarities across whole evolutionary trees, enabling loss or gain of evolutionary abilities (wings, feathers, second membranes etc) to be identified and polarised. As this is a comparison of many species, it allows potential mistakes from the arguments made in transition analysis to be found.
• Sequence trees. Sequence trees are ... problematic, but at the same time indispensably useful. They are formed by taking DNA sequences from a range of organisms and then using algorithms to tell the 'relatedness' between sequences and using these 'relatedness' levels to make evolutionary trees. They tend to be biased towards your sample distribution, undirectional, unable to properly account for generation times, and go somewhat screwy when you try to introduce horizontal gene transfer. Nevertheless they were instrumental data in showing that archaea and bacteria are two very distinct super-kingdoms (and I will freely admit that most of my distrust for them occurs because I can't get the damn things to work whenever I try them)

So...using these techniques can we get a clearer idea what was happening with bacterial membranes during those 1 billion-odd years before the arrival of eukaryotes? On the face of it; positive-to-negative seems to make more sense: start with one membrane, gain a second, possibly by gene duplication.

However like many evolutionary stories, that one falls apart a little when closer examined. Because Gram positive bacteria are not simply 'one cell membrane' they also have a massive cell wall surrounding them. Developing a second cell membrane on top of that seems absurd. And then why would the cell wall shrink? And how would anything get through this suddenly developed cell membrane. Transport proteins for the outer membrane tend to form a protein structure called a beta-sheet, while those for the inner membrane form an alpha-helix. That's a whole new system of protein folding that has to evolve pretty quickly, because otherwise the bacteria will starve, nothing can get through its outer membrane (which is balancing precariously on top of the huge cell wall...)

In view of this, the schematic seen on the right starts to make a little more sense (figure taken from the reference below). 'Murein' means 'peptidoglycan cell wall' and the cytoplasm denotes the inside of the cell. In this scenario, the double-membraned proto-bacteria (which has spend the last half-a-billion years or so evolving a well adjusted double membrane system) suddenly looses the outer membrane. A very simple genetic change would lead to a massively overgrown cell wall, which would rip the outer membrane away. The cell looses all it's outer membrane porins, and signal systems, but in return gains a highly protective cell wall, which potentially allows it to survive in different niches. How these aspects are lost genetically is another matter, and the paper rather hand-waves away by saying that unused genes tend to get lost eventually. Which is true in bacteria, they have such a small genome they don't want it getting filled up with unnecessary genes, but I have a feeling genes tend to leave something behind. Even so, the question of where the now-unnecessary genes go is possibly one of the weaker parts of this arguments (to my untrained student eyes at least.).

One thing that would really support this hypothesis would be to show that Gram positive bacteria formed a 'mono-clade' i.e came from a single universal common ancestor. Unfortunately this data is proving hard to pin down, not helped by the bacterial trick of swapping DNA around with all and sundry. Another confounding factor is the sheer space of time. Trying to determine whether a range of different modern bacteria all came from the same blob several million years ago is a daunting task. You can sort of get RNA sequence trees that support the mono-cladal Gram positives, but only if you close one eye and squint, which is not generally accepted scientific practise.

I don't think I'll ever end up going into taxonomy, even of bacteria. but it does produce fascinating ways to look at the world; how it changed, how it evolved, and how it finally turned into the way it is now. Orwell wrote, fairly famously, "He who controls the past commands the future", and when you're trying to figure out how bacterial resistance works, and preferably how to stop them getting it, that phrase takes on a whole new meaning beyond the political.

(It's not a perfect quote for this post. "Understands the past" would work better. But I'm not quite pretentious enough to go trawling through the quote archives to find something better. Any suggestions would be appreciated :p )

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Cavalier-Smith T (2006). Rooting the tree of life by transition analyses. Biology direct, 1 PMID: 16834776

Lab Rat guide to Fourier Transformations

Protein crystallography is one of those mysterious things that I always feel I should know more about. It starts with protein crystals, fair enough, and then heads rapidly out beyond the Magical Event Horizon leaving me with a picture of set of small fuzzy dots which mysteriously resolve themselves into an electron density map. Somewhere in all this someone will mention the dreaded Fourier transformation, at which point I know all hope is lost and a might as well stop listening.

With the help of a good lecture and an utterly outstanding website though, I'm starting to get the hang of it. X-ray crystallography works by shooting X-rays at a protein crystal. As the crystal is in a regular lattice structure, the X-rays are scattered in a regular way. Each scattered ray can be characterised by the amplitude and the phase, as shown below:Each dot on the resulting image (which usually looks something like the figure shown on the right) corresponds to a scattered X-ray. The position of the dot gives (by way of a set of clever equations) the amplitude of the wavelength. However in order to calculate the position of an atom you need both the amplitude and the phase, and you can't get the phase from just the position of the dot. And here is where the dreaded Fourier transformation comes in, to help give information about the phase.

The Fourier transformation is an equation, which gives reciprocal information about a molecule, for example if the molecule is a single small point, the Fourier transformation (i.e what it looks like after plugging its positional coordinates into the Fourier equation) will be a large fuzzy blob, as shown below (all pictures from now on are taken - with permission - from Kevin Cowtan's Book of Fourier) . Molecule is on the left and its Fourier transformation on the right:
For the more mathematically minded, what the Fourier transformation actually does is take a function and express it as the sum of a set of different sine and cosine waves. Apparently this can be done for any continuous function. By using this transformation you can get not only the amplitude, but also the phase of any given molecule inside an atom. You can also make the equation go backwards as well, turning the red fuzzy blob on the left back into the individual point. As most molecules contain many, many atoms there are various tricks you have to do in order to make this easier for large molecules, but first a quick proof of this concept using my favourite pictures on the Cowtan website.

Here is a picture of a duck:
And here is the Fourier transformation of the duck, with the amplitude represented by the brightness of the colour and the phase represented by the actual colour:
In order to show that the Fourier transformation really does show the phase, this transformation is mixed with the Fourier transformation of a picture of a cat. Rather than mix them both equally, the amplitude (brightness) of the duck transformation is mixed with the phase (actual colour) of the cat to give the following Fourier transformation:Performing the Fourier equation on this blob (to turn it back into an animal again) gives the following result, hopefully not unsurprising if I've managed to explain this alright:
It's a cat! A rather blotchy cat, true, with a fairly trippy background, but nevertheless the Fourier transformation has faithfully reproduced the phases, rather than the amplitudes.

So how does this help when looking at molecules? It turns out that the fuzzy black-and-gray-dots picture that is the end result of X-ray crystallography (shown above and reproduced here on the right) is the Fourier transform of the atomic electron clouds inside the protein crystal. That picture is like the fuzzy coloured blobs that the duck and cat images came out of. In the same way that those turned into animals, this picture can be turned into the approximate shape of the electron clouds surrounding a molecule. For low resolution images this can show the secondary structure of a protein, the positions of alpha helices and beta sheets and a general idea of protein shape. For high resolution images, individual amino-acid residues can be seen, allowing a much more detailed view of the structure to be generated.

It isn't always perfect. Sometimes you do get the equivalent of a blobby cat with a trippy background and have to play around with homologous comparisons and allowed bond-angles to get a meaningful structure. There are plenty of strategies that exist to help you get a better image as well, particuarly for larger molecules which need more help resolving phases. From what I've heard though, once you've actually got the crystal, the rest seems like childs play in comparison. I know people who have spent their whole PhD's, and longer, just trying to isolate and concentrate a single protein crystal...

Mitochondrial Networks

Having taken a brief look at some plastids last week, I thought I should probably balance things out by writing about mitochondria; the energy generating centres of the (eukaryote) cell. Like plastids, mitochondria are thought to originate from endosymbiosed bacteria-like organisms and are often shown as looking something like the picture on the right. As well as creating energy in the form of ATP mitochondria are also involved in the B-oxidation of fatty acids (producing energy from fats), Iron-sulfur cluster generation, oxygen metabolism, cell death control and Calcium ion buffering and signalling.

Mitochondria are surrounded by two membranes, an inner (the Inner Mitochondria Membrane - IMM) and an outer (OMM). The inner membrane is the main barrier to the outside world and contains most of the energy-making apparatus either embedded in it or present in the inter-membrane space. The outer membrane is used to coordinate function with signalling, and plays a part in apoptosis, or organised cell death.

In the cell however, mitochondria are often not found in the neat little packages as shown above, but instead fuse together to form long cell-wide tubular arrangements. Usually, they can be found in both vesicle and tube like states, constantly fusing or breaking up depending on cell circumstances, the type of cell, or the functional state of the mitochondria. The result is shown below; a network of mitochondria within the cell:Two processes are involved in the creation and maintenance of this tubular network. Fusion; the joining of mitochondria, and fission; where they split apart.

Fusion: mitochondrial fusion is controlled by large GTP proteins, which use the hydrolysis of GTP to produce energy to join the two membranes together. OMM localised proteins Mfn1 and 2 carry out the initial tethering and joining of the OMM by forming coiled-coil type interactions of their C-terminals which acts to bring two mitochondria together. Once the outer membranes have fused, OPA1 (a soluble protein found in the inter-membrane space) fuses together to join the inner membranes. The fusion of both the outer and inner membrane is usually highly synchronous, although studies in yeast have shown the two fusions can be decoupled.

Fission: in contrast, fission is controlled not by GTPases, but by a protein related to dynamin called Drp1. Drp1 is usually found in the cytosol, but it can localise to the mitochondria in clumps which lead to active fission sites. The Drp1 forms polymers which wrap spirally around the mitochondrial tubule and lead to it splitting. It is thought to be recruited to specific sites by the mitochondrial-bound protein Fis1, although this still requires further study.

As aberrations in mitochondria dynamics are generally associated with neurodegenerative disorders, the control and organisation of these processes are vital. A variety of cofactors and inhibitors have been found that can regulate the Mfn proteins involved in fusion, and OPA1 is thought to be controlled by alternative proteolysis to create different isoforms. There are a number of different protein kinases that are able to phosphorylate the Drp1 fission protein, which not only allows a large degree of control, but means that this control can be synchronised with different intracellular signaling pathways, giving more integrated cellular control.

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Benard G, & Karbowski M (2009). Mitochondrial fusion and division: Regulation and role in cell viability. Seminars in cell & developmental biology, 20 (3), 365-74 PMID: 19530306