Meera Senthilingam: Welcome to the Diamond Light Source Podcast with me, Meera Senthilingam. This month we’re probing down to the atomic scale as we get an insight in to the world of structural biology. We’ll be finding out just what structural biology is, the molecules it can enable us to see and investigate how visualising molecules such as proteins can find treatments for conditions like pre-eclampsia.
Robin Carrell: We’ve shown, specifically, the change in angiotensin into the more active oxidised form takes place in pre-eclampsia. It opens prospects for the treatment of, what has otherwise been, a very resistant condition, that is the high blood pressure of pregnancy that is the accompanying and prime feature of pre-eclampsia.
Meera: Cambridge University’s Robin Carroll will be discussing the role of the protein angiotensinogen in hypertension and pre-eclampsia and how knowing the structure of this protein could result in a whole new class of drugs against the conditions. We’ll also be hearing about the proteins involved in repairing our DNA and guiding the development of our Nervous System. So all that insight into the proteins that keep our bodies functioning plus the latest news and events from Diamond coming up in this November edition of the Diamond Light Source Podcast.
Meera: Investigating and visualising the structure of molecules such as proteins and DNA can provide a great insight in to just how our cells, and therefore our bodies, actually work and knowing just how we work can help us solve the problems that arise when something stops working, A knowledge that the field of Structural Biology is helping to provide, as
Gwyndaf Evans explains.
Gwyndaf Evans: So Structural Biology is really a subset of biochemistry, structural biology and biophysics that really aims to get a structural understanding, hopefully at the atomic level, of the most active molecules in cells and in the body in general and they’re principally proteins and nucleic acid, things like DNA that are responsible for the majority of functions going on in the body and indeed in any organism. The importance of understanding the structure is then to try relate that to how these molecules function then we can understand how mechanisms for diseases come about and we can think about designing drugs and curing disease through an understanding of that.
Meera: So the key here is to visualise and see the structure of these particular proteins and molecules. We are now at the
Microfocus Macromolecular Crystallography Beamline where you are the Principal Beamline Scientist; tell me a bit more about this beamline. It’s an interesting setup here, tell me how does this process actually work to visualise a protein structure?
Gwyndaf: Generally the method we deal with here is fundamentally crystallography, it’s x-ray diffraction from crystals. So it relies on scientists in the home labs producing crystals of the molecules that we are interested in looking at. What Diamond does, what Diamond Light Source does, is it produces these incredibly fine beams of x-rays that we can then use to sort of hit these crystals. What happens when an x-ray beam interacts with any sort of ordered lattice or a crystal is that it sort of generates a very intricate pattern of spots that we have to record. We measure those on large area detectors that we put behind the crystal and analysing those spots gives us the ability to reconstruct what those molecules look like at an atomic level hopefully.
Meera: Ok, so tell me a bit more about the setup here. We’re about 50 meters away from the actual synchrotron here in the Sample Area, there’s a series of metal boxes and we’re out by the final one where I imagine the beam actually comes out to hit the sample. The sample is actually placed on a sample pin which, looking at it, is miniscule, it’s less than a millimetre wide.
Gwyndaf: Yes, if we looked at this under a microscope, at the end of it, would be a tiny little nylon loop, a fifth or a tenth of a millimetre in size, and they have to use these loops to literally fish the crystals out of a small drop that has been used to sort of grow the crystals in. They’ll than immediately freeze those crystals in liquid nitrogen to very low temperatures which is necessary really for them to survive in the x-ray beam a little bit longer than they normally would. Then they’ll come with anything from maybe 10 or 20 up to several hundred crystals to the synchrotron. One by on they’ll be mounting those crystals using a robot, the samples are actually mounted on this rotation, this motor that simply rotates the crystal around as well, so the actual experiment that is going on here is something called a ‘rotation method’, where while the x-rays are hitting the crystal, we rotate it around and that allows us to measure really a complete sphere of data as we call it, so giving us a full 3D structure.
Meera: So you’ve got this sample set up here, the beam is hitting the sample and just about a foot away you’ve got a large, maybe half a metre squared box, which I’m assuming will pick up the scattered x-rays?
Gwyndaf: Yes, this is a solid state pixel array detector and it’s built up of many, many silicon sensor modules and there are loads of those put together to make this square-looking face, if you like. This thing works at very high speeds so what we can essentially do is rotate that crystal and just keep it rotating and this thing virtually records a movie of these x-ray spots that are being generated or being produced by the crystal and the x-rays. That data is then analysed off-line, so computer programmes will just run through that stuff and work out what the molecule looks like from there.
Meera: What are you looking for when you get a picture of a protein?
Gwyndaf: What we actually see if sort of a cloud representation of the electrons. We’re not really sort of seeing positions. We get something called an electron density map. Atoms are surrounded by clouds of electrons and it’s the x-rays that interact with those clouds of electrons that we’re measuring. So the final structure, the final module that we’re building up is a point representation of where atoms are within that structure. One of the biggest applications is now in the drug industry in terms of pharmaceuticals and drug discovery, but recently what synchrotrons and what these new sort of developments, these big establishments and fine beams have given us the ability to do is work on larger and larger and more complex structures.
Meera: What are the challenges now and what perhaps is this beamline designed to overcome in particular challenges?
Gwyndaf: The case for building this beamline literally came out of wanting to have a better understanding of particular classes of proteins; firstly Membrane Proteins, also viruses and lastly more generally, these large macromolecular complexes. The importance of Membrane Proteins as a class is that as the potential source of targets for drugs they represent virtually half of the interesting targets. Over the last sort of 50 or 60 years we’ve built up a library of protein structures which now contains getting on for 60,000 and possibly more, protein structures, but the structures of membrane proteins are massively underrepresented in that library. We only have, say, just over 200 membrane protein structures that we’ve solved. The way that structural biology is evolving now is by trying to build up a more comprehensive picture of how these smaller structures that we’ve solved over the last 50 years interact with one another and how they go together to form these larger structures and how then this complex picture of many, many proteins and complexes interact with one another and how they go to form the cell, basically a living cell.
Meera: Gwyndaf Evans, Principal Beamline Scientist on Diamond’s Microfocus Macromolecular Crystallography Beamline. Now, as Gwyndaf mentioned, more insight is needed into the structure and function of cell membrane proteins and one scientist working on a class of these involved in our neural development is
Yvonne Jones from the University of Oxford.
Yvonne: So for the last couple of years I’ve become particularly interested in the cell surface receptors that are responsible for allowing cells to be guided to the right positions in the body and having got there, stay there. So they’re really determining the balance between adhesion and mobility. And there are several families of these that are particularly well studied at the moment in the nervous system because of their roles in the development of the nervous system. In particular; in guiding the growing axons in nerves to their correct location during development. One of the big families that had turned out to be very important in this is made up of the semaphorins, the proteins that come in and trigger the activity of the receptors, and the receptors are called plexins. So particular flavours of semaphorins bind to particular flavours of plexins and in general the result of that binding is that a cell moves away from the direction that the semaphorin is coming from. So it’s a ‘repulsive’ signal.
Meera: And where do these semaphorins come from, how are they released in the first place to then come along and bind to the plexins and repel the cells?
Yvonne:They’re include in our genome and at appropriate times their expression is turned on and many of these semaphorins are actually still attached to cell surfaces but some are secreted. The ones so far that I’ve worked on are naturally cell-attached ones.
Meera: And your more recent work has been trying to look at the structures of these proteins, these receptors and these molecules.
Yvonne: I want to actually look at the atomic level details of how a semaphorin interacts with a plexin and what it is about that interaction that allows the plexins to know that it should be signalling.
Meera: How do you use Diamond, how do you use a synchrotron to visualise the interactions that are taking place?
Yvonne: So we’ve been using several of the macromolecular crystallography beamlines, the
MX beamlines, to see the detailed shape and surface characteristics of the plexin receptor and the semaphorin and to understand how the two things recognise each other and lock together. The exciting thing is that we now know that a semaphorin isn’t just one molecule, it goes around in pairs and that is actually very important to the way it functions. The plexin, on the other hand, is tending to be just by itself. Once the semaphorin comes in a binds it, it will actually serve to pull together 2 copies of plexin and in fact we think what’s happening is that the plexins actually start to cluster and it’s those large groupings of plexin that are needed to start the signal going.
Meera: So these pairs of semaphorins are what is attracting more than just a pair of plexins, so lots of plexins grouping together to ...
Yvonne: What we can see initially and what we can see at the crystal structure is a pair of plexins, but once we start looking at things back in the context of the cell surface, we, and others, suspect that actually they’re then beginning to form clusters, whole crowds, and it’s that that is important in sending the signal.
Meera: Knowing this about this interaction and just how it’s controlled, could this be perhaps used for any medical applications?
Yvonne: If we can understand how these systems work then there may turn out to be incidences where we want to go in and specifically influence that signalling. Our one example where we think it may be important to go in and stop the semaphorin/plexin interaction is where we want to be able to regenerate nerve growth where there has been damage, say, to the spinal chord. This isn’t the only system where it may be important to influence and we’re working on several such systems in the lab to understand how these very specific protein-receptor interactions could be potentially targeted with the design of new drugs that might then allow nerves more easily grow back to repair damage in things such as spinal cord injury.
Meera: Yvonne Jones, Professor of Protein and Crystallography at Oxford University.
Did you know that Diamond is so powerful, it produces more light than a hundred million suns?
Meera: You’re listening to the Diamond Light Source Podcast and this month we’re probing down to atomic scales to visualise the structure of our body’s most crucial molecules. And still to come; we discover the workings of a protein involved in hypertension and pre-eclampsia and find out how understanding the workings of this protein could provide a whole new class of drugs against the conditions. But before that, let’s join Sarah Bucknall from the Diamond’s Communications Team for a round up of the latest news and events from Diamond; starting with good news in the form of funding.
Sarah: Yes, last month we found out that
Diamond’s going to receive capital funding for its phase 3, which basically means that we’ll get 69 million pounds from the large facilities capital fund over the next 4 years. That means we’re able to build 10 additional beamlines by 2017 and that will bring the total to 32 and maximise Diamonds potential to support UK science and industry.
Meera: But phase 2 is currently in operation isn’t it as well?
Sarah - Yes, so work continues on the final 4 phase 2 beamlines and they’ll be ready by 2012.
Meera: So what’s phase 3 going to offer?
Sarah: Well after consultation with the scientific community, we’ve
identified the first few beamlines. There will be an innovative macromolecular crystallography beamline to enable structural determination at longer wavelengths and we want to improve imaging capabilities to enable imaging of materials at levels unprecedented for the UK. There’ll be more facilities that allow for the study of electronic structure of the most complex and intellectually challenging materials of the physical world and with phase 2 we also aim to increase high throughput access.
Meera: As well as looking into the future, Diamond has been around now since 2007 and you’ve reached quite an important milestone in terms of the paper that have been published as a result.
Sarah: Yes we’re really excited to reach our 1000 publications milestone. So we actually went into operation in July 2007 and in that time have been producing peer-reviewed journal and conference papers published by researchers using experimental data collected at Diamond, and by our own staff scientists and technicians.
Meera: So 1000 publications in just under 4 years, that’s quite impressive actually.
Sarah: Yeah, it’s really good, I mean we started with 7 beamlines and whilst we were producing papers we were adding on more beamlines, so we have 18 now and it’s with those 18 that we’ve been able to produce these papers. And the most recent is the first user paper from our latest beamline to go into operation; that’s
B18, the Core EXAFS beamline. Other highlights include Josep Sulo-Suso’s work on using infer-red light to identify cancer cells, there was also a paper about moving closer towards hydrogen fuelled cars by improving gas storage, and Parkinson’s research, engineering materials, it covers a lot.
Meera: Many of which we’ve covered in the Podcast before as well and these are all available online as well?
Sarah: Yes we have a
publication database on our website so anybody can just have a look at that and see the papers we’ve produced.
Meera: And sticking with the website, people can also go there to find out more news?
Sarah: Yes, we’ve just released the
Autumn 2010 edition of Diamond News, that’s our newsletter that we release twice a year. This issue covers; understanding nickel and how it bonds with its surrounding elements to aid extraction; there are updates to existing beamlines; stories on modifications to the storage ring for upcoming beamlines; and we had a really busy summer so the newsletter also gives a roundup of events and workshops over the last few months; and we cover a particular piece of research that led to a breakthrough in pre-eclampsia. Pre-eclampsia is a condition that leads to the death of 10 women and 1,000 babies in the UK every year. Researchers from the Universities of Cambridge and Nottingham recently published a paper in Nature which has identified the processes involved in the condition.
Meera: And one of those researchers was Robin Carrell who we’ll actually be hearing about later in the podcast. Well just to finish off Sarah, there must be another Diamond day coming up?
Sarah: Yes, our Inside Diamond open day areas popular as ever and the places are just getting snapped up so the next available date is Saturday 26
th March in 2011 and what people can do if they come along to Inside Diamond is they hear a talk about the facility and its different applications. They then get to go on a tour and actually look around and go inside the machine and meet some of our scientists and engineers, so it’s a really good day out. You do need to register, and you can
find out more online.
Meera: Thanks Sarah. Sarah Bucknall from Diamond’s Communications Team, who’ll be back in the next edition with more news from Diamond, but in the meantime visit
www.diamond.ac.uk for more news and updates and also to sign up for the March Inside Diamond Day before it books up. Now, moving back to the visual world of structural biology, we now focus on the repair of our DNA. Due to various factors such as UV, our DNA is constantly being damaged and organisms need to repair that damage on a regular basis in order to survive. There are many proteins involved in this repair, one of which is XPD, a protein that
Jim Naismith at the University of St Andrews has been looking into.
Jim Naismith: The protein that we studied is a protein called XPD and what that does is it’s a helicase and that helps you unwind the DNA around the damaged lesions and that’s one of the very first steps in repairing DNA.
Meera: If this protein for any reason becomes dysfunctional, what are the consequences?
Jim: In humans there are a number of diseases, the most famous, or infamous, is called Children of the Moon syndrome. These are children who have to be kept away from sunlight because they are unable to properly repair their DNA damage. What this means is that these children often develop cancers and fairly significant other illnesses. There’s also other phenotypes to do with hair. You get a brittle hair disease and you also have some cases of deformities during development.
Meera: So what are the malfunctions that happen in these proteins that result in them not working, what happens in these diseases?
Jim: Well, you’re unable to unwind the DNA and start the repair and what we were able to show is that the mutations for one disease cluster in one part of the protein and the other phenotype, the brittle hair disease, clusters in a different section of the protein.
Meera: How have you set about looking at the structure of this XPD protein?
Jim: Humans are quite similar to bugs called archaea. There are 3 trees of life if you like; so, bacteria tree of life; the eukaryotic tree of life, which is us, or we’re in that tree, and then there’s a 3rd branch called Archaea. Eukarya and Archaea are diverged later than split from bacteria, so what that means is that we’re actually quite similar in some ways to these archaea organisms and one of the easy in which we are similar is in DNA repair. Now the human enzyme is extraordinarily difficult to work on, it’s longer, bigger. The archaeal equivalent of XPD is much more tractable to biophysical methods and so we were able to clone that, purify it, crystallize it and determine the structure at the Diamond synchrotron. But the key residues that are mutated in the human disease can be mapped very easily on to the structure of the archaeal organism and in fact you can test those by biochemical methods to see if you can recapitulate defects in the protein.
Meera: So, having looked at the structure of this protein in the archaeal versions of the proteins, what have you found about the structure of this particular protein?
Jim: We’ve found that one set of mutations are clustered in what is called the ratchet. Helicases work by expending ATP and as they do so they ‘drag’ a duplex DNA over its space and break it up into separate strands. And so one set of new mutations exert their effect by working there and that’s actually at the interface between two parts of the protein. The other set of mutations are distant from that and what they will do is affect the ability of the helicase to recruit other proteins into the cluster because in all organisms, but especially in higher organisms, such a complicated thing as DNA repair often brings other proteins in and you get these multi-protein complexes and in fact the other set of mutations cluster exactly where you would predict other proteins would dock. Quite different biochemical phenotypes; one will activate helicase activity; and the other one the helicase activity is normal; but what it does is it stops that helicase to recruit other proteins.
Meera: So there are 2 factors really, so there is a 2-step process almost in this early stage of DNA repair?
Jim: Yes, and if you have the inability to unwind the DNA, you get the very severe illness, the cancer, the Children of The Moon. If you have the ability to unwind the DNA, but not to bring other proteins to the party, you get the other very severe, but not quite as severe, diseases like Brittle Hair. Brittle Hair, you think ‘Oh, I can just live with that’ but there are actually a lot of abnormalities to go with that, that are significant.
Meera: So what real insight has this structural insight really provided in terms of really understanding the diseases and what other insight could be achieved through this?
Jim: So what this structure enabled us to do is segregate why one set of mutations caused a particular disease and why another set caused an apparently totally different disease. So that wasn’t known, they couldn’t segregate them before but with the structure you can. Now the fact that the cause of one of the pathways only affects the recruitment of other proteins and leads, in some ways, to a more subtle, but as I emphasised earlier, a rather serious illness, this may help us disentangle or may help others disentangle the pathway involved that goes wrong in DNA repair in those people/ That type of pathway that goes wrong in DNA repair is often seen in cancer. So these mutations may allow, or may permit some guidance to see what other mechanisms are involved in DNA repair and possibly help in understanding cancer.
Meera: Jim Naismith, Professor of chemical biology at the University of St Andrews. Now, that’s almost it for this month, but let’s have one more bit of structural insight. This time into a protein that could help in the fight against hypertension and pre-eclampsia. Both of these conditions are unfortunately quite common, with 10 women and 1000 babies dying of pre-eclampsia in the UK every year. There are various factors involved in inducing hypertension; our heart rate; the volume of our blood; and also the constriction of our blood vessels, causing them to narrow and therefore increase our blood pressure. This latter cause is what
Robin Carrell from the University of Cambridge is looking into, and he told me more about the proteins that are behind this constriction.
Robin Carrell: A major factor is a peptide hormone called angiotensin which directly results in a tightening of vasoconstriction of small blood vessels. Angiotensin originates from a large protein that circulates in our blood called angiotensinogen. The hormone is released by an enzyme from the kidney called rennin, and the renin cleaves off the terminal part of this circulating protein angiotensinogen to give this very small peptide hormone, angiotensin.
Meera: You’ve been looking at the actual structure of this angiotensinogen protein to see what happens to it to release angiotensin.
Robin: Exactly, and there was new information, and in this case, 2 surprises that gave us an insight into processes relating to the control of blood pressure.
Meera: So I’m intrigued, what did you find, what were these surprises?
Robin: The first and very satisfying finding is that angiotensinogen is far from being a passive source of the hormone. It interacts in a very active way with the enzyme renin. The cleavage site that renin acts to release angiotensin is buried within the angiotensinogen molecule and we demonstrated this, and were also able to demonstrate the way that the interaction with renin results in the cleavage site becoming accessible, changing from the interior of the molecule to the exterior of the molecule. So we’re now seeing that angiotensinogen plays an active and positive part in what is the initiation of the main process of controlling blood pressure.
Meera: What actually happens to it for it to become exposed say, there’s also a second surprise!
Robin: The surprise to us and to all was the presence of a ‘hidden’ switch, a fine tuning mechanism that controls the activity of the interaction between renin and angiotensinogen that release the hormone and this switch is based on a disulphide bond; a bond between 2 sulphurs. Now in the case of angiotensinogen, with this disulphide bond they are liable, or susceptible, to being broken as the molecule changes from an oxidising to a reducing environment. In a reducing environment, the bridge between the 2 sulphurs is broken and the two parts of the molecule can move apart. In an oxidising environment, the 2 sulphurs are bonded and held together and they hold the molecule in a more active shape, that is they more readily release the hormone angiotensinogen, that controls blood pressure.
Meera: If the angiotensinogen is in an oxidised environment, does that mean it is therefore more exposed and therefore more likely to be cleaved by the renin and for the hormone to be released?
Robin: That is right, that the molecule exists in two forms and they switch from one to another as the protein moves from a reducing environment to and oxidising environment.
Meera: Why would taking a therapeutic approach using this early mechanism be better than current treatments for hypertension?
Robin: It needs to be emphasised that what we’re looking at is the initiating stage in the release of the hormone angiotensin. There is a second stage where the hormone is refined into the form that actually interacts with the arteries and this second stage is controlled by an enzyme for which there have now been designed a series of inhibitors, people may well know them as the ACE inhibitors, that are used very effectively to treat blood pressure. But the problem is, one problem is, that the use of ACE inhibitors is contraindicated, for the very best reasons, in pregnancy. And it is in pregnancy that we get one of the greatest challenges for hypertension for people in the prime years of their life. The condition of pre-eclampsia, which is so common as it affects 2-7% of all pregnancies, is still not well understood. So what we’ve done is add a facet of understanding because we’ve shown specifically the change of angiotensin to the more active, oxidised form takes place in pre-eclampsia. It opens prospects for what was otherwise has been a very resistant condition. That is, the high blood pressure of pregnancy that is the accompanying and prime feature of pre-eclampsia.
Meera: So I can imagine this has a great deal of benefits if it were to come into practice. So I guess, what actual stage is the research at now, how far away do you imagine we are from that being a therapeutic use?
Robin: The quick answer is that now we’re beginning to understand the mechanisms, it opens various approaches, theoretical and otherwise, to treatment and that could be many years ahead. But there’s been an intensive effort made to look at therapies for hypertension and now as we look at the various treatments that have been tried or being given in trials in early stages, there are some that we feel might be relevant to the basic process that’s occurring and we hope our findings will be an encouragement to these people and eventually pharmaceutical companies to follow this up.
Meera: Robin Carroll, Emeritus Professor of Haematology from the University of Cambridge. Now that’s it for this edition of the Diamond Podcast, but do join us again in the new year 2011 for more insight into the science taking place at Diamond. In the meantime though, if you have any questions about the research or science taking place at Diamond, the email address is
podcast@diamond.ac.uk, you can also listen to previous editions of this program online at
www.diamond.ac.uk/podcast or
www.nakedscientists.com/diamond. You can also subscribe to the Diamond podcast on iTunes. Thank you this month to Gwyndaf Evans, Yvonne Jones, Sarah Bucknall, Jim Naismith and Robin Carroll. But until the New Year, thank you for listening I’m Meera Senthilingam.
The Diamond podcast is brought to you by Diamond Light source and produced by the Nakedscientists.com. There’s more information on our website at www.diamond.ac.uk/podcast
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