Tuesday, 29 April 2014

Haemoglobin: Molecule of the Month for May

Hb molecule with the porphyrin
rings  indicated  by red discs:
each ring contains an iron atom
Haemoglobin (or hemoglobin if you are in the USA, often abbreviated to Hb) is one of the most well characterised proteins in the canon of Biochemistry and Physiology. It was the first protein to have its Molecular Weight calculated (nearly 200 years ago) and its bright red colour is a result of the electronic state of the porphyrin ring (or haem) which is found as one mole per mole of Hb subunit: Hb comprises 4 polypeptide units, or subunits and is therefore called a tetramer. Our understanding of the molecular properties of Hb was perhaps driven by its importance in human physiology and, as you might imagine became of great interest during the first world war which coincided with the discovery of sodium citrate as an effective anti-clotting agent and an appreciation of the phenomenon of blood group matching.

Hb carries oxygen from the blood to the tissues and achieves this by an elegant an well understood molecular mechanism. The binding of oxygen to Hb was described by Hill and Adair around 50 years before the structure of Hb was determined by Max Perutz, one of the most successful scientists to come out of Austria, via  Cambridge, where he arrived just before the second world war. Perutz and John Kendrew made pioneering discoveries in the determination of protein structures, working on Hb and Myoglobin (a similar protein to Hb found in muscles) respectively. Perutz (left) shared the Nobel Prize with Kendrew in 1962, after a distressing period of confinement in Canada via Huyton! Perutz, like most Germans and Austrians were held in confinement during the early stages of the second world war and Huyton boasted one such internment camp.

The oxygen binding curve is probably the first encounter a Biochemistry or Physiology student has with a complex curve. The sigmoidal (or S-shaped) plot shown right illustrates that as the concentration of oxygen (pO2) is slowly increased a short phase of very rapid binding occurs, followed by a flat phase (or saturation). In other words Hb prefers to capture oxygen molecules over a small range of concentration, a range that is adapted to our own physiological characteristics. We refer to this process as cooperative, since it turns out that the binding of one molecule of oxygen makes it easier for the second, third and fourth to bind to each Hb subunit (remember Hb is made up of 4 subunits, above). You have seen this type of curve before in monitoring bacterial cell growth. Biology has a limited number of mathematical strategies in its evolutionary tool box, it would seem! If the binding of oxygen was additive and simple, the graph would be a straight line, which would probably flatten off as the binding sites are filled up.

Now consider this conundrum: if each Hb subunit is identical, then each Hb subunit should bind oxygen with the same affinity, but it clearly doesn't (from many observations). How then is this observed co-operativity achieved? I have told you that Hb is a tetramer of identical protein chains each containing a porphyrin ring, or haem molecule. How does differential binding take place? This is where Perutz and an army of scientists working in different fields across the world, came together to provide an explanation. In the absence of oxygen (deoxyHb), Hb molecules exist in subtly different forms. That is they can adopt slightly different 3D shapes. Hb has intrinsic flexibility, and when oxygen binds to one site, a shape change is transmitted through the molecule which increases the second oxygen binding site's affinity for oxygen. Then the third is even more likely to bind oxygen, until the fourth site is finally filled. It is a little like zipping up your jacket: it is slow at first, but once the zip is engaged it gets easier as you complete the process, until you reach the top. The ability of certain proteins to shape-shift (or as we say in Biochemistry, undergo a conformational transition) is one of the most important phenomena in Biology. It is part of our understanding of key molecular changes in cancer and some forms of dementia. You must appreciate however, that these various shapes exist in an equilibrium that is determined by the amino acid sequence of the protein, the conditions in vivo (or in vitro) and by the molecules that interact with Hb. We will return to these important phenomena later.   

Sunday, 27 April 2014

Our own model organism!

Thanks to a small award from the Royal Society, we are able to realise our ambition to make Tenebrio molitor, the humble meal worm, the first model organism for our Innovation labs. This is just a short post to whet your appetite, but we shall begin this week in the Y10 labs, to generate materials, begin to document the Biology, the Biochemistry and Molecular Biological characteristics of the insect at various stages of its life cycle. The Greenland Biodesign team have been maintaining our "crop" of larvae and as of last week we have taken delivery of our first three beetles, which I would like to call John, Paul and George: Ringo hasn't arrived yet, but then he was a latecomer to the Band! More on this as we get underway in the labs this coming week, when we make our first stab at curating the genome data already available for T.molitor.

More thoughts on interactions

Before I leave our discussion of fertilisation, here are a few more thoughts on molecular interactions. In particular think about the way proteins are drafted in to bring cells together, compared with the way in which small molecules interact with enzyme active sites. It is instructive to consider the interaction of an enzyme with its substrate. What is the normal concentration ratio between a metabolic enzyme (for example) and its substrate? It will of course vary, but I would expect it is often 1000:1 (substrate concentration to enzyme). Take a glycolytic enzyme such as hexokinase which activates six-carbon sugars for entree into the energy generating pathway of glycolysis. 

Hexokinase requires ATP (a common source of phosphate) and glucose to come together in the correct orientation at the active site of the enzyme. At this point there will be one molecule of each: enzyme, sugar and ATP. Recall that in the case of an interaction between two molecules (A and B) the on rate is largely determined (in Biology) by diffusion through an aqueous medium. Therefore, you might expect the correct spatial encounter between 3 molecules to be less frequent than between 2. What I want to point out is that the next stage, in which one or more chemical steps take place, can sometimes be broken down into stages (one of the ways we think enzymes achieve catalysis). The total conversion (in this case) of the two substrates into glucose-6-phosphate and ADP, may not be achieved after every three way encounter. In other words productive interaction may not be 100% efficient (in fact we know that so called abortive complexes form at the active sites of many enzymes). This is not dissimilar to the case of sperm egg recognition. 

We should consider molecular encounters as a series of phased processes that sometimes lead to a productive outcome: a sugar phosphate in the case of hexokinase, or a fertilised egg in the case of Reproductive Biology. But sometimes the pathway is aborted or diverted and in some cases enzymes can give rise to alternative outcomes. The evolutionary pressures that are brought to bear on molecular interactions are therefore not just simply those that ensure specificity and affinity of encounter: sometimes downstream consequences form part of the story. I shall return to this when we consider the landmark experiments on the proteases and the enzymes that ensure the correct amino acids are added to growing protein chains: the aminoacyl tRNA synthetases, which have informed a great deal of our thinking on enzyme mechanism. But for now, think about thermodynamics in the context of evolution when rationalizing new discoveries, such as sperm egg recognition.

Thursday, 24 April 2014

Binding, specificity and molecular interactions: lessons from reproductive biology

My previous post on the sperm egg recognition molecules gave me some initial concerns. As a Biochemist brought up to believe that specificity of interactions goes hand in hand with high affinities (usually reflected in dissociation constants in the nano-molar range... which needs some explanation!). 

We measure the interaction between two molecules (such as two proteins, or a small molecule [a metabolite, a drug or an enzyme substrate]) using the simple relationship below, where A and B are two interacting molecules and the rate constant kon defines the binding or association step  and the rate of dissociation of the AB complex is given by koff

This is an equilibrium which is simply defined by an equilibrium constant under the experimental, or physiological conditions.The equilibrium constant (Ka) is directly related to the proportion of free A and B versus complexed AB and is given by the ratio of on and off rates:

Biochemists, often use the dissociation constant (Kd) which is the reciprocal of the equilibrium constant, and has units of concentration. Importantly the units of kon are M-1.sec-1 and koff units are sec-1. This means that the Ka is in units of M-1 and therefore Kd is expressed in molar terms and is used because it is possible to relate its significance to a concentration term. So we talk of Kds as being "in the nanomolar range" or "in the micromolar range". As a rule of thumb I think of protein:protein, or protein DNA interactions as nM; substrate interactions as mM; and cofactor interactions with enzymes as μM. These concentration units are also a reasonably good approximation to the physiological concentrations of proteins, substrates and cofactors respectively. So, the lower the (concentration) units of the dissociation constant, the higher the affinity. I shall defer a discussion here of the forces that underpin the interactions (hydrophobic, ionic, H-bonds etc.) until a later Blog.

The on rate of encounter
The association rate constant (kon) is largely determined by the rate of diffusion of two molecules (eg an enzyme and its substrate) and will therefore have units that express the rate of encounter of two molecules depending on their concentration. This is referred to as a bimolecular rate constant and has typical units of M-1.s-1. The off rate will be defined as per unit time (sec-1). What are the practical consequences? Well, an interaction characterised by a nanomolar Kd, will result from an encounter (or collision) that is generally the same for most interactions (weak or strong), but once contact is made, it will be very difficult to dissociate. Think of the fly landing on a Venus fly trap (left), where access to the interior is simple (on rate is diffusion limited), but once inside, the doors are shut (right): the off rate is very slow. The encounter between two molecules
A slow off rate!
with perfect fit (see the lock and key above) has been promoted for over 100 years (by the second Nobel laureate in Chemistry (Emil Fischer, who incidentally would have shared a platform in Stockholm with Ronald Ross from Liverpool, in 1902). We now see this as a simplification, since the intrinsic flexibility in molecular interactions is better described by a "hand in glove" phenomenon. There are occasions when the on rate is unusually low, in which case there is a barrier to interaction which must first be overcome before productive binding takes place, but the most common situation is a modified Venus fly trap where binding triggers a shape change which secures the ligand in the active site (or binding pocket).

Returning to Juno and Izumo! Why do some critical cellular interactions involve weak binding events? We must now move beyond the simple model I have described above, to consider the reproductive system. Male ejaculation releases on average 250 000 000 sperm, per egg. It is also know in humans that following a single productive encounter, no further sperm interactions lead to fusion. By comparison, consider the interaction between a bacterial repressor protein and its DNA target sequence: here encounter will block gene transcription, but eventually the repressor is likely to be released, often in response to ligand binding and the gene is expressed. Such systems are also intrinsically leaky and there may always be a level of transcription allowed to break through. However, in fertilisation the sperm egg interaction sets in motion an irreversible process and breakthrough is not acceptable!. Maybe we need to make sure this productive binding occurs therefore at a low frequency, but once it has occurred, the egg must prevent a second interaction. Weak binding can work when there is a massive excess of ligand (the sperm, in this case). But why not make a smaller number
of sperm and utilise high affinity recognition? Clearly this is not favoured in evolution. I want you to think about a system that has evolved to incorporate massive redundancy: 25m sperm, when just one would do! And then begin to calculate the dissociation constant that is likely to represent a system in which one egg (B) encounters 250 000 000 molecules of A to create an AB (fertilised egg) complex. You should assume 1000 sperm can bind to a single egg, but only one sperm triggers fusion. Think about the calculations and the problems in experimental challenges in detecting these interactions. I am glad I went back to basics and tried to appreciate the significance of this landmark discovery. I will discuss productive and non-productive binding in the context of enzymes later, but sperm egg recognition has made me think harder about specificity and binding phenomena in Biology.

Thursday, 17 April 2014

The birds and the bees: Juno and Izumo

The recent identification of the two molecules that trigger fertilisation in mice is a key moment in Biology. Sexual reproduction is thought to be over 1bn years old and is the cornerstone of genetic variation, since asexual reproduction provides a limited basis for generating genetic diversity. (In simple terms, the gene pool is much bigger if you reproduce by sexual means). The search for these molecules has been going on for some time and it worth thinking for a moment about the problem. The molecules (if indeed there are just two) will be different in all species, since crossing the species barrier is an important aspect of sexual reproduction. (However, they may show similarities which define their overall biochemical characteristics). So are model organism studies valid and is the mouse an adequate model organism? Then then there is the ethical issue of obtaining sperm and eggs from human donors in order to isolate and characterise the molecules in humans. Is this acceptable? Is it even possible from a technological standpoint?

The Izumo1 protein from mouse sperm was identified in 2005 by a Japanese team from Osaka. The name refers to a wedding shrine in Japanese, and is essential for sperm egg interaction in both mice and humans. I immediately used BLAST to look at the sequence identity between Murine (mouse) and human versions of both  Izumo1 and Juno. Juno, as some of you may know is the Roman goddess of fertility and marriage. The Juno proteins from mice and man are just under 70% identical (although this isn't definitive since the folr4 gene [the previous name forJuno] has isoforms and I haven't read the papers in detail yet) and the Izumo proteins are around 50% identical (the same caveat applies here). So my first though is that the core functions are similar and there is sufficient difference to block cross fertilization. So far so good. 

How did the group at the Sanger Institute (see the interview on you tube) identify Juno? Gavin Wright's group at the Sanger have been developing an elegant method for the identification of weak affinity receptor ligand interactions. This may seem a contradiction: why would a highly specific interaction be weak and often transient? Most previous methods have assumed that such key interactions are strong and stable. I shall return to this issue later. The method also recognises that cell surface molecules are amongst the most challenging from a biochemical isolation perspective and so libraries of expressed molecules on cells are screened by a technique called AVEXIS. The extracellular component (ectodomain) of a particular protein is coupled to a plastic dish and cells are passed over the immobilized protein until they bind. These interactions can then be identified (for details see the Wright lab link). 

How do we know that the Wright lab are correct? The key pieces of data are the ability of specific antibodies targeted at Izumo1 and Juno to block sperm and egg interactions. However, a key finding is that around 30 minutes after the interaction of I and J, J has disappeared, which is consistent with the observation that one fertilisation event blocks all subsequent sperm binding interactions. This explains the block to polyspermy and opens the doors to an investigation of the mechanism behind the loss of J after 30 minutes. This discovery and its relationship to this phenomenon in other species will open a new window on evolutionary biology and suggests some important avenues for the Yin and Yang of childbirth: infertility and contraception.

Tuesday, 15 April 2014

The privilege and joy of Science

Today I had the enormous pleasure of watching a group of UTC Y12 students attend their first scientific meeting. The event was the Liverpool School of Tropical Medicine's annual postgraduate symposium, held in one of Liverpool's many famous iconic buildings: the Adelphi Hotel. The day was organised by Professor Richard Pleass and Eleanor Carr, who had kindly incorporated a poster competition for the UTC students. The meeting is unusual in the breadth of material covered in the seminars; all of which are presented by postgraduate students who are well into their projects. Much of the material was of a high level, nevertheless, the students all showed great patience and tenacity in concentrating through the day's talks and, after a number of discussions with me and LSTM scientists,to clarify points, they were able to hold thoughtful discussions and begin to frame questions, showing considerable scientific maturity. I was incredibly impressed, as were the academics at the symposium. 

When it came to assessing posters, the students were asked to make individual choices and then to reach a consensus. They approached the challenge, not only enthusiastically, but with professionalism and in a highly logical way. There was a majority for one poster, with another coming close second. They prioritised the clarity and accessibility of their choice, after working their way around the 30 posters on display. They engaged with the postgraduates in discussing their work and really entered the spirit of the day as if they were postgraduates themselves. They ended the day by composing a joint "tweet" and we selected a winner that best described the reason for their poster choice from Rachel Winrow, which included Einstein's quote on communicating your results:  

"If you can't explain it simply, you don't understand it well enough."

Monday, 7 April 2014

Hungary for Biochemistry? [Part 1]

How do we begin to analyse the characteristics of Science in any country, and in this case Hungary? I should make it clear that I am speaking from my own perspective and experience. However, I believe it is important to appreciate the International nature of Science and scientists, including historical and political perspectives. For the purposes of this Blog I will not consider contributions made before 1900 (although I shall return to this important point later in the future). One convenient measure of achievement or impact of a country, is to look at the number of Nobel Prizes awarded on the basis of nationality. This is convenient because this award is recognised as a universal indicator, although there is the caveat that the Nobel Prize has certain restrictive criteria that impose some limitations on the validity of this as a sole source. Another measure might be the total number of peer reviewed publications and patents that appear in the scientific literature each year. And finally, how much money does a country spend annually on Science (which must be considered when comparing value for money of a particular country). It is also important to acknowledge cultural, political and economic factors that can all influence the productivity of a given nation. It would of course be unrealistic (and arrogant) for me to provide an authoritative account of Hungarian Science without devoting appropriate time on investigating such sources. However, I think it is possible to obtain a good level of insight by speaking with individuals who have lived and worked in that country and by extracting data from reliable sources such as NCBI, the Economist etc. and the above graphic is taken form an edition of Nature devoted to metrics. This is therefore a taster of Science in Hungary and it is hoped it will stimulate a few of you to find out more!

Hungary is a land-locked Central European country that shares borders with the historically important nations of Austria, Serbia Croatia, Romania, Ukraine, Slovakia and Slovenia. It has a population of around 10 million (the UK is 63m) and covers an area that is about half of the United Kingdom: it is a member of the European Union. Politically, Hungary was subsumed by the Soviet Union until the collapse of soviet communism at the end of the 1980s. Currently, Hungary is ruled by the conservative Fidesz party and it has been a member of the European Union for around 10 years. The capital city is Budapest and we will be looking at an academic group for Szeged, which lies south of Budapest and is famous for its paprika, which is a key ingredient in Hungarian goulash! Szeged is Hungary's third largest city, but Szeged is also home to the most prestigious University in Hungary.

Hungary has had no fewer than 9 Nobel Laureates, which represents around 9 per 10m population: compare this with 10 per 10m in the USA (the world's most successful nation in terms of total number of Nobel Prizes) and 19 per 10m for the UK. Hungary has a strong tradition of mathematics; you may have heard of Rubik's cube, but also the elaboration of the foundations of non-Euclidian geometry. Personally, when I think of Hungarian Science, I always think of Albert Szent-Gyorgyi (shown right), famous for his work on vitamin C and metabolism in general, who was awarded the sole recipient of the Nobel Prize for Physiology or Medicine in 1937. So far this has been an historical view of Hungarian Science and we shall hear from Dr. Antal Kiss in part two of this Blog. 

Biotechnology forms an important element in the future economic development of Hungary, which is itself built on strong teaching and research traditions in areas like Mathematics and Biochemistry. The work in Antal Kiss's laboratory (that's Antal, left) has been of interest to me for some time, since his group's early involvement in the Biochemistry of DNA restriction and modification enzymes. Publications from Antal's group are characterised by their attention to detail, impressive creativity and the care taken over the experimental data. I have enormous respect for the work from Antal's group, but I also have fond memories of the hospitality and kindness shown to me and my colleagues when we visited his institute as part of a European Research collaboration network. It is these experiences and the intellect, commitment and humanity of Antal and his colleagues that makes science so rewarding. In the next Blog, Antal will tell his own story, I hope this whets your appetite.

Friday, 4 April 2014

Primers, templates and another class act!

Our first attempt at classroom Synthetic Biology is on track as we completed the first stage of our attempt to develop a controlled system for peptide synthesis using Synechocystis genomic data and the help of Sarah, a graduate student in Professor Neil Hunter's research group at the University of Sheffield. The inspiration for the project grew out of discussions with scientists at Croda, one of our commercial partners. Last week, we worked through the first principles of PCR and following this success, we set out to develop a robust method for genomic DNA extraction to generate a supply of PCR template. The class demonstrated that addition of low concentrations of SDS dramatically improved DNA yield prior to boiling, but that these high yields did impact on successful PCR. (We shall revisit the DNA extraction methodology later in the project, since we wanted to produce the most efficient way of going from cells to PCR product). As you can see (top left), amplification of a control Synechocystis gene,was a success for the majority of the class (80 students on 20 groups: Y12s will be able to access their own data via Edmodo). One of the most important aspects of this experiment is the value of a group approach to solving an experimental problem. I will discuss this in a separate Blog, but suffice to say, Sarah had warned us that PCR success with Synechocystis is more sensitive to enzyme buffer and conditions than most colony PCRs; so, I am even more delighted by the results!

The aim of the class today was two-fold. The first was to establish the methodology for the second half of the project: amplifying target Synechocsytis genes and cloning them. But the other aim was to teach everyone how to design PCR primers. Last week, I made my first attempt to explain the logic behind primer design at the end of the day in the lab, but 15 minutes in, with lots of blank expressions, I thought, this needs thinking through more carefully. I was also wrestling with explaining the concept of pi to the younger class (more on this later), and how to move away from from the widely held idea that p is the name of a button on a calculator! I took a small group aside and asked them to design two primers for a short duplex. Those of you who teach this will know that virtually everyone gets the orientation wrong first time. So this week, I went through the background that I felt that was necessary: DNA base pair complementarity rules, the linkage of 5' and 3' to the orientation of the sugar phosphate backbone, the nomenclature behind the numbering of atomic centres in organic ring structures, the sensitivity of the active site of DNA Polymerases to strand orientation etc. Then asked them to have a go. Yes everyone got it wrong the first time. Then, after explaining why they had got it wrong....60% got it right the second time! By the end of the session, everyone I spoke to in the group (60%) had got it. 

So what did I learn from this session. Asking a large lab class to develop a method (in this case for DNA extraction) to meet a set of criteria is a great way to speed up scientific discovery. That concepts like primer design can provide an opportunity to teach pattern recognition, chemical nomenclature and elements of active site chemistry as well as molecular genetics. In summary, synthetic biology is proving to be an excellent vehicle for teaching science in schools and I hope to report on the next stage of the project in about six weeks time.

Tuesday, 1 April 2014

The fruits of evolution: viruses and organelles?

The structures of macromolecules, their assembly into larger (supra-molecular) assemblies is part of a continuum in Nature that extends through to viruses, organelles and cells. And much of the underlying architectural features are found again in the plant kingdom, on a much larger scale. So when I was thinking about a graphic to explain phage infection of bacteria (or viral infection of mammalian cells), the pomegranate came to mind. Not only is it packed in a manner that is reminiscent of phage particles, it is also possible to use simple volume analysis to appreciate the upper limits on phage infection and the need for the cell to accommodate both replicating and assembling particles as well as its own genome, transcriptome and proteome: not forgetting intracellular membranes in some cases. 

On the right, is an example of a densely packed pomegranate with the casing removed. When looking through some of the original bacteriophage literature, it seems that the maximum occupancy of the host by phage particles is approximately 25%. Which is not surprising since the host has other things it needs to accommodate even when it is invaded.

Encouraged by the similarities and the ease with which students can begin to appreciate the mathematical and physical principles that underlie the size limitations (proportionality of spherical volume to internal contents and an appreciation of the structural aspects of biopolymers) and the stability of such structures. Not forgetting the imposition of structural organisation on the evolution of biosynthetic strategies: an important aspect of developmental biology and our appreciation of it in evolutionary terms. The maize husk, corn on the cob is another excellent example of a regular structure that is reprised in the rabies virus (compare left and right images).

TMV showing the helical
arrangement of subunits
 In contrast, the helical pitch of the subunits that form the outer casing of Tobacco Mosaic Virus illustrates that assembly pathways can influence the arrangement of subunits in a way that is distinct from the maize arrangement of its kernels. It is clear that evolution has settled (as usual) on several ways of packaging material either for protection (as in the viral capsid and nucleic acids) or for access (in the case of maize). Moreover, in contemplating assembly, the TMV arrangement has more in common with a certain Swedish domestic furnishing organisation, than a classical sculptor! Although the occurrence of natural helical growth is observed in some climbing plants (above right)?

Those of you over 50, or interested in space exploration, will recall the LEM (Lunar Excursion Module) which was delivered to the moon in 1969 via Apollo 11. For many of us currently active in Science, this was surely a landmark event. The general features of the LEM are strikingly similar to those observed through many elegant Electron Microscopy studies of Bacteriophage T4, and more recently through the superb crystallographic work of Professor Michael Rossman's group at Purdue University. I think the comparisons speak for themselves!

Finally, the organelles that drive energy production and harnessing in Biology are the mitochondria and the chloroplast. These structures tend to be less packed internally than say the pomegranate model above. Instead, they are characterised by invaginations of membranes, which form the scaffold and medium for electron transfer proteins. I'll let you be the judge of my Passiflora edulis analogy?