Principles of Protein Structure

BCA Winter Meeting: Structures of Supramolecular Assemblies

2012-01-06T05:39:00.001-08:00

The British Crystallographic Association (BCA) is a national organisation set up to support all types of crystallography in the UK. It is affiliated to the International Union of Crystallography and has five special interest groups, one of which, the Biological Structures Group, is devoted to the study, by crystallography, of proteins and other biological macromolecules. One of the Biological Structure Group's main activities is its annual Winter Meeting, which conventionally takes place just before the Christmas break. This is always well attended by students and post-docs; the speakers, however, are generally senior scientists presenting some of their most exciting recent research in protein structure.

The 2011 BCA Winter Meeting was held at the Diamond Light Source, the country's national synchroton facility (which is located at Harwell, near Didcot in Oxfordshire. When it opened in 1997 it was the largest scientific facility to be built in the UK for over thirty years. Synchrotons like Diamond generate highly intense, monochromatic (single-wavelength) beams of electromagnetic radiation that are used for many types of scientific experiment, including X-rays for crystallography. The theory behind how X-ray diffraction is used to solve the structuresof proteins is not covered in PPS, but it is in both the courses that can be taken for the second year of the MSc: fairly briefly in the general Techniques for Structural Molecular Biology course and very extensively in a specialist course.

This meeting took "Structures of Supramolecular Assemblies" as its theme and featured nine distinguished speakers, discussing complex structures, biological mechanisms and protein-protein interactions. And the first speaker was none other than the head of the Department of Biological Sciences at Birkbeck, Professor Gabriel Waksman. His ground-breaking work on the structure and mechanism of fibres that form at the outer membrane of bacteria such as E. coli to attach to the host cells has been discussed previously on this blog (e.g. in June 2011) and so will not be described in more detail here.

Waksman's talk was followed by two more describing proteins and protein complexes that are embedded in the membranes of bacterial cells. The first of these was given by Colin Kleanthous from the University of York, who described proteins involved in signalling through porins, proteins that form pores in the outer membranes of Gram negative bacteria. Porins were the first transmembrane proteins to be discovered where the membrane-spanning region forms a beta barrel rather than a bundle of alpha helices. Very many alpha-helical membrane proteins pass signals into cells from their environment, with the signal arising when ligand binding triggers the receptor to either dimerise or make a subtle change in its structure (conformation). Colicins are protein antibiotics that are synthesised by bacteria, often to kill very similar bacteria that occupy the same ecological niche. They do this by "parasitizing" the porins through which nutrients enter the target bacteria, a particularly difficult task as porins act as filters that generally allow only "nutrient-sized" molecules (less than about 600Da) to enter cells. Kleanthous described the structures and mechanisms of the "outer membrane translocation" domains of several colicins bound to porins; his group is now trying to solve the structures of intact colicin-porin complexes using both crystallography and electron microscopy.

Leo Sazanov from the MRC Laboratory of Molecular Biology in Cambridge then described the structure of respiratory complex I, which is embedded in the bacterial inner membrane.. This is the first enzyme in the respiratory chain, and it is found in mitochondria as well as bacteria: the respiratory complex I in human mitochondria has been implicated in the pathology of Parkinson's disease. The bacterial enzyme complex is smaller and simpler than the human one and is often used as a model system. Sazanov and his group crystallised the complete complex from Thermus thermophilus, a "thermophilic" bacterium that can live in extremely hot conditions. Proteins from thermophilic bacteria are often more stable and easier to crystallise than their equivalents in other species. The complete structure was found to contain no fewer than 63 transmembrane helices, 14 in each of three similar subunits. Sazanov discovered that this protein's mechanism involves a quite substantial conformational change between its oxidised and reduced form; an analogy with coupling rods has led the protein to be described as the "steam engine of the cell". It featured, in similar terms on the front cover of the issue of Nature in which the structure was described (Efremov et al. (2010), Nature 465, 441-5).

Nature cover illustrating the structure of respiratory complex I

About 5% of all proteins in an "average" bacterium are synthesised in response to heat or other stress signals. Stresssosomes are large multi-protein complexes found in bacteria and that control this stress response through the release of another protein, known as the sigma factor. Rick Lewis from the University of Newcastle described how the overall structure of a stressosome from Bacillus subtilis has been solved by electron microscopy and structures of some of the individual components by crystallography. He is using these structures to explore the mechanism through which the stressosome senses the presence of stress conditions. Although several pieces of the stress response pathway still remain to be discovered, his group has shown how the system could respond to differences in levels of light and oxygen (the latter through the presence of a globin domain in one of the stressome proteins), how it could regulate the production of diguanylate cyclase, and how the system is re-set through the action of a serine/threonine phosphatase (the structure of which was solved at Diamond).

Helen Walden of Cancer Research UK described how her group's structural studies are shedding light on the mechanism of a DNA repair pathway that is damaged in Fanconi anaemia, a rare genetic disorder that causes, among other things, a greatly increased susceptibility to one form of leukaemia. This repair pathway, which fixes cross-links in DNA, is triggered by the single ubiquitinylation of a DNA repair protein. (This is the fusion of a small protein known as ubiquitin with a target protein.) The process is triggered when cross-links cause DNA to stop replicating; the first step is the assembly, in the nucleus, of eight proteins into a "core complex" in the nucleus. The core complex then activates another protein, known as FANCL, and this catalyses the fusion of ubiquitin with the DNA repair protein, activating it. The structure of FANCL was recently solved in Walden's group by Ambrose Cole, who is now a post-doc at Birkbeck (PDB 3ZQS). Interestingly, the structure of this protein is not the beta-propellor that was predicted by sequence analysis; instead, it contains two domains similar to the ubiquitin conjugating enzyme UBC. Mutations that abolish ubiquitin binding are known to cause disease.

These are only a few highlights of a fascinating day's science. Other, no less interesting, structures presented there included several viral proteins: the HIV integrase bound to some of its inhibitors, presented by Peter Cherepanov (Imperial College London); the nucleoprotein from the virus that causes Lassa fever, described by Chang-jing Dong (University of St. Andrew's); and the NS1 protein from the influenza virus, described by Phil Kerry, also from St. Andrew's

Structural Secrets of an Ancient Viral Plague

2011-11-29T10:14:00.001-08:00

Research in Biological Sciences at Birkbeck, and several related departments at neighbouring University College London, is combined into the Institute of Structural Molecular Biology. The Institute holds a regular seminar programme - every Wednesday lunchtime during termtime - in which it invites excellent scientists, many with links to the colleges, to present their research. A few weeks ago, the seminar speaker was an electron microscopist, Sarah Butcher, who is based at the University of Helsinki in Finland. Her group has been investigating the structure of a virus that causes a very well-known disease: measles.

Measles has been known of for millennia. The disease (although of course not its cause) was first described in ancient Egypt. It is one of the most infectious viruses known, but people who encounter measles (if at all) as an unpleasant childhood affliction are often surprised to learn that it is a killer. About 164,000 people lost their lives as a result of measles infection in 2008, most from lingering immunosuppression rather than the acute infection. Most deaths occur in Africa and south Asia; a smaller epidemics have recently arisen in the UK when the MMR vaccination lost popularity over the MMR autism scare.

The measles virus is a paramyxovirus; an enveloped virus with a single strand of RNA as its genome, and closely related to the viruses that cause mumps, respiratory syncytial virus (RSV) infection and para-influenza in infants and children. It has two surface proteins and iis thought to attach directly to the membranes of the cells it infects via one of these.

Until recently, structural studies of the measles virus have been fairly limited. Many groups have studied it using an electron microscopy technique called negative staining, but that can only see the virus' surface. Structures of one intact measles virus protein and domains of three others have been deposited in the Protein Data Bank; the haemagglutinin (e.g. PDB code 2RKC); two separate domains of the phosphoprotein (1OKS and 2K9D) and a structure of fragments of two proteins simply called P and N bound together (1T60).

Sarah Butcher and her group used a technique called cryo-electron microscopy, which allows the interior of viruses to be visualised, to study the measles virus. Their results led them to focus on the matrix protein, which is thought to be important for the assemby of the virus (the protein coloured cyan in the images below). All previous models had placed the matrix protein covering the inner part of the viral membrane. What the Butcher group saw, however, was completely different. They could see a protein surrounding parts of nucleocapsid - the viral RNA and its associated, protective protein - and further analysis identified this as the matrix protein. The matrix binds tightly to parts of the nucleocapsid to make rod-like structures, and these fold into anti-parallel units that are somewhat remniscent of antiparallel beta sheets in proteins. This model suggests that the process of virus replication will be more complex and yield more potential drug targets than has previously been thought.


Two models for the organisation of proteins and RNA in the measles virus. Top: the old model, with the matrix protein (cyan) surrounding the virus coat. Bottom: the Butcher group model, with the matrix protein surrounding parts of the nucleocapsid. Figure credit: Proc. Nat. Acad. Sci. USA (2011)

Structures of proteins from other viruses, particularly HIV and influenza, will be covered quite extensively later in the PPS course. We don't study the technique used in this study, cryo-electron microscopy, in PPS but it is covered in one of the options for the second year of the PPS course, Techniques in Structural Molecular Biolog

Welcome to new students!

2011-10-03T08:20:00.000-07:00

A warm welcome to the Principles of Protein Structure blog to all students who have just started studying Birkbeck's Principles of Protein Structure course!

I run this blog to link the material that you will be studying in the course to new research developments in the areas of protein structure and function and related aspects of biotechnology and medicine. Throughout the taught course (but more often in the later part of the course) I will post reports of recent developments. I might, example, report on talks given in the ISMB seminar series run jointly by the Department of Biological Sciences at Birkbeck and research departments in neighbouring University College London. The overall title of the programme for Autumn 2011 is "“Proteins ofthe Future: Mechanism, Evolution and Design” which is closely connected to the content of the PPS course. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas.

Do, if you get a chance, look through some of the earlier posts on the blog to see the kind of topics that we will be discussing. However, don't be discouraged if at this stage of the course you find the science presented there difficult to understand. I can assure you that it will get easier!

And the best of luck for the 2011-12 PPS course and for your studies at Birkbeck! We hope that many of you will go on to complete our MSc in Structural Molecular Biology.

Best wishes,

Dr Clare Sansom

Senior Associate Lecturer, Biological Sciences, Birkbeck and Tutor, Principles of Protein Structure

Waking Up to Structural Biology

2011-07-05T05:26:00.000-07:00

Professor Nicholas Keep (known to generations of Birkbeck students as Nick) was appointed to a chair in biomolecular structure in 2009. It was June 2011, however, before he gave his inaugural lecture at the college. In this lecture he gave an overview of the techniques he uses as an experimental structural biologist and some of the discoveries he has made through them.

Nick’s career so far has been a glittering one. He took both his degrees at Cambridge and did postdoctoral work at UCL and back in Cambridge at the prestigious Laboratory of Molecular Biology (LMB) before being appointed as a lecturer at Birkbeck in the mid-90s. Since then he has risen steadily through the ranks and is now not only Professor but Executive Dean, with academic and financial oversight of the whole Faculty of Science. It is perhaps not surprising that it took him two years to fit in his inaugural lecture.

He began his lecture with a whistle-stop tour of the history of structural biology, beginning at Birkbeck and with the first Professor of Crystallography here, J.D. Bernal (see June 2008 post). Before he even arrived in London, however, Bernal had published (with Dorothy Hodgkin) the first ever diffraction pattern to be obtained from a protein crystal. Very much later, Nick as an undergraduate Cambridge student was inspired by two Nobel laureates, Max Perutz (who published the first-ever three-dimensional protein structure, that of myoglobin, in 1958) and Tim Hunt , to specialise in this aspect of molecular science. His PhD research was on the structure of methylmalonyl-coA mutase , an enzyme that binds to vitamin B12. This provides another link back to Dorothy Hodgkin, whose Nobel Prize in 1964 was awarded partly for determining the structure of this important vitamin.

He then spent a few minutes going through the essential principles of protein crystallography. This involves purifying and crystallising a protein and then exposing the crystals to a parallel beam of X-rays. These X-rays are deflected from the atoms in the crystal in a regular way to produce a diffraction pattern, and this can be interpreted to give first a map showing the density of electrons in the molecule, and then a model of the positions in space of all atoms in the molecule (missing out, in most cases, the lightest atom, hydrogen). In an ideal case, this whole process can now take a month or so, but this is rare: many structures still take years to solve. Birkbeck now has excellent facilities for purifying proteins and growing crystals. We can collect the X-ray data on site but often go to more powerful machines – synchrotrons – located in the UK and beyond. Britain’s first synchrotron is still in use, but in Jordan; it was replaced about five years ago by a state-of-the-art facility, Diamond , near Harwell in Oxfordshire. Birkbeck’s scientists also use synchrotrons elsewhere in Europe; Nick’s favourite is in Grenoble.

Nick then went on to describe some of the research projects that he has led or contributed to at Birkbeck. Working with Lin Field and Jing-Jiang Zhou at Rothamsted Research in Hertfordshire, he solved the structure of an insect protein that binds odorant molecules, which has led to some useful insights into the mechanism behind insects’ extremely sensitive sense of smell. Much of his work, however, is and has been in proteins that are involved in one way or another with human disease. Duchenne Muscular Dystrophy is a progressive, muscle-wasting genetic disorder that almost always affects boys and that is caused by mutations in a large muscle protein called dystrophin. Nick and his colleagues have solved the structures of a part of this protein that binds to another muscle protein, actin, and of related proteins.

It was only when he reached some of his final examples that the pun in his title, “Waking up to Structural Biology”, became clear. The bacterium Mycobacterium tuberculosis (see also April 2011 post ) infects the lungs of about a third of the world’s population. In most people, however, it remains in a wholly benign, dormant condition. In about 5-10% of cases, however, the dormant bacteria will “wake up” when an infected individual is under stress (for example, by exposure to another infection) and overt tuberculosis (TB) develops. Bacteria in the dormant stage are untreatable by any current TB drugs. Nick and his group first studied the structure and function of a protein known as resuscitation promoting factor that is involved in this “waking up” process,. He observed similarities between its sequence and that of a very well-known protein, lysozyme , which breaks down bacterial cell walls, and later, when the structure was solved, it was seen to have a lysozyme-like fold. This led to his identification of a glutamate residue as key to this enzyme’s activity. He is now looking at other proteins involved in M. tuberculosis resuscitation including a small heat shock protein (a protein that helps keep other protein structures stable under stresses such as raised temperatures), Acr1. This is the most abundant protein in dormant TB. He ended with a glimpse of a new unpublished TB protein structure.

Nick concluded a fascinating lecture by thanking his lengthy list of co-authors and particularly his research group, stressing the collaborative nature of science, and Gabriel Waksman, head of the Department of Biological Sciences, closed the proceedings by praising his achievements in teaching and administration as well as research.

Both second-year options in the MSc Structural Molecular Biology programme are concerned with the techniques used to study the structures of biomolecules. Techniques in Structural Molecular Biology (TSMB) is a general course covering crystallography, NMR, electron microscopy and some of the molecular biology and bioinformatics techniques associated with them, whereas Protein Crystallography (PX) is, as its name implies, a more specialist course.

The Structural Biology of Pilus Biosynthesis: Or, How Bacteria Man the Pumps

2011-06-02T06:45:00.000-07:00

Science Week at Birkbeck College was celebrated for the second time in early May 2011 with presentations from each of the college’s three science departments. The two lectures from researchers in Biological Sciences were linked by the common theme of nano-machines in biology. Just as a car engine, for example, is built up from many interacting parts, so some proteins work together in large complexes to do particular jobs within cells. Professor Helen Saibil, whose ground-breaking work in electron microscopy has featured earlier in this blog (see November 2010 post) presented some of her research into chaperones, protein machines that carry out “quality control” work enabling other proteins to form into and stay in the precise three-dimensional shapes they need to function.

The other speaker from Biological Sciences was Professor Gabriel Waksman, a distinguished structural biologist who combines a very successful research career with running both Birkbeck’s Biological Sciences department and the Research Department of Structural and Molecular Biology at UCL. Professor Waksman’s work for many years has focused on the complex structures through which bacteria interact with the outside world. Pathogenic bacteria cause problems for their hosts only when they interact with them, by secreting toxic substances into their environment or attaching to host cells. Now, when bacteria are rapidly developing resistance to many traditional antibiotics and more antibiotics, particularly with novel mechanisms of action, are desperately needed, some of these mechanisms are at last becoming understood.

Gram negative bacteria, which have double cell walls, often carry hair-like fibres or filaments known as pili on their surfaces. Some bacteria use these to bind to receptors on the surfaces of host cells, a process that can trigger the host cell surrounding and engulfing the bacteria in infection. Different forms of bacteria even from the same species carry different pili that bind to different cell receptors: for example, some E. coli bind to and infect bladder cells causing cystitis, while others infect kidney cells causing pyelonephritis. E. coli can also use pili to bind to each other, forming colonies around the bladder that are particularly difficult to treat.

The type of E. coli that infects the kidney carries a large number of so-called P pili on its outer membrane. These consist of a relatively thick rod near the cell wall and a thin filamentous tip. The whole pilus is made up of thousands of similar protein subunits encoded by genes within the Pap gene cluster. Almost all are identical PapA subunits, and these form the rod: the tip consists of just a few homologous PapE subunits, tipped by the sensor, PapG, which recognises and binds to kidney cells.

Pilus subunits, like all proteins, are synthesised in the cytoplasm; they need to be transported to the outer membrane and polymerise to form the pilus structure, and this complex task is achieved by other Pap proteins. When a pilus subunit is synthesised it is first translocated across the inner membrane into the periplasm, where it will be immediately degraded unless it can bind to a PapD protein. This acts as a chaperone, moving the subunit to the outer membrane where it docks with the membrane-bound PapC. This latter, or “usher” protein, is the core of the pilus biosynthesis molecular machine, and PapC and PapD together give the process its name: the chaperone-usher pathway. PapC has a large central pore through which the intact pilus is secreted.

Over many years, Gabriel Waksman and his group have solved the structure of many of these Pap proteins, and they have now built up an accurate picture at atomic resolution of how the chaperone-usher pathway works. The first structure to be solved was that of a binary complex of one pilus subunit, PapK, bound to the chaperone PapD (PDB 1PDK). PapK – and, subsequently, each of the pilus subunits – was found to have an immunoglobulin type fold, but with one beta-strand missing. This structure can only be stabilised when another protein, either the chaperone or a second pilus subunit, completes this fold with a strand of its own. One by one, starting with the tip subunit PapG, chaperone-subunit complexes migrate to the PapC usher, where the chaperone strand is replaced by a strand from another subunit in what has been termed a “donor strand exchange” model of polymerisation. The pilus fibre therefore forms from a series of “typical” immuno-globulin-like subunits in which each subunit is completed with a single strand from the next nearest subunit. The pilus biogenesis process only stops when a PapH, or “terminator” subunit is incorporated.

The usher forms a wide pore in the outer membrane (PDB 2VQI) and acts both to synthesise and to secrete the pilus polymer. It is a long, multi-domain protein. Using X-ray crystallography, Waksman’s group first determined that the pore comprises a very large beta barrel derived from the central domain of the usher, with a small sub-domain embedded within this domain forming a “plug” that blocks the pore when it is not being used. The usher also has a short N-terminal domain that dangles down into the periplasm and grabs on to chaperone-subunit complexes. The function of the C-terminal domain, however, remained unknown until the group solved the structure of an intact usher-chaperone-subunit complex.

It was only when this intact structure was solved that Waksman’s group really began to understand the mechanism of this complex “molecular machine”. For this, the group used a homologous system in which the usher is a protein known as FimD, the chaperone is FimC and the bound subunit FimH. This structure was a “first” in several ways, not least because it was the first time that an intact, folded protein was observed inside the pore of another protein structure. In this structure, the C-terminal domain of the FimD usher was seen to bind to the chaperone-subunit complex. It appears that, once the N-terminal usher domain has grabbed on to a chaperone-subunit complex and moved it into the usher, that complex will move up the usher structure to the binding site on the C-terminal domain, freeing the N-terminal domain to capture the next subunit.

This work, the culmination of fifteen years’ study of this secretion system, has just appeared in Nature (published online ahead of print 1 June 2011). More importantly, however, this elegant piece of structural biology may be exploited in the war against bacterial infection. A drug that bound to the usher and prevented pilus biosynthesis – a “pilicide” – would not kill the bacteria, but it would prevent them from binding to their target cells and also from forming the antibiotic-resistant colonies that can remain in the urinary tract for years and that lead to persistent infection.

Structural Biology in the Fight against TB

2011-04-04T06:08:00.000-07:00

About a third of the world's population - more than two billion people - are believed to be infected with Mycobacterium tuberculosis, the bacterium that, as its name implies, causes tuberculosis (TB). In most people the infection remains latent, but about 10% of cases develop into causes almost two million deaths a year. Strains of extensively drug-resistant TB (XDR-TB), which are resistant to two of the most effective first-line drugs and to at last three 0f the second-line drugs used against TB, have been found in many countries.

The Stop TB Partnership marks March 24 each year as World TB Day. 2011 is the second year of a two-year campaign to inspire innovation into TB research and care, On the move against tuberculosis. On March 24 2011 the Department of Biological Science at Birkbeck held an afternoon symposium featuring some of the department's tuberculosis research. This was organised by Dr Sanjib Bhakta, head of the ISMB Mycobacteria Research Laboratory and a senior lecturer in the department. Dr. Bhakta's research focuses on the discovery and validation of novel drug targets within the Mycobacterium tuberculosis proteome (link is to the TB proteome page in section 5 of PPS). Structural biology forms a crucial part of this work.

Birkbeck's Stop TB Day research symposium started with a keynote lecture given jointly by Dr Bhakta and Professor Edith Sim of Kingston University and the University of Oxford. Professor Sim is a member of the core group of TBD-UK, an organisation of UK researchers involved in the discovery and development of novel drugs for tuberculosis. After an introduction by Dr Bhakta, she described research in her group into the characterisation of a group of proteins that are necessary for the survival of the M. tuberculosis bacterium within cells. The enzyme NAT metabolizes and inactivates isoniazid, which is one of the first-line drugs used against TB. Researchers in Sim's group have developed inhibitors of this enzyme, some of which have been licensed to pharma company Eli Lilly for further development.

Sim's group is now focusing on a related family of proteins encoded by the Hsa genes which are involved in the metabolism of cholesterol and are also necessary for the bacterium to survive in macrophages. They have recently solved the structure of one of these enzymes, HsaD, which catalyses the cleavage of a carbon-carbon bond in one of the breakdown products of cholesterol. Structures of a mutant form of this enzyme alone (PDB code 2WUD) and with inhibitors (e.g. PDB code 2WUE) are yielding important insights into the mechanism of action of this enzyme. Both NAT and HsaD may prove useful targets for the design of anti-TB drugs that are likely to have novel mechanisms of action and that may therefore be active against resistant strains of the bacterium.

The keynote address was followed by some short talks by members of Dr Bhakta's research group at Birkbeck. Two of these, by Dimitrios Evangelopoulos and Dr Antima Gupta, described novel methods for testing drug susceptibility and for screening potential inhibitors respectively. Two others, however, focused again on the structural biology of potential drug targets. Dr Tulika Munshi described the Mur ligases, a family of proteins that are involved in synthesising the bacterium's complex cell wall. This cell wall is extremely rich in peptidoglycan; it is essential for the growth of Mycobacterium tuberculosis and has no homolog in the human proteome, both features that are important in a good drug target. Munshi and her colleagues have solved the structure of a member of this family, the ATP-dependent ligase MurE (PDB code 2XJA), in collaboration with Birkbeck structural biologist Professor Nicholas Keep (who is also the director of the MSc in Structural Molecular Biology) and identified amino acids that are essential for its activity. Another speaker, PhD student Juan David Gusman, described screening compounds recently isolated from Columbian plants as potential inhibitors of this enzyme. This work, published last year in the Journal of Antimicrobial Chemotherapy (link to PubMed) identified 3-methoxynordomesticine hydrochloride as a potential lead compound.

The scientific presentations were followed by a poster session and by an interesting panel discussion on some of the political issues involved in tackling this important public health issue. The take-home message from the day was that important steps are being taken - particularly in the academic and not-for-profit sectors - in elucidating the metabolism of this bacterium and developing badly needed treatments for the disease it causes and that Birkbeck researchers are playing an important part. If these treatments are to make it into clinical use, particularly in the developing world, however, political will as well as research insights will be needed.

Computational Biology - and Bioinformatics - at UCL

2011-02-24T05:44:00.000-08:00

People not uncommonly mix up the term "bioinformatics" with "computational biology", or use the two terms interchangeably. This is an easy mistake to make as both disciplines involve a computational approach to life sciences, and both terms have evolved over time. The consensus, however, is that there are significant differences: bioinformatics refers to the analysis of large quantities of (generally molecular) biological data, whereas all research fields that involve the development and use of computational approaches to them study of biological problems can be grouped into coomputational biology. Thus, although this is not precisely correct, it is more accurate to think of bioinformatics as a sub-division of computational biology than the other way round.

Birkbeck's much larger neighbour, University College London (UCL) has over twenty research groups working in areas that fall within the remit of computational biology, scattered across a number of departments and locations. Last week the college's whole computational biology community came together in a one-day symposium to make connections between these diverse research groups and to promote and celebrate the wide range of computational biology research that is carried out at UCL. This post gives a very brief overview of some of the work presented there, with links to the research groups involved.

The symposium was chaired by David Jones, director of the Bloomsbury Centre for Bioinformatics. which includes researchers from both UCL and Birkbeck. It started with a keynote lecture from Steve Oliver of the University of Cambridge, describing his group's ambitious project to understand and model completely the metabolism and behaviour of a simple, single celled organism, the yeast Saccharomyces cerevisiae.

The first UCL speaker was Christine Orengo of the Research Department of Structural and Molecular Biology. In the 1990s, she, with Janet Thornton (now director of the European Bioinformatics Institute) developed the CATH protein structure classification database which is very widely used in PPS. Since then, researchers in her group have built more databases of protein structure and function and prediction tools, and have moved on to the analysis of complete genomes and (as she presented here) functional protein networks. Domenico Cozzetto of the Department of Computer Science then described novel methods of predicting protein function from multiple data sources.

The research presented in both these talks clearly falls within the remit of "bioinformatics", in that it is concerned with the analysis of large quantities of molecular data. The next speakers, however, illustrated just how widely the term "computational biology" is being applied. Peter Hammond trained as a mathematician but is now working at UCL's Institute for Child Health, using imaging techniques and mathematical models to determine the subtle effects of genetic differences on human face shape. These models are already being used to aid early diagnosis of developmental disorders, facilitating both early intervention and genetic counselling. His presentation was followed by two more with a medical focus, by Angus Silver, a neuroscientist who develops mathematical models of neuronal signalling in the cortex, and a physician, Malcolm Finlay from the Heart Hospital, who described the computer simulations that his group has developed to predict the electrochemical responses of individual patients' heart muscles during periods of abnormal heart rhythm (arrhythmia).

A later talk by Sally Price of UCL's Department of Chemistry illustrated the value of computational biology to the pharmaceutical industry. She described the use of inter- and intra- molecular forces (to be covered in PPS section 9) to determine which crystalline structures of chemicals, including prescription drugs, would be most likely to form. For me, however, one of the highlights of the day was a talk by Mark Girolami of the Department of Statistical Science that linked computational biology, biostatistics and genetics to archaeology and anthropology. Mark described how a genetic mutation that allows some Europeans to digest milk as adults spread through the continent. His models traced the emergence of the mutation (which would not have remained in the population if it had not conferred significant evolutionary advantage) to a time and a place - about 7-8,000 years ago and in what is now central / Eastern Europe - when cattle replaced sheep and goats as the main domesticated animals.

I hope that this partial overview of a fascinating day's science will give you some idea of the breadth of computational biology research, and the depth of its coverage at UCL.

Peter Murray-Rust, PPS and a Semantic Molecular Future

2011-01-18T08:53:00.000-08:00

Yesterday (17 January) I attended a symposium at the Department of Chemistry, University of Cambridge, that was held to celebrate the career and ideas of one of the founders of the PPS course: Peter Murray-Rust. Since 2000, Peter has been based in Cambridge, where he is a Reader in Molecular Informatics.

The symposium was opened by Professor Sir Tom Blundell, a former head of the crystallography department at Birkbeck and now emeritus professor of biochemistry at Cambridge. Tom told the audience that when Peter came to Birkbeck in the mid-90s he already had a distinguished career in molecular science behind him. He had been a PhD student at Oxford in the 1960s, working with Keith Prout on the crystal structures of inorganic molecules. Tom, who worked with Dorothy Hodgkin on some of the early crystal structures of insulin, was a fellow student. Peter then worked as a lecturer at the University of Stirling, Scotland, and at pharma giant Glaxo (now part of GSK). He joined Birkbeck at a time when the Web was just beginning to open up the world of the Internet to the wider community. He, with David Moss, then a reader in crystallography at Birkbeck, and research associate Alan Mills, saw the potential for the web to extend the department's specialist teaching beyond the reach of those who could readily commute to central London, and PPS was born. The first, experimental course was delivered, free of charge, in 1995; within six years it would be incorporated into the full online-only MSc course.

Peter has never been one to let the grass grow under his feet. While he was busy creating and launching the PPS course, he was already thinking about how the still infant Web could be harnessed to allow data and information to be manipulated and understood rather than simply displayed. With Henry Rzepa from Imperial College, London, he developed Chemical Markup Language (CML) as a version of XML ("extensible markup language") for chemists. This is now very widely used. The first scientific paper written entirely in XML was published in 2001, although the editors of the journal concerned described it as "an interesting exercise, but [not] easy to deal with by any means". It is now being used for Microsoft's new chemistry add-in for Word: Chem4Word.

CML is described as a semantic language. The term "semantic web" was coined by WWW developer Tim Berners-Lee as "a group of methods and technologies to allow machines to understand the meaning – or 'semantics' – of information". Peter's wide interests include, besides the automatic analysis of data in scientific publications - the development of virtual scientific communities, and he campaigns passionately for all scientific data to be freely available to all. He was one of a small group who drafted the Panton Principles (named after a Cambridge pub) which state that future scientific advances will depend crucially on all science data - not necessarily its interpretation - being made freely available on the Internet. Later presentations, including one by Henry Rzepa, developed these ideas in more detail. The symposium ended with a presentation of software being developed in Peter's group, including an application where a student manipulated an image of a molecule by waving his arms. This may have looked like a fun gimmick, but it must be potentially useful for disabled students who have difficulty with using a mouse.

There was one other delegate at the symposium to whom PPS students owe a debt, although they are probably unaware that they do: Roger Sayle, the developer of Rasmol. Today, we rely on molecular graphics programs that are fast, free, and easy to install and use on any desktop machine. Roger's Rasmol, developed in the mid-90s, was the first of these.

If you would like to know more about Peter and his ideas, and how some of them have been applied in Birkbeck's web courses, you can visit his blog.

Metals in Proteins: A British Crystallographic Association Meeting

2010-12-22T09:00:00.000-08:00

The British Crystallographic Association (BCA) is the UK's national society for crystallography- the study of molecular structure using X-rays - applied in physical sciences and in engineering. It has four discipline-specific special interest groups and a further group for young scientists. Protein crystallography is, obviously enough, the main concern of the Biological Structures Group. That group always holds a one-day scientific meeting in mid-December, just before the Christmas break. The 2010 Winter Meeting was held on 15 December in Reading University, hosted by Dr Kim Watson and her colleagues at the School of Biological Sciences there. Its main theme was the role that metals play in protein structure and function, particularly in signalling and transport proteins.

Before the main business could begin, however, the delegates heard two interesting short talks. The first was by Martin Welsh, coordinator of life sciences research at Diamond, the UK's synchrotron facility (located in the Oxfordshire countryside). This provides the most intense radiation sources for X-ray crystallography (and other analytical techniques) in the country, and although it has been open since 2007 it is still complete. The UK scientific community is still deciding the range of problems to be addressed by the last set of beam lines, which in turn will determine how they are constructed. He called for the structural biology community to become fully involved in this process, as it risks losing out to the physical scientists.

The second introductory talk was by the BCA's President, Professor Elspeth Garman from the University of Oxford: first structural biologist to hold this position for many years. She explained how the relationship between technological developments in X-ray crystallography and the biological insights that it provides is as strong now as it was when Dorothy Hodgkin (Nobel Laureate in Chemistry 1964) was solving the structures of penicillin, vitamin B12 and (in 1969) the peptide hormone, insulin.

Highlights of structural biology research were then presented in three main sessions. The first of these featured three keynote lectures by distinguished structural biologists. The only common feature of the biological systems described was the fact that metal ions formed a crucial part of their mechanism of action.

First, Chris Schofield from the University of Oxford described his group's work elucidating how mammals sense and respond to changes in the concentration of oxygen. This work can be said to date back to the nineteenth century, when climbers, and people living at high altitude where oxygen concentrations are lower, were first observed to have increased numbers of red blood cells. It is now known, in part thanks to Schofield's work, that cells respond to low oxygen levels through a transcription factor called Hypoxia Inducible Factor 1 (HIF-1), which in turn regulates the production of another protein, erythropoietin, which is involved in the production of red blood cells. HIF-1 is upregulated if oxygen concentrations are low and broken down by the proteasome when concentrations are high. Schofield has used crystallography to determine the structural basis of the "switch" between the stable (low oxygen) and unstable (high oxygen) forms of this enzyme. The stable form contains two key proline residues (link is to section 2) and these are oxidised to hydroxyproline to give the unstable, high oxygen form. The enzyme that catalyses this oxidation reaction, HIF prolyl hydrolase (PHD) binds an iron ion in its active site unusually tightly, which causes it to react unusually slowly; it is this slow reaction that enables it to react to long-term changes in oxygen concentration.

Ben Bax, from GlaxoSmithKline (GSK) in Stevenage (and formerly a Birkbeck postdoc), then described how X-ray crystallography is being used to design novel inhibitors of bacterial proteins called DNA topoisomerases. These are enzymes that catalyse the winding and unwinding of DNA molecules, a process that is different enough in bacteria for inhibitors of these enzymes to be specific and effective antibacterial drugs. Bacteria have two forms of this enzyme, topoisomerase II (or DNA gyrase) and topoisomerase IV. Both these, confusingly, are known as type II topoisomerases; these are the target of the fluoroquinolone antibiotics, which have been in clinical use since 1962 and had sales of over $7Bn in 2009. Bax's group at GSK has solved the structure of a complex between the antibiotic moxifloxacin and topoisomerase IV from Acinetobacter baumannii, revealing that the enzyme-inhibitor interaction involves a magnesium ion that is not involved in the catalytic reaction (PDB entry 2XKK). They have also solved the structure of DNA gyrase from Staphylococcus aureus in complex with a novel bacterial topoisomerase inhibitor (PDB entry 2XCS), which sheds new insight into the enzyme's mechanism and the role of manganese ions in it.

The final talk in this session was given by Wyatt Yue, from the Structural Genomics Consortium in Oxford, and concerned the structure and function of an enzyme, methylmalonyl-CoA mutase (but known, thankfully enough, as MUT) which is involved in the metabolism of vitamin B12 (also known as cobalamin). This vitamin is essential for life; bacteria can synthesise it, but mammals need to take it in through their diets. Its structure was solved by Dorothy Hodgkin in 1954. It can bind cobalt in any of its oxidation states: Co(I), Co(II) and Co(III). Although only two enzymes including MUT require vitamin B12 as a co-factor, its deficiency can cause severe disease. Yue and his group have solved the structure of MUT bound to vitamin B12 (PDB entry 2XIJ) and that of an associated protein, a GTP-binding protein known as MMAA (PDB entry 2WWW). This protein is believed to act as a "gatekeeper", necessary for binding MUT to the appropriate, adenosyl form of vitamin B12. Two copies of the same MMAA mutation, which abolishes its interaction with MUT, have recently been identified in a patient with the metabolic disorder methylmalonic aciduria.

There were two further sessions at the conference, which I don't have room to blog about: one covered metal ion transport and catalysis, and the other metals in proteins that function as transporters, receptors and chaperones. There is some more information on the conference website.

Merry Christmas!

Microfilament structure highlighted in new paper

2010-11-25T06:38:00.000-08:00

Microtubules are long protein filaments that form an important part of the cell cytoskeleton. They are formed from polymers of a protein called tubulin - first tubulin forms dimers, each consisting of one alpha and one beta subunit, and then these dimers polymerise head-to-tail to form structures called protofilaments. The microtubules are hollow tubes formed from assemblies of parallel protofilaments. Several proteins collectively known as microtubule associated proteins (MAPs) associate with these microtubules and stabilise - or occasionally destabilise - different forms.

Doublecortin is a MAP that stabilises the most commonly found type of microtubule, which is composed from thirteen protofilaments. Mutations in both proteins that destabilise the interaction between them can cause devastating neurological diseases. A recent paper by Carolyn Moores, an electron microscopist working at Birkbeck, has highlighted details of the interaction between these proteins.

You can read a summary of this paper here (PDF format). This is one of a series of summaries of high impact papers by Birkbeck scientists that I have written for the website of the Institute of Structural and Molecular Biology, which links researchers in these disciplines at Birkbeck with those at UCL.

If you have time once you have read the summary you may explore the structures of these proteins further by downloading the atomic coordinate files that were "docked" into the electron density in this paper from the PDB. The PDB entry used for the alpha-beta tubulin dimer structure is 1JFF and the one used for the doublecortin domain that is bound to tubulin is 1MJD.

Protein Structure Highlighted in Birkbeck Science Week

2010-11-05T05:43:00.000-07:00

At the end of October Birkbeck College hosted its first Science Week, featuring lectures from distinguished researchers in all science departments. Two talks by members of the Department of Biological Sciences highlighted the beauty and importance of protein structure: the topic of the PPS course. Both lecturers came originally from North America; both moved to Birkbeck about twenty years ago; and both have achieved very significant honours as researchers in structural biology since.

Proteins, DNA and the Components of Life

Professor Bonnie Wallace’s research focuses on the structural analysis of proteins and peptides bound to membranes, and particularly on those involved in ion transport and cell signalling. Her talk in Science Week was entitled “Proteins, DNA and the Components of Life”. She began by describing proteins and nucleic acids as large molecules – “macromolecules” – that are essential components of all living cells. They are both polymers of smaller units; as you should know by now, proteins are polymers of amino acids and nucleic acids similarly are polymers of nucleotides. Amino acids combine as “beads on a string” in many different ways to form the vast array of known proteins; the functional – structural, metabolic or transport among many others – of each is determined by its three-dimensional structure. The techniques used for determining protein (and DNA) structure, including X-ray diffraction, which have developed from fairly crude beginnings about fifty years ago, now allow even large structures to be determined quickly. The section of Professor Wallace’s talk that introduced basic protein structure was illustrated with a physical model of a protein from the 1970s that was then compared with modern molecular graphics representations of protein structures. “Cartoon”, “ball and stick” and “space-filling” protein models, as well as stereo views that can give the illusion of three-dimensionality, are all useful for different purposes.

DNA molecules also are linear polymers, and these – particularly in eukaryotic cells – are extremely long and need to be packed very tightly if they are to fit into the tiny cell nuclei. The packing mechanism involves interactions between the DNA molecule and proteins called histones, to form a structure resembling “beads on a string” that condenses further to form fibres and eventually chromosomes.

The final part of Professor Wallace’s talk concerned the evolutionary differences between DNA in different individuals and in different species, celebrating the tenth anniversary of the publication of the first draft of the human genome. Genomics has provided a useful correction to human arrogance by revealing that many organisms have larger, if not necessarily more complex, genomes than ours. More importantly, differences between bacterial and human cells, between cancer cells and normal ones, and between dysfunctional (mutated) and normal versions of disease-linked proteins, are enabling us to design and develop drugs against a wide range of diseases. Important drugs for infectious diseases including influenza and HIV have been designed using “structure-based” techniques. Professor Wallace ended her talk by describing some of her group’s work on the structure of a protein that is embedded in a cell membrane and provides a channel for sodium ions to pass into cells. Anti-epileptic drugs such as lamotrigine work by binding to and blocking these channels, which will be discussed in detail in the section on Membrane Proteins towards the end of the PPS course.

Model for the structure of a human sodium channel

The Arms Race between Man and Pathogen

Professor Helen Saibil studies proteins using electron microscopy, which can determine the structure of very large proteins and of protein complexes embedded in cell membranes. Her talk, intriguingly titled “The Arms Race between Man and Pathogen”, focused on two pieces of her group’s research into proteins that punch holes, or pores, in membranes. These independent studies led to the discovery of an unexpected similarity between a group of bacterial proteins that cause damage during infection and one of human (and other mammalian) proteins that help protect against viral infection and blood cancers.

Pore-forming protein toxins are “weapons” that bacteria can use in order to break holes in the cells of their human or other animal hosts. They are used by a wide range of bacteria, including some very dangerous ones; one example is Streptococcus pneumoniae, which, as its name implies, causes pneumonia. Unusually, these proteins can exist in two forms, one that is water-soluble and another that is membrane-embedded. The change between these forms requires a considerable rearrangement of the protein’s shape, with one hydrophobic (“water-hating”) segment that is located deep in the core of the soluble protein uncurling to form a “beta-hairpin” structure that embeds in the membrane.

In 2005, Professor Saibil’s group used electron microscopy to determine the structures of two forms of the pore forming toxin from S. pneumoniae, pneumolysin, that together illustrate its mechanism of action. Initially, the protein associates into ring-shaped “pre-pores” of about forty monomers that sit on the membrane surface; the pore itself is formed when part of each monomer changes shape and burrows deep into the membrane. They were able to fit the more detailed X-ray crystal structure of a related protein as a soluble monomer into the shape of the pre-pore protein, and show the type of rearrangement that would be necessary for part of that protein to “collapse” into the membrane.

Another protein that has been studied by Professor Saibil and her colleagues recently is perforin, which is secreted by some lymphocytes (white blood cells that form an important part of the immune system) and which also punches holes in membranes: this time, in the membranes of infected or cancerous cells. This allows the lymphocyte to deliver toxins to the affected cell that trigger the process of apoptosis, or programmed cell death. People with a defective form of this protein suffer from severe viral infections and have a greatly increased risk of blood cancers.

There is no detectable similarity between the sequence of perforin and that of pneumolysin. However, when the first structure of a human perforin-like molecule was discovered in 2007, researchers were surprised to discover that its fold was similar to that of the bacterial protein. It became clear that human and bacterial pore-forming proteins must have evolved from a common ancestor. Protein structures are more tightly conserved than gene and protein sequences, and the structures of distantly related proteins are often much more clearly similar than their sequences.

Hoping to understand more about the mechanism of this protein, Professor Saibil’s group then used electron microscopy to solve the structure of perforin assemblies in contact with membranes. They observed pore structures that were similar, but not identical, to those of pneumolysin. In particular, the perforin structures indicated a much smaller conformational change between non-bound and membrane bound forms than the one seen with pneumolysin. Also, the perforin molecule seemed to be arranged “inside out” in the pore structure as compared to that of pneumolysin, an observation that the group was able to verify with biochemical tests. This finding was very unexpected, but turned out to be consistent with a twist between two parts of the pneumolysin molecule that does not occur in perforin. Both molecules have “arms” that extend down into the membrane to form the sides of the pore, but perforin’s arms are much longer and do not require as big a conformational change to reach down into the membrane.

View of the cryo-EM reconstruction of a perforin pore, with the front half cut away to show how modelled perforin molecules fit into the EM electron density.

The grey surface is the EM map and the subunits are coloured by domain / subregion, as follows. N-terminal region, blue; central beta sheet, red; C2 domain, yellow; modelled beta hairpins (pore lining), orange; EGF domain, green; C-terminal region, magenta. The membrane is seen as a double layer of grey electron density at the base of the pore.

This story of two related proteins, one on each side of the battle between man and pathogen, features many aspects of protein structure research that are covered during the PPS course. These include the beta-hairpin structure, the interatomic forces that determine the way proteins fold, and the structures of membrane proteins. If, however, you have been inspired to learn more about the way electron microscopy is used to study protein structure, you should aim to take our second-year MSc module, Techniques in Structural Molecular Biology.

Welcome - and Birkbeck Science Week

2010-10-05T09:24:00.000-07:00

Welcome PPS 2010-11 students to the PPS blog!

I have run this blog in some recent years to help students understand how what they study relates to current research in protein structure and in areas related to it such as drug design and development. This year, I will be posting accounts of lectures, conferences and new papers in the areas covered by the course, and link them to the scientific literature more widely. Comments are welcomed and if any of you would like to have a go at blogging I can set you up as an author.

We have a weekly seminar programme shared between Biological Sciences at Birkbeck and the other departments in the Institute of Structural and Molecular Biology, and later in the course I will occasionally post reports of these seminars when they seem particularly relevant to the course material. There is a theme for the seminars each term, and this term (Autumn 2010) this is "Cellular and Structural Biology of Infection".

Before then, however, I will be blogging from Birkbeck Science Week at the end of October. There will be two lectures then that are very relevant for this course, both given by senior professors in the Department of Biological Sciences: Prof. Helen Saibil on the structures of bacterial protein toxins and Prof. Bonnie Wallace on proteins and DNA. Do check back on this blog afterwards as I hope that will give you an idea of both the beauty and the elegance of our subject. Or - if you are within reach of London on either of those days - do come and hear what they have to say! Science Week lectures are free and open to all, although registration is required.

Enjoy the blog - and do let us know what you think of it!

Clare Sansom

Fragment-Based Screening and Drug Design

2009-06-29T07:35:00.000-07:00

The last Monday seminar of the Summer term at Birkbeck was given by Dr Rob van Montfort of the Institute of Cancer Research. Van Montfort, a former postdoc in the School of Crystallography here, spent six years in industry, at the biotech company Astex Therapeutics before joining the ICR two years ago. There, he is developing high throughput, structure-based drug screening techniques and using them to design novel compounds as candidate anti-cancer drugs.

Van Montfort first described the problem that techniques such as his have been designed to solve: that of attrition in drug discovery. Not only does it often take well over 10 years to move a potential drug compound "from the bench to the bedside", but the attrition rate is immense and not sustainable. A typical drug discovery programme will involve the testing of millions of compounds, and result in, say, half a dozen candidates for Phase I clinical trials and, if all goes well, a single registered drug. And even if a compound reaches the clinic, it may well not recoup the millions of dollars that have been spent on its development.

Drug companies and adacemic groups have been turning to novel technologies to help address this problem and reduce the time and cost of drug development. The technique de Montfort described, fragment-based screening, is one of these. It was originally developed by Wim Hol's group at the University of Groningen, the Netherlands, and developed for commercial use by companies including Astex and Plexxikon.

Historically, X-ray crystallography has been used mainly in the mid stages of drug development, in modify promising lead compounds into drug candidates. Now - thanks largely to high-throughput crystallography or structural genomics programmes - X-ray technology has improved to the point where it can be used at a much earlier stage. In fragment-based screening, groups of small molecules taken from a "fragment library" are soaked into protein crystals and the resulting structures examined by X-ray crystallography to see which fragments have bound to which parts of the protein's ligand-binding sites. These fragment hits generally bind very weakly but may be "joined together", if they bind into different pockets within the binding site, and modified further to generate tight-binding inhibitors and, eventually, candidate drugs.

De Montfort went on to describe a few published case studies of Astex' protein drug targets, including the P38a MAP kinase. This kinase is involved in cellular responses to stress, and its inhibitors may be therapeutically useful in a variety of inflammatory and auto-immune diseases. Fragment-based screening identified a lipophilic fragment that bound into the selectivity pocket of this kinase (Hartshorn et al. (2005), J. Med. Chem. 48, 403-413; PDB 1W7H) and modified it to produce a larger, tight-binding inhibitor (Gill et al. (2005), J. Med. Chem. 48, 414-426; PDB 1W82 & 7 others).

Thrombin, a serine protease involved in blood coagulation, is an important therapeutic target for stroke and deep vein thrombosis (DVT). Van Montfort was involved in the development of thrombin inhibitors, a process that was particularly hard because the thrombin active site is charged, and the charged compounds that would be expected to bind there are unlikely to work as oral drugs. Howard et al. (2006) published a fragment-based screen against thrombin using a library of uncharged compounds, finding small fragments that bound to one pocket within the substrate-binding site and larger ones that bound to a different one (J. Med. Chem. 49, 1346-55; PDB 1WBG). Combining the chemistry of these fragments into a single, larger molecule produced a potent series of uncharged, non-peptide inhibitors with structures that had not previously been seen in protease inhibitors.

Kinases are signalling proteins that control many biochemical and physiological processes, including the cell cycle, and cell-cycle kinases are very important as targets for anti-cancer drugs. Both Astex and the ICR have extensive programmes developing kinase inhibitors, and thehe two organisations have collaborated on the discovery of compounds that inhibit protein kinase B (see e.g. Saxty et al. (2007), J. Med. Chem. 50, 2293-6; PDB 2UW3).

Van Montfort has now set up a fragment-based screening lab at the ICR and developed a library of over 1800 fragments. His group is investigating potential protein targets for drugs against cancer, including kinases.

All the journal references in this blog post are in the Journal of Medicinal Chemistry, which should be available via the Birkbeck e-library.

Analytical Ultracentrifugation: Structures of Unstructured Proteins

2009-06-18T09:50:00.000-07:00

Dr David Scott of the National Centre for Macromolecular Hydrodynamics, based at the University of Nottingham, has been visiting Birkbeck for a few days to give a course. He also gave a seminar in which he explained one of the biophysical techniques used in the Centre, analytical centrifugation, and how it is used to help determine something of the "structure" inherent in unstructured, or partially structured, proteins.

The analytical ultracentrifuge was invented by Theodor Svedberg in 1923; three years later, he won the Nobel Prize in Chemistry for research using it. It is simply a centrifuge that spins very fast (from about 1,000 - 60,000 rpm); the sample being spun is monitored optically over a period of time. The normal settling of particles in solution under gravity (sedimentation) is speeded up by spinning in a centrifuge, which essentially replaces the gravitational force by a centrifugal force. The speed of sedimentation depends on the masses and shapes of the particles involved. The maths is far too complex to be described here.

One of the first uses of ultracentrifugation was in the determination of molecular mass. Sedimentation times are measured in Svedberg units (S); 1S is exactly equivalent to 10^-13 seconds. These times are related rather inexactly to molecular mass and often used to characterise large proteins and protein complexes; you have come across these in the PPS course in our discussion of ribosomal subunits. The small subunit of the Thermus thermophilus ribosome, illustrated there, is described as "30S".

Ultracentrifugation is now used routinely to determine whether a sample is homogeneous; if it is, all particles will have the same mass and shape, and therefore the same sedimentation time. A plot of sedimentation velocity for a sample can show whether the solution is homogenous or heterogenous, and whether protein is aggregated (in which case, aggregates will consist of different numbers of molecules and have different masses). Sedimentation equilibration experiments, which investigate the final steady state where sedimentation is balanced by diffusion, can be used to determine ligand binding and chemical reactions.

Scott has used ultracentrifugation and other biophysical techniques to investigate the structures of unstructured regions of a bacterial DNA-binding protein, KorB. This protein has a DNA-binding domain and a dimerisation domain each with a known structure, and it is known to interact with RNA polymerase as well as with DNA. Other parts of the protein, however, are only known to be "intrinsically unstructured". A combination of ultracentrifugation with other techniques useful for studying unstructured proteins, including circular dichroism and small angle X-ray scattering, were used to investigate the range of structures adopted by these unstructured regions. Results so far indicate that KorB forms a range of relatively compact structures when isolated in solution but that the unstructured regions extend when it binds to a related protein, KorA.

Analytical ultracentrifugation, CD spectroscopy and other biophysical techniques will be described in much more detail in the second year MSc module, Techniques in Structural Molecular Biology (TSMB).

Targeting Mycolic Acid Biosynthesis - Towards New TB Drugs

2009-06-18T07:42:00.000-07:00

The Department of Biology and Chemistry at Birkbeck recently hosted as a seminar speaker Dr Geoff Coxon, from the University of Strathclyde, Glasgow. Dr Coxon described a series of compounds that his group had synthesised, some of which are promising candidate drugs against tuberculosis. These are inhibitors of the enzyme beta-ketoacyl-ACP synthase (otherwise known as FabH) with a novel chemical scaffold.

Mycobacterium tuberculosis now infects over a third of the world's population, and tuberculosis kills two million people a year. The threat of tuberculosis is growing largely because of two reasons: a synergy between HIV and TB infections, and the growth of drug resistant strains of the bacterium. Multi-drug resistant (MDR) TB is defined as TB that is resistant to the two most commonly used anti-TB drugs, and extensively drug resistant (XDR) TB as TB that is resistant to four drugs including a fluoroquinolone. The latter is particularly hard to treat. More information on this is available from the TB Alliance.

In order to combat drug resistance it is essential to keep introducing new drugs, and preferably drugs with novel targets and mechanisms. Ideally, a drug should be active against active, replicating TB and persistent TB, which can resist treatment by remaining dormant in macrophages. Much work in anti-TB drug development has focused on enzymes involved in synthesising the very complex cell wall of the bacteria.

The genome sequence of M. tuberculosis (Camus et al., 2002, and material in PPS section 6) revealed a number of enzymes involved in cell wall synthesis. Coxon's group is focusing on the synthesis of one cell wall component, mycolic acids. These are long chain, 2-alkyl 3-hydroxyl fatty acids with between 60 and 80 atoms in their hydrocarbon chains. Two enzyme systems are involved in their synthesis: FAS-1 synthesises the main chain and FAS-2 adds the alpha branch. FAS-2 is found only in plants and mycobacteria.

The FAS-2 system includes a large number of enzymes, all involved in the complex, cyclic process of synthesising a long lipid chain. Inhibiting any of these enzymes will prevent the synthesis of the mature long chain mycolic acids. Coxon's group has been targeting one key enzyme in this process, known as FabH, which connects the FAS-1 and FAS-2 systems. The reaction it catalyses is an extension of the lipid chain by two carbon units.

This enzyme is a homodimer with a tunnel-shaped active site; the chain extension takes place after the substrate has moved into the tunnel. Its structure was first solved in 2001 by Scarsdale and co-workers (PDB code 1HZP). Coxon and his co-workers started their search for a specific inhibitor of this enzyme from the natural antibiotic, thiolactomycin (TLM). This, however, is a relatively weak inhibitor and a chiral compound that is extremely challenging to synthesise. He used fragment-based chemical libraries to develop a range of likely inhibitors with similar skeletons but that would be simpler to synthesise.

Some of the first compounds investigated were better inhibitors of the enzyme but not active against the whole FAS-2 system or M. tuberculosis itself. It appeared that another enzyme, known as KasA, may sometimes take the place of FabH if that is inhibited. They have now produced a series of compounds based on a 2-aminothiazole-4-carboxylate scaffold that includes some very active ones. However, the compounds in this series that are the most potent inhibitors of the enzyme are not the most active against the intact bacterium, and vice versa - there are complex interactions going on that are not yet entirely understood. It is likely, even, that FabH is not their most important protein target.

A good medicine against tuberculosis must be active against both resistant and dormant forms of the bacterium, with few side effects and few interactions with other drugs, and preferably orally available, and it must be cheap, and therefore easy, to synthesise. These 2-aminothiazole-4-carboxylates are easily synthesised and some are effective against M. tuberculosis in vivo, but much more work is needed to determine whether they will fulfil the other criteria.

See Al-Balas, Q. et al. (2009) PLoS ONE 4(5) (open access) for more information about this work.

Dr Coxon also works in TB Drug Discovery UK, an alliance of scientists involved in developing new treatments for tuberculosis.

Structure of the trypanosome microtubule cytoskeleton

2009-03-09T10:09:00.000-07:00

Twice a term, the School of Crystallography hosts a seminar for the whole of the Institute of Structural and Molecular Biology, which consists of research departments in related disciplines from both Birkbeck and neighbouring University College London. Tom Blundell's (see previous post) was an ISMB seminar; today, we heard one from Professor Keith Gull of the Sir William Gunn School of Pathology, University of Oxford.

Much of Keith Gull's work concerns the single-celled parasite Trypanosoma brucei, which is endemic in large parts of Africa and causes African trypanosomiasis, otherwise known as sleeping sickness. Diseases like this one attract relatively little research funding even though they are important causes of morbidity and mortality in many poor countries; they are classed as neglected tropical diseases. Keith and his colleagues in Oxford are studying many aspects of the molecular biology and genetics of this parasite.

The trypanosome cell surface is covered by a dense coat made up of very many copies of a single protein, called variable surface glycoprotein (VSG). A glycoprotein is a protein with carbohydrate (sugar) groups attached to one or more amino acid sidechains; the word "variable" is used because there are about 1000 variants of this protein. Each of these variants is encoded by a different gene, so the VSG genes account for about 10% of the trypanosome's genome. However, only one of these is expressed at any time, so the trypanosome coat (almost) always consists of multiple copies of a single protein. (There will be times when two proteins are present because the expressed variant is in the process of changing.) Although the sequences of VSG proteins differ considerably, their structures are very similar: they are anchored to the cell membrane and the large extracellular domain consists of an antiparallel coiled coil of alpha helices (see PDB file 2vsg).

Most of Keith's talk, however, concerned the structure of the trypanosome cytoskeleton, which consists of microtubules. Very basically, these are composed of polymers of a protein called tubulin and they are involved in maintaining the structure of components of many types of cells. Keith and his group have used the technique of electron tomography - a type of transmission electron microscopy - to obtain exquisite pictures - if at much lower than atomic resolution - of the structure of this cytoskeleton and begin to understand its function and role in trypanosomal cell division. Structures of these multi-protein complexes are not - yet - available in the PDB.

Trypanosomes have flagella - whip-like structures attached to the cells that can propel them through the host bloodstream. Flagella are found in many cell types, and if they have receptors bound they can also be used to sense cells' surroundings. The trypanosome flagellum is attached to the cell body via a filament and this point of attachment is within a pocket that, interestingly, is the end point for all vesicular traffic within the cell. This means that this pocket needs to contain a large number of proteins vital to the parasite's survival, including its transferrin receptor and haemoglobin receptor. Imaging the structure of the trypanosome cytoskeleton, particularly in the pocket where the flagellum attaches, has also given insight into the rather unusual process through which this cell conducts mitosis (cell division).

Genomes, Structural Biology and Drug Design

2009-02-13T09:32:00.000-08:00

Professor Sir Tom Blundell, head of the School of Crystallography at Birkbeck from 1977 to 1995, returned last Monday to give an extremely well attended seminar - a tour de force of the relationship between structural biology and drug design Tom's distinguished career has also included a time as the head of the research council that funds non-medical biological research in the UK, BBSRC; he moved from Birkbeck to become head of Biological Sciences at Cambridge; and he is a director of a biotech company, Astex Therapeutics, which he founded in 1999.

Tom started his talk with a brief history of structural biology and its role in drug discovery. His personal involvement in the discipline goes back to the 1960s, when he, as a Ph.D. student, visited companies such as Eli Lilly, which manufactured insulin, with his supervisor, Dorothy Hodgkin - who solved the insulin structure, but won her Nobel prize (Chemistry, 1964) for structures of penicillin and vitamin B12.

The intervening decades have seen trends in drug discovery come and go. In the 1990s, it seemed that increases in the speed of synthesising and screening large numbers of small molecules against drug targets had made the more targeted approach of the academic structural biologists redundant. However, even the millions of compounds that can now be screened represent the tiniest fraction of "chemical space": the number of potential molecules of a size to bind to a drug target is larger than the estimated number of atoms in the universe.

And now, in the so-called age of the genome, structural biology has become an integral part of drug discovery, involved in all steps: target identification and validation, screening, and lead compound identification and optimisation. The organisation of information about sequences and structures in databases - some of which were mentioned in the PPS Bioinformatics section - began when Tom was at Birkbeck, and was spearheaded by his co-workers and collaborators, particularly Janet Thornton (now head of the EBI). The databases set up and curated by members of his Cambridge group - too many to describe properly here - are available from this page.

Some particularly useful insights arise from the relationship between the single changes in nucleic acid sequence (known as Single Nucleotide Polymorphisms, or SNPs) that are collected into databases and the structural biology of drug targets. Sometimes, with simple Mendelian diseases, one such change is sufficient to cause disease; more complex diseases arise (anything from hypertension to breast cancer to bipolar disorder) arise from interactions between many such changes that increase the chance of disease. Mapping changes in protein coding brought about by an SNP to a protein's structure can give insight into disease-causing changes in protein mechanism and lead to the identification of novel drug targets. See Burke et al. (2007), BMC Bioinformatics 8, 301 (this is an open access journal with full text available free of charge).

The drug discovery programme at Astex Therapeutics is based on experimental structural biology, on a technique known as fragment screening. In this, small chemical fragments that bind to drug targets are identified by fast X-ray crystallography. Knowing both the structures of these small compounds - which are too weak as binders to be identified by chemical means - and where they sit in a drug target's binding site enables chemists to build them out to form larger tight-binding molecules that fit into the whole site. The company's pipeline focuses on kinase inhibitors as anti-cancer drugs, and some of its lead compounds have reached early clinical trials.

Designing Magic Bullets

2009-01-20T06:08:00.000-08:00

This week's Monday seminar at Birkbeck was given by Jose Saldanha, who works in Willie Taylor's group at the National Institute of Medical Research at Mill Hill, London. Both Jose and Willie are Birkbeck Ph.D. graduates. Jose described his work in using molecular modelling and bioinformatics to design specific antibodies for therapeutic applications.

These antibodies are the "magic bullets" of the title of this blog post. Interestingly, the term was invented by Paul Ehrlich, who won of one of the first Nobel prizes for Medicine - in 1908 - for his work on immunity. The magic bullet, he suggested, would be a molecule that could be targeted directly to a part of the body needing drug treatment, and which a drug molecule could be attached to.

Antibodies (or immunoglobulins) are secreted into the bloodstream in response to the presence of "foreign" molecules (e.g. from bacteria or viruses) and target those molecules in order to destroy the "invader". They do this by binding very specifically to the targeted molecule. Therefore, molecules with essentially the same structure must be able to bind an almost infinite variety of targets.

The mechanism that has evolved for this is a very elegant one. The commonest type of immunoglobulin, Immunoglobulin G (IgG) is a Y shaped molecule made up of four chains, two heavy and two light. Heavy chains contain four copies of the same domain fold (a type of beta barrel) and light chains contain two. The binding site is at the "top" of the "arms" of the Y; the domain of each chain that is closest to the binding site is much more variable in sequence than the others, and is termed the variable domain (the others are constant domains). Each variable domain has three regions that are particularly variable in sequence, termed complementarity determining regions or CDRs, and it is these that bind the target molecule (the antigen). There is a more about immunoglobulin structure on this page from the Arizona Biology Project, and it will be covered in depth in section 11 of PPS.

The only antibody that can be successful as a "magic bullet" is a completely homogenous sample, where every molecule has the same sequence and structure and binds the same antigen. Such identical antibodies, or monoclonal antibodies, can be produced by fusing myeloma (cancer) cells with spleen cells produced by an infected mouse, and selecting and cloning those resulting cells that secrete the required antibody. However, these are mouse antibodies that do not work in humans; they can also cause an immune response because they themselves are seen as "foreign".

In the 1980s, Greg Winter at NIMR developed a process called "CDR grafting" to overcome this, in which the CDR regions from the mouse antibodies are grafted on to a human antibody framework. An alternative technique involves the fusion of a few residues from the mouse antibody with a human one to form a "humanised" antibody. Jose's work at NIMR involves sequence and structure analysis to work out exactly which mouse antibody residues to fuse with which human antibodies for different indications. A "successful" antibody must express well, bind specifically to its antigen and cause no immune or other adverse reaction in human patients. Since starting the project, he has designed more than 30 different humanised antibodies, some of which are in clinical trials.

There are many structures of immunoglobulins and immunoglobulin fragments in the PDB. The most commonly crystallised fragment is the four domains that make up one of the arms of the Y, which is known as Fab (standing for "Fragment, antibody binding"). Look at this one (1BM3), which binds a peptide antigen. And remember - we will be coming back to this topic later in the course.

An Enzyme in Histidine Biosynthesis

2008-12-11T09:53:00.000-08:00

The School of Crystallography at Birkbeck has a regular programme of research seminars held on Monday lunchtimes during term. Many of these describe recent developments in protein structure, and, from time to time, I will be reporting these here and linking in to course material where relevant. This week's seminar was by Adrian Lapthorn, a protein crystallographer based at Glasgow University, who just happens also to be the external examiner for the MSc in Structural Molecular Biology as well as the second year module TSMB. Adrian's research is concerned with solving the structures of enzymes, including those involved in the biosynthesis of the amino acid histidine.

Histidine is known as an essential amino acid; that is, it cannot be synthesised de novo in humans, but must be supplied from the diet. However, bacteria, fungi and plants all have enzymes that enable them to synthesise histidine from simple chemical precursors. The enzymes in the histidine synthesis pathway are therefore, at least potentially, good targets for novel antibiotics and herbicides as there are no equivalent human enzymes for them to inhibit, so they should be relatively free of side effects.

In bacteria, the histidine synthesis pathway consists of 10 steps, catalysed by a total of eight enzymes (some of which are bifunctional). The first step in this pathway is synthesised by an enzyme called HisG (or ATP-phosphyribosyltransferase) which catalyses the following reaction:
ATP + PRPP ---> PR-ATP
(PRPP is Phosphoribosyl pyrophosphate; PR-ATP is phosphoribosyl-ATP. The action of the enzyme is, therefore, to transfer a phosphoribose group on to the ATP molecule.

This enzyme is interesting for several reasons, besides the pharmaceutical and biotechnological interest in its inhibition. For one thing, unusually, there are no specific active site residues; the substrate is stabilised in the active site cleft by binding to magnesium ions.

Adrian's talk was subtitled "the long and the short of it", because some bacteria have a "short" form of this enzyme, and others a "long" form with an extra 80-odd residues at its C-terminus. All bacteria with the short form also have an additional enzyme, HisZ, which binds to HisG in an equivalent position to the C-terminal domain of the long form during catalysis. The long form of the enzyme consists of three discrete folded units called domains. There are two similar ones at the N terminus, followed by a long alpha helix and the C terminal domain, which is absent in the short form and has a similar structure to that of the small protein ferredoxin. The active site is between the two similar domains.

You will learn much more about domains and their folds in the next section, Towards Tertiary Structure. For now, look at this structure (PDB 1Q1K) of the long form of HisG (from E.coli) and try to identify the three domains and the active site. You might find it helpful to look at a single chain only.

Welcome 2008-9 Students to the PPS Blog

2008-12-08T09:45:00.001-08:00

Yes - welcome!

I run this blog to help PPS students understand how what they study throughout the course relates to recent research, mainly in structural biology but also in bioinformatics and some other related subjects. I plan to update it every few weeks (or more frequently if there is enough to say) between the end of the Autumn term and the end of the course, with reports of lectures, conferences and new papers in the areas covered by the PPS course, and link these to the course material and the wider scientific literature. Some other lecturers at Birkbeck may also occasionally add posts.

The first blog post linked to the 2008-9 course will appear before the end of this week, and will describe crystal structures of enzymes presented at one of the talks in our weekly seminar programme here at Birkbeck. In the mean time, do scroll down and look at some of last year's posts, although you won't be able to follow the links to the course material there.

You will be encouraged to comment on blog posts and, if you wish, to make your own - you will all be added as authors in the New Year. Anyone who can view the blog should be able to comment, but only authors can make posts.

Enjoy the blog - and let us know what you think of it!

Clare Sansom

London Structural Biology Club meeting

2008-07-29T10:07:00.000-07:00

We at Birkbeck have just hosted a meeting of the London Structural Biology Club. This is a network of students and researchers in structural biology based in London and the South-East of England. Members get together for a couple of afternoons a year to hear research presentations, and the talks are followed by further informal discussion over refreshments (usually pizza and beer).

Four talks were given at the Birkbeck meeting, with each presenting not only new structural studies but also novel insights into molecular function and mechanisms derived from those structures. First to talk was Carien Dekker from the Institute of Cancer Research in London. She described the protein interaction network - known as the "interactome" by analogy with "genome" and "proteome" - of a eukaryotic cytosolic chaperonin, CCT. Chaperonins are a sub-class of chaperones, the proteins that assist other proteins in forming their stable three-dimensional structures, and they consist of two ring-like structures that associate back-to-back forming a cavity in which their substrate proteins fold. Dekker and her co-workers have used a number of different proteomics techniques, including the insertion of a long internal tag into a loop of the protein, to discover the range of substrates for this chaperonin. Proteins involved in functions as diverse as protein import into the nucleus, protein degradation, and chromatin remodelling. CCT is also necessary for the formation of the septin ring complex, and thence for cytokinesis (the last stage of cell division).

This work was published very recently in the EMBO Journal. Dekker and her colleagues are now working on the structure of CCT, which they hope will reveal more details of the function of this chaperonin: watch this space.

The second talk was given by David Komander, who has just moved from the Institute of Cancer Research to set up his own lab at the prestigious MRC Laboratory of Molecular Biology in Cambridge. He described some intriguing details of the ubiquitin system, through which proteins can be tagged for degradation. Ubiquitin (mentioned briefly in section 7 of the PPS course material) is a small protein (only 76 amino acids) with an alpha+beta fold (see PDB entry 1UBQ). Its C-terminus can be covalently linked to lysine side chains or N termini of other proteins. As ubiquitin itself has seven lysine residues (as well, of course, as an N-terminus) it can polymerise to form short chains. Proteomics has shown that all possible combinations of ubiquitin linkages can exist, but linkages in which the molecules are connected through lysines K48 and K63 are the most common. Poly-ubiquitin tags composed of different linkages have been linked with different functions; for example, binding a K48-linked ubiquitin chain to a protein will tag it for proteasomal degradation, whereas a K63-linked chain will tag a protein for signalling. The structures of these two forms of poly-ubiquitin have been shown to be very different, with K63-linked poly-ubiquitin forming an extended chain and K48-linked poly-ubiquitin a compact fold.

Ubuquitinlyation is a reversible process, and Komander has been studying the enzymes (deubiquitinases) that catalyse the hydrolysis of the peptide bonds between two ubiquitins, or between ubiquitin and another protein. These DUBs are analogous to the phosphatases that remove phosphate groups from protein side chains; their specificity , however, is more complex than that of phosphatases. Earlier this year, Komander and David Barford published the structure of the N-terminal domain of one such protein, A20 (Komander & Barford (2008), Biochem. J. 409, 771-785l; full text available). This is a cysteine protease domain known as the ovarian tumour (OTU) domain. These structural studies suggest both a novel architecture for the protein's catalytic triad and a novel mechanism - reversible oxidation - for the regulation of protein ubiquitinylation.

The "home team" at Birkbeck contributed a talk from Han Renaut, in Professor Gabriel Waksman's group. Waksman's own account of this work, on the structure of bacterial secretion systems, was blogged back in May and will not be described in more detail now.

Lastly, we heard from Erhard Hohenester who described an unpublished structure of SPARC, a protein that binds collagen. About 30% of the dry weight of the human body is composed of fibrils of this structural protein. It has a unique structure, being composed of three strands wound round each other in a triple helix. Every third residue of each strand must be a glycine, and the protein also contains a high percentage of proline. Some proline residues are post-translationally modified with the addition of an -OH group to form hydroxyproline. Besides being the major structural component of animal tissue, collagen binds to and forms complexes with many proteins including integrins and some tyrosine kinases. However, until now the only structure of a complex of collagen with another protein was with integrin (see PDB entry 1DZI).

SPARC, or osteonectin, is secreted by osteoblasts during bone formation, and binds calcium as well as collagen. Its structure as an isolated protein has been known for over ten years; it has two domains, one alpha-helical and the other containing many disulphide bonds (PDB 1BMO). Details of the new crystal structure of the collagen-SPARC complex, solved by Hohenester's group, must wait until the paper is published, but it is possible to say that it binds the hydrophobic sequence GVMGFO (which is a rare sequence in collagens, although one often involved in protein-protein interactions) into a hydrophobic pocket on the PARC molecule. [Note that that "O" is not a mistake; it is the single letter amino acid code for hydroxyproline.]

This London Structural Biology Club meeting was sponsored by Alpha Laboratories Ltd.

Cell invasion by the malaria parasite

2008-07-01T04:10:00.000-07:00

Last Monday's seminar in Crystallography (and the last of the summer term) was given by Dr Mike Blackman of the National Institute of Medical Research (NIMR) in Mill Hill, near London. His title was "Protease involvement in host cell invasion and exit by the malaria parasite. The following short report is contributed by Christine Slingsby:

Dr Blackman's lecture and discussion was on the topic of the
characterisation of several serine proteases, identified from the
genome of the malarial parasite, Plasmodium falciparum. One appears to operate in the membrane and one in the cytoplasm.

These are key enzymes used by the parasite to invade a host cell. Although they cleave with great precision certain proteins on the surface of the merozoite (blood stage of the parasite), it is unclear at the molecular level how the enzymes recognise their substrates. In other words, unlike, say, trypsin, which cleaves on the C-terminal side of a lysine of arginine, these subtilisin-like serine proteases have little sequence specificity.

Dr Blackman used the analogy of the success of HIV treatments based on the HIV aspartic protease, to enthusiastically push his work forward to try and discover inhibitors of these new enzymes as potential anti-malarial drugs.

Much more information is available on his website: http://www.nimr.mrc.ac.uk/parasitol/blackman/

Insights into arginine methylation in histones

2008-06-27T03:34:00.000-07:00

This week's seminar at Birkbeck was given by another alumnus of the School of Crystallography, Wyatt Yue, who studied for his PhD here from 1999-2003 working with Susan Buchanan. Wyatt is now at the Structural Genomics Consortium at Oxford University, but the work he presented was all done when he was a post-doc with Lawrence Pearl at the Institute of Cancer Research in London. He described some elegant work on the structure and function of one member of a family of enzymes that catalyse the addition of methyl groups to arginine residues in histones.

Histones are proteins that are involved in the coiling and compacting of DNA, so the long DNA molecules fit into the cell structures. In eukaryotes, DNA is first wound round an assembly of eight histone molecules to form a structure called a nucleosome. The DNA then resembles beads on a string; simplistically, this structure is further compacted by coiling into a chromatin fibre, from which the chromosome structures are formed. (Links here are to Wikipedia.) The histone molecules have N-terminal "tails" which protrude from the folded structure and are typically not seen in crystal structures. Residues on these tails can be chemically modified by e.g. phosphorylation, acetylation and methylation. These modifications are dynamic and form the so-called "histone code" which is one of the processes that control gene expression.

One of the most important of these changes is the methylation of nitrogen atoms on the side chain of arginine residues; this process is catalysed by a family of enzymes called protein arginine methyltransferases (PRMTs). Six members of this family are found in mammals; the protein that Wyatt has been working on is PRMA4, otherwise known as coactivator-associated methyltransferase 1 (CARM1). All these proteins share a common, catalytically active core domain; CARM1 is the only member with an additional C-terminal domain. The structure of the core is known (PDB 2oqb); it has two domains, an N-terminal Rossman fold, with two extra helices, and a C-terminal beta barrel with an insertion of a helix-turn-helix motif known as the arm. Core structures of two other PRMT proteins have also been known; compared to the others, the CARM1 core has a piece of ordered structure at the end of its C-terminal beta strand (the beginning of the C-terminal domain) and the helices of the arm are longer. Therefore, the cavity between the monomers of the crystallographic dimer is larger, leaving space for the C-terminal domains.

Lawrence Pearl's group, including Wyatt, has now solved the structure of the core domain bound to its co-factor, S-adenosyl methionine (PDB 2V74). This has revealed that it is only the co-factor binding that creates the cavity into which the arginine substrate is bound, indicating that the co-factor must bind first. This binding order has also been demonstrated in kinetic studies. The arginine pocket is close to the methyl group that is transferred during catalysis and is lined by negatively charged residues to attract the positively charged arginine.

CARM1 methylates three arginine residues in the N-terminal tail of the histone H3: R2, R17 anad R26. However, the mechanism of each methylation is not exactly the same. Studies with chimeras have shown that the pre-core region is necessary for methylation of R26 but not R17; Arg 17 methylation is faster if the neighbouring residue Lys 18 is acetylated.

You can read more about this work in the publication: Yue et al. (2007), EMBO J. 26(20): 4402-12. (Link to PubMed). The online version of EMBO Journal is available in the Birkbeck e-libarary.

J.D. Bernal and Crystallography's Beginnings

2008-06-10T09:39:00.000-07:00

Last week at Birkbeck we held the annual lecture celebrating the life and work of J.D. Bernal, the founder and first head of the School of Crystallography at Birkbeck. Bernal - "Sage" as many of his contemporaries knew him - was one of the most influential figures in the early development of structural biology. In 1934, while at Cambridge University, he and his student Dorothy Crowfoot obtained the first X-ray diffraction pattern from a protein. That protein was pepsin; its structure was only solved many decades later. (The link is to a structure of porcine pepsin by a group led by another head of Birkbeck Crystallography, Professor Tom Blundell.)

Dorothy Crowfoot - as Dorothy Hodgkin - went on to win the 1964 Nobel Prize for Chemistry, for her work on the structures of vitamin B12 and penicillin. She also solved the structure of insulin. Bernal's mind was no less brilliant than his student's, but the ultimate prize still eluded him. Most commentators agree that he is one of the greatest scientists never to win the Nobel, although several of those he worked alongside and inspired, including Francis Crick, Aaron Klug and Max Perutz, did so.

It is possible that Bernal simply never allowed himself to stay focused on any one area for long enough to win the Nobel. This was essentially the view of Sir Lawrence Bragg, himself a Nobel Laureate (Physics, 1915): "if one traces back almost any fruitful line of crystallographic work, it will be found that Bernal assisted at its conception but left the child to be brought up by foster-parents. This is particularly so in the case of molecular biology and in the analysis of protein crystals. Immediately on seeing the first x-ray differentiation pictures from protein crystals...he assumed that protein structures would sooner or later be solved, and handed out problems to his students and to anyone whom he could persuade to take them up."

Bernal's politics may also have disenchanted him from the Establishment. He was a life-long and well-known Communist (in an era when this was far more common among intellectuals than it is today) and an internationalist, and visited the Soviet Union many times. The painter Picasso was among the giants of the European Left who knew him well; one meeting between the two, after which Picasso left behind a mural on the wall of Bernal's flat in the college, has even been made into a play. (The Wellcome Trust now own the mural, and have recently installed it in their London headquarters.) He was also a pacifist, and chaired the World Peace Council from 1959-1965.

Bernal's contributions to scientific thought go far beyond the practice of what is known as "hard science". He gave much attention to the interaction between science and society, and how science should be managed and funded. One of his many books, The Social Function of Science, published in 1939, is considered the earliest text on the sociology of science. And this year's Bernal Lecture focused on an earlier book still: The World, the Flesh and the Devil (1929): subtitled An Enquiry into the Future of the Three Enemies of the Rational Soul .

There is not enough space in a blog post to do justice to this fascinating man. Those of you who would like to know more about him are referred to a biographical memoir written in 1980 by Dorothy Hodgkin and available online.

Gabriel Waksman on bacterial secretion systems

2008-05-14T09:54:00.000-07:00

Gabriel Waksman, head of the School of Crystallography at Birkbeck, has given a seminar in Birkbeck's School of Biological and Chemical Sciences describing his group's work bacterial secretion systems. Gabriel studied at the University of Paris and then spent the first part of his academic career in Washington University in St. Louis, Missouri, before joining Birkbeck in 2003. He now combines his position here with a chair in Biochemistry at University College London and directs the new Institute of Structural Molecular Biology which links the research in the two colleges.

Gabriel's research group works mainly on elucidating the structure and function of bacterial secretion systems, which produce hair-like appendages, or pili, on the surfaces of Gram negative pathogens. These are very important for infection as they allow bacteria to recognise, and then attach to, host cells. Mutant bacteria in which these proteins are not expressed are not pathogenic.

Two of the best understood of these systems are found in variants of E. coli which infect the human urinary tract. The P pilus recognises and binds to kidney cells, causing the kidney infection pyelonephritis, and the type I pilus recognises bladder epithelial cells and causes urinary infections. Both pili can be found on the same bacterium. The structures of both pili are similar, consisting of many protein subunits; it is the tip subunit that recognises its target cell types by binding to different cell surface sugar residues.

All subunits associated with a particular pilus are encoded by genes within a gene cluster and named accordingly: the P pilus genes are Pap genes and the type I genes Fim genes. Structures of at least one representative of each type of subunit have now been solved, many by Gabriel's group and its collaborators. Knowing these structures has enabled the group to understand the mechanism through which the pili are formed.

The pilus subunits that polymerise to form the main part of the fibrous structure all have similar structures. They are immunoglobulin-like, mainly-beta structures in which one sheet is lacking a central beta strand, and they are therefore unstable independently unless they are bound to a chaperone protein. A strand from the chaperone fits into the gap, forming regular hydrogen bonds with the neighbouring strands, and this stabilises the chaperone-subunit complex. This is then transported to the growing pilus, where the N terminal peptide from a subunit already in the structure replaces the chaperone strand in the new subunit, adding it to the polymer via a mechanism called "donor strand exchange". The resulting fibre therefore consists of a string of similar subunits, with the N terminal peptide of one subunit forming a strand in the central beta sheet of the previous subunit in the assembly.

The Waksman group's most recent structural studies concern the protein through which the pilus is assembled, known as the usher. This is a mainly beta membrane protein (link is to material in PPS section 11, which will be released next week) which is embedded in the E. coli outer membrane. The structure of the E. coli P pilus usher, solved by X-ray crystallography, shows the beta barrel and a middle or plug domain which interrupts the main beta sheet of the barrel. With 24 strands, it is the largest outer membrane beta barrel protein structure to be elucidated so far. In its inactive form, the plug domain fits inside the barrel, completely blocking it. They also used cryo-electron microscopy to isolate the structure of a type I pilus complex during pilus assembly. The usher forms a dimer within the cell membrane but, interestingly, the EM studies show that a pilus is secreted through only one monomer of the dimer.

This is very complex work which can only be touched on in a blog post. If you would like to know more, have a look at a few of these papers (links to abstracts in PubMed):

Sauer et al. (1999), Science 285, 1058-61
Sauer et al. (2002), Cell 111, 549-51
Remaut et al. (2006) Molecular Cell 22, 831-42
Remaut et al. (2008), Cell 133, 1-13 (to be published May 2008)

Predicting RNA binding from protein sequences

2008-04-21T06:17:00.000-07:00

The first Birkbeck seminar of the new term was given by Sue Jones, from the University of Sussex. Sue is no stranger to Birkbeck as she did her Ph.D. with Janet Thornton at University College, and later worked with her at the European Bioinformatics Institute and the biotech company Inpharmatica. Today she described a piece of software that she and her colleagues have developed for predicting motifs in protein sequences that are likely to bind to RNA.

Proteins function largely by interacting with other molecules - they are "social" molecules. Protein interaction partners include other proteins, carbohydrates, "small" molecules and ions, and the focus of today's talk: nucleic acids. The structures and functions of RNA molecules are diverse and include protein coding (mRNA), protein synthesis (tRNA and ribosomal RNA), splicing, hydrolysis of nucleic acid bonds (in RNA enzymes or "ribozymes") and control of gene expression (the so-called "micro-RNAs or miRNAs). RNA-binding domains in proteins include RNP domains, dSRNA binding domains, and K homology (KH) domains - all these are mixed (alpha and beta) structures.

Jones and her colleagues surveyed known structures of protein-RNA complexes and marked residues that were in close contact (through van der Waals or hydrogen bonding) with the RNA. They described each amino acid in terms of predicted accessible surface area, conservation within the family of homologous proteins, and chemical properties. Not surprisingly, positively charged and polar amino acids were favoured in binding to the negatively charged nucleic acid over negatively charged and hydrophobic ones; glycine, which is flexible, and tryptophan, which can form base stacking interactions were also favoured.

Jones then built these features, averaged over a "window" of 5-25 amino acids, into a support vector machine to predict RNA binding features in proteins of unknown function. (This technique is a form of "machine learning"; you don't need to know about it for this course, but if you're interested in knowing more and can cope with maths at a relatively high level, see the Wikipedia entry.) This was found to be at least as reliable as any similar tools that are publicly available.

There will be more about protein-nucleic acid binding in the next section of course material, Protein Interactions and Function, which is due to be released at the end of April.

British Crystallographic Association Spring Meeting

2008-04-14T04:47:00.000-07:00

I spent Tuesday - Thursday of last week at the British Crystallographic Association Spring Meeting. The meeting has 4 strands Biological, Chemical, Industrial and Physical Crystallography.
Each contribute a plenary and then have their own separate sessions. The Biological Plenary was the Bragg Lecture where one famous crystallographer speaks about another usually older crystallographer and their work. This year Tony Crowther from the MRC Cambridge talked about his work and that of Michael Rossmann from Purdue University. Both made seminal contributions to the method of molecular replacement in protein crystallography. More on that in TSMB. Michael Rossmann when a postdoc for Max Perutz at the LMB in Cambridge was the first person to realise that the chains of hemoglobin looked like the chain of myoglobin and hence that you could solve structures of related proteins by molecular replacement. Michael then developed the mathematics and early software for molecular replacement. Tony Crowther did a Ph.D. with David Blow at the LMB and developed an improved form of the translation function. While working on natural language processing inEdinburgh, Tony also realised how to give a much faster and more acccurate version of the rotation function, which was the basis of the molecular replacement method for a long time. Tony's career was actually mainly in electron microscopy, he returned to the LMB from Edinburgh to work for Aaron Klug and became a group leader in his own right. Both he and Michael Rossmann have done most of their work on viruses and he talked about Michael's work on bacteriophage and his work on Hepatitis.
The Biological Group sessions were on Membrane proteins. Chris Tate from LMB in Cambridge described work they have been doing to stabilise membrane proteins by mutation. They search for alanine mutations that increase the stability of the protein in detergent and then carry out mutations in combination until the protein is stable for half an hour at a temperature 15-20 degrees hotter than the original. They have succeeded in crystallising beta-1 androgenic receptor, which will give important comparison to the beta-2 published just before Christmas.
The most interesting talk for me was from Thomas Sorenson, now at Diamond, on the work he and colleagues had done in the group of Poul Nissen in Aarhus. The group have published structures in several states of eukaryotic ATPase transproters (Calcium, sodium, proton). Interestingly these proteins were discovered by a Dane, Jens Skou http://www.pumpkin.au.dk/en and the group used proteins provided by various groups in Denmark that have worked on the systems for many years. They used natural sources and did not purify the proteins down columns, but just used differential extraction. This means that they isolated the membrane fraction that contained most of the protein and then extracted with detergents and this material was pure enough to crystallise in the presence of the right combination of detergents and lipids. The other biological sessions were on neutron diffraction, probing fast biological reactions, complementary methods, and ligand binding and drug design. Neutron diffraction gives the position of hydrogen atoms as both hydrogen and even more so deuterium diffract neutrons much more relatively than they do X-rays and you get density for hydrogen atoms. The catch is that neutron fluxes are much weaker and you need crystals that are 0.1- 1 mm3 compared to 0.0001 mm3 for a protein crystal. Studying reactions in crystals often means trapping intermediates by freezing out. Arwen Pearson from Leeds gave a good talk about a redox system that she had worked on in Minnesota where the reaction cycle can be carried out in the crystal, even changing space group between states. The catch, and this is common, is that X-rays themselves generate free radicals which can reduce redox centres so by collecting the data the redox state is altered. This meant that they had to collect data from several crystals before they became too damaged. The highlight for me of the Ligand and Drug session was a talk from Chris Phillips at Pfizer about their new non-nucleoside HIV Reverse Transcriptase inhibitor. These target a hydrophobic pocket in the protein, and tend to be rather 'greasy'. The Pfizer group had carefully designed a ligand that was both smaller and more hydrophilic and hence a better drug in terms of bioavailability.
There were many more great talks, but I hope this gives you a flavour of the meeting
Nick

Greetings from Poznan

2008-04-14T01:24:00.000-07:00

This is just by way of an apology for my relative silence on PPS blogs and forums lately.

I am half way through two weeks' teaching at Adam Mickiewicz University, Poznan, Poland, funded by a grant to Birkbeck through the EU Erasmus programme (formerly known as Socrates) which funds student and lecturer exchanges between EU countries (and some others). I am teaching a two-week course on bioinformatics mostly, this year, to postgraduate Physics students but I have also taught in other departments.

I have known my host here, Professor Mariusz Jaskolski, since we were both working in the same lab in the States, NCI Frederick, in the early 90's. Mariusz was involved in some of the early work on the structure of HIV protease which is covered extensively in the PPS course. Since then he has gone on to found the first X-ray crystallography group in central-eastern Europe and to solve the structures of viral integrases, asparaginases and others... and many of his students, and others at AMU, have taken PPS and/or other distance learning courses from Birkbeck.

Normal service will be resumed next Monday.

Diamond Beamlines

2008-03-17T10:26:00.000-07:00

Today's seminar was by Dr Liz Duke from the Diamond Synchrotron. Liz was the first scientist employed to develop the protein crystallography beam lines over 5 years ago and now the first 3 beamlines are taking users. She outlined the design and some of the highlights (the first time she went into the experimental hall, the first beam and the first diffraction) and the ongoing issues that they want to improve. For example the sample robot takes 4 1/2 minutes to mount a new sample and be ready to collect data, which people find slow (although in tests the quickest a person could do it was 6 1/2 minutes). By really understanding the steps where corners can be cut they hope to take another minute or so off the time. They are now finding the balance between giving users access and finding enough time for themselves to implement the improvements that people want to see. However Diamond like all large projects is under some threat from the STFC (Science and Technology Facilities Council) review of its spending commitments. They do not have enough money in the next Comprehensive Spending Review to continue all the projects currently being funded and have launched a consultation http://www.scitech.ac.uk/STFCConsultation/comment.aspx?ci=1 which closes at the end of this week. Although Diamond is a High Priority there may not be as much money for the ongoing running and development costs as are needed.

Regulation of the EGF receptor

2008-03-07T04:10:00.000-08:00

The epidermal growth factor (EGF) receptor is (as its name implies!) a receptor that sits at the surface of cell membranes. Like many other such receptors, its intracellular region contains a tyrosine kinase; the extracellular part binds to a small, soluble protein, epidermal growth factor (EGF). When EGF binds it stimulates a conformational change that leads to a dimerisation of two EGFR molecules. This activates the tyrosine kinases so they pass a signal - essentially saying "EGF has bound here" - through the cell in a cascade of phosphorylation reactions.

EGFR is one member of a family of four similar receptors, known as the ErbB family; it can also be known as ErbB1. You may have heard, indirectly, of another member of this family, ErbB2; this is over-expressed on the surface of breast cancer cells in about a quarter of breast cancers. It is the target of the drug herceptin, which has transformed the lives for many women with so-called "herceptin receptor positive" breast cancer.

This week, Professor Mark Lemmon from the University of Pennsylvania gave a seminar at University College, London, about the structural basis for the regulation of the EGF receptor. This was one of the regular seminars organised through the Institute of Structural Molecular Biology, which brings together researchers at Birkbeck and UCL working in structural biology, chemical biology, biophysics, proteomics and bioinformatics.

Lemmon's research is concerned with the structure of the extracellular, EGF-binding regions of these receptors. These are made up of four domains - two "L-domains" and two cysteine-rich domains, arranged in the order L-C-L-C starting from the N terminus of the protein. EGF binds between the two L-domains, and the two C-domains form the interface between the monomers in the dimer. In the absence of ligand, the extracellular region adopts a "tethered" conformation in which the dimerisation domain is occluded. However, this inactive, auto-inhibited conformation can also exist in the presence of EGF; Lemmon and colleagues solved the structure of the entire extracellular region, with ligand bound and in an inactive conformation (PDB file 1nql).

Lemmon and his colleagues have now studied the transition between the inactive, tethered state and the active, extended one using the technique of small-angle X-ray scattering (SAXS), which is used for observing large conformational changes in molecules. They found, importantly, that introducing mutations into the "tether" region of the protein cannot drive the transition to the active conformation. Rather, in this protein (but not in the apparently ligand-less herceptin receptor) it is only EGF binding that can cause the transition to the active form.

A useful (if a few years old) review of tyrosine kinase structure and function is Hubbard & Till (2000), Annu. Rev. Biochem. 69, 373-398. This is accessible from the Birkbeck e-library with your username and password.

The bioinformatics of the 'flu

2008-02-19T09:34:00.000-08:00

What was the most lethal epidemic of infectious disease in modern times? AIDS perhaps? You might think so, but you would be wrong. Between the beginning of the epidemic and the end of 2007, AIDS killed people 25 million; the influenza epidemic of 1918 killed 40 million. This vast figure is also about double the number who died in the First World War, which ended the same year. Furthermore, a high proportion of those deaths were of healthy individuals in the prime of life. Yet most of the time we think of influenza as little more than a very nasty nuisance...

However, there has recently been a renewed interest in past influenza epidemics as a result of the result of the virulent strain of influenza currently sweeping through populations of wild and domestic birds worldwide. The influenza virus is endemic in birds, and strains tend to spread periodically from them to mammalian hosts: pigs as well as humans. Mapping genetic changes in the influenza virus, and how these affect its spread, is an important research area. Richard Goldstein of the National Institute for Medical Research, based in Mill Hill, London, gave a fascinating Monday seminar on this topic yesterday, looking into the past to see how genetic changes could have led to the lethal 1918 epidemic.

Influenza viruses contain two proteins on their spherical surfaces: a neuraminidase and a hemagglutinin. These proteins come in various forms: 16 different hemagglutinins are known, and 9 different neuraminidases. Any influenza virus can be characterised by these variants - for example, the most common type of influenza currently afflicting humans is H3N2, and the feared bird flu H5N1. Recent flu epidemics appear to have been caused by reassortment events, where the genomes of different viral subtypes combine to form an entirely new one that will not be recognised by human immune systems. An epidemic in 1957, for instance, coincided with a shift from influenza H1N1 (which had been circulating since 1918) to H2N2.

Influenza virus hemagglutinin and neuraminidase are both mainly-beta proteins, and their structures are described further on this page of PPS section 5.

So, what happened in, or before, 1918 to cause the epidemic of H1N1 flu? Molecular geneticists, such as Goldstein, study this by reconstructing phylogenetic trees showing the evolutionary distance between viral isolates taken in different places at different times. Yet most of these calculations can only show evolutionary distance, not the direction of change - in the jargon of phylogeny, they produce unrooted, rather than rooted, trees (there is no known "top"). The research was at rather an impasse until postdoc Mario dos Reis (a Birkbeck Ph.D.) noticed that the GC content of viruses infecting humans, but not of those infecting birds, decreased over time. This enabled the group to add an evolutionary "clock" to the phylogenetic tree for each of the influenza virus' 11 genes. This showed that some of the genes had entered the human population at different times, indicating that the variant that caused the 1918 flu had arisen from several recombination events. Interestingly, only one gene (neither H nor N) could have made the jump in 1918; most viral proteins were present in the human population in their 1918 forms well before that year. M1, like some other proteins, appears to have made the shift in 1899.

So, what did happen in 1918? There do appear to have been changes to the H gene then. But it may also be possible that the world population was so debilitated, and susceptible, after four years of war that a variant that had already been around for a few years was, unusually, able to cause such an epidemic...

Function of vFLIP - a protein from the virus associated with Kaposi's sarcoma

2008-02-11T06:24:00.000-08:00

Today's Monday seminar was given by Professor Mary Collins from the Division of Infection and Immunity at University College London's medical school. She described some of her studies of the function and mechanism of vFLIP, a protein from the human herpesvirus 8 (HHV8). This is a cancer-causing (oncogenic) virus and is responsible for the AIDS-associated tumour, Kaposi's sarcoma.

While very many people - about 10-15% of blood donors in London, for example - have been exposed to, and have antibodies for, this virus, it only causes problems in people with defective immune systems. In these people, however, it can cause lymphoma or multicentric Castleman's disease as well as Kaposi's sarcoma. This, however, is now - thanks to HIV and AIDS - one of the commonest cancers worldwide, in fact the commonest in some African countries. AIDS-related Kaposi's sarcoma is fairly rapidly fatal if untreated; however, it can now be controlled very well (although not completely cured) with anti-retroviral therapy.

HHV8 is a large (by viral standards!) double-stranded DNA virus. The protein vFLIP is one of a cluster of proteins expressed while the virus is latent. It binds to a protein called i-kappa kinase gamma in virus-infected cells. This causes the kinase to phosphorylate inhibitors of the cytokine NF-kappa B, leading to the release of NF-kappa B from inhibitor complexes and ultimately the degradation of the inhibitors. This leads to a cascade of gene expression that is essential for the prevention of apoptosis; in contrast, knockdown of vFLIP levels by siRNA will lead to induction of apoptosis. vFLIP is therefore implicated in the survival of virally infected cells.

Endothelial cells that have been infected by HHV8 adopt a characteristic "spindle cell" phenotype. Microarrays have shown that the virus induces complex re-programming of gene transcription in these cells, which is likely to induce this dramatic change in their morphology. Future work will elucidate the precise role of the signal transduction cascade induced by vFLIP in this "morphological reprogramming".

There is no structure available for vFLIP, but there are a large number of kinase structures known. From the UniProt database, the closest to i-kappa kinase is the intracellular Ser/Thr protein kinase domain of Mycobacterium tuberculosis PknB (PDB entry 1MRU).

Seminar by Prof Armitage, Oxford

2008-02-05T03:11:00.001-08:00

This weeks Monday seminar was by Prof Judy Armitage from Oxford (http://www.bioch.ox.ac.uk/aspsite/research/brochure/Armitage/) . Do go to her website as it will help you to see some of the pictures. She was interesting to us as a biologist (microbial physiologist) heading up a Systems Biology centre. Systems Biology is a trendy phrase but has almost as many meanings as there are centres. Systems Biology is really putting biology on a quantitative basis by developing mathematical models that accurately predict experiments. This can be at various scales, whole organisms, individual organs, whole cells or just a particular pathway. Prof Armitage has been studying the photosynthetic bacteria Rhodobacter sphaeroides for many years. It has now become more significant as Craig Ventner has found that a close relation is the most abundant organism in sea water and therefore central to photosynthesis in the sea.
However what Judy is currently studying is the chemotactic response where these bacteria swim towards their food source. In contrast to the well studied E.coli system where there is only one set of proteins, there are 4 operons in R.spheroides. Although in vitro the proteins were able to cross react and phosphorylate, they did not compensate for each other when deleted.
Her group showed by attaching fluorescent proteins (GFP/CFP) etc to the various components that unlike E.coli where the chemotactic proteins were all at the pole of the cell, there were two systems in R.spheroides, one at the pole but the other in the mid cell. One to sense the internal "happiness" of the cell- ie was it well fed and the other to go and find new food sources when it was not. She pointed out the importance of a number of their experimental details. Firstly they add the fluorescent protein in frame with the protein of interest in the genome so as to get natural abundance not overexpression. Secondly they only pay attention to those mutants where the pathway still functions as the fusions can sometimes disrupt the true localisation and give a false result, but when this happens the pathway does not work. The group has also done work on the flagellae that drive the bacteria and using a special fluorescent microscope were able to count the numbers of the subunits that drive the motor- 22 per complex.
Her take home message was that you cannot assume that all things work the same as E.coli!

2008-02-02T21:34:00.001-08:00

Dear Dr. Clare;

Thank you for inviting us to this blog. It should be a interesting experience! Take care, bye!

Best regards,
Dr. Nadia

Structure and mechanism of Hsp90, a heat shock protein

2008-01-21T08:51:00.000-08:00

Heat shock proteins are over-expressed when cells are exposed to heat or stress. Their function is to help other proteins to fold "correctly" into their mature, functional forms and, as such, they are classified as "chaperones". The structures and functions of these proteins will be described in much more detail in section 8 of the PPS course ("The Protein Lifecycle").

Today (21 January), in the School of Crystallography's Monday seminar programme, Maruf Ali from the Institute of Cancer Research in London spoke about his research on the structure of the heat shock protein Hsp90. This protein is found in all kingdoms except for the Archaea; it interacts with many other proteins (known as "client proteins" to help them enter their mature, active structural forms. Hsp90 client proteins include kinases that are important targets for anti-cancer drugs - hence the ICR's interest.

Hsp90 is a three-domain protein. The N-terminal domain binds ATP, which is necessary for the protein's activity; the middle domain binds client proteins; and the C-terminal one is involved in dimerisation. Structures of each domain separately were already known when Maruf started his post-doc a few years ago; each of these domains has a fold in Scop's alpha+beta class. Maruf's work involved solving the structure of a mutated form of the intact protein bound to a co-chaperone, (Ali et al. (2006), Nature 440, 1013-1019; PDB code 2CG9). This structure gives a clear picture of a complex structure, showing how a "lid" of structure closes to enabl the client protein to bind, and supports a previously proposed model in which the N-terminal domain is also involved in dimerisation.

Try downloading the structure, loading it into Jmol or a similar program, and seeing if you can identify the three domains.

CCP4 2008 Study Weekend

2008-01-15T06:03:00.000-08:00

CCP4 (Collaborative computing Project 4) is responsible for one of the main X-ray crystallography computer packages. It holds a study weekend the first weekend in January each year attended by 3-500 delegates. It is the social event of the UK protein crystallographers calendar as well as being the best methods meeting each year certainly in Europe. This year the topic was "Low Resolution Structure Determination and Validation" inspired by the retraction in Dec 2006 of five high profile structures of membrane proteins, which had been wrongly determined due to the author using an incorrect piece of software (not CCP4!) which meant that his maps were inverted ie his helices were all left handed rather than right handed. Because the resolution was low, as membrane protein crystals often are, this mistake was not immediately obvious from the maps and he forced through the refinement of right handed helices into left handed density by some dubious methods. Less was said about another structure published in Nature which essentially does not have the copies of the protein touching in the crystal as this is still under investigation by the American University from where this structure originates. Analysing these mistakes should help the community to avoid them themselves and possibly spot them as referees- all these papers had got past expert referees.
On a more positive note the first example of an ab initio (ie without a sequence homologue) prediction of a protein structure that was good enough to solve a crystal structure was presented. The main conclusion of the meeting is that the resolution of the structure is pretty much the only thing that determines the quality of the structure. Big structures at the same resolution are just as good as small structures. However big structures tend not to be at as high a resolution so that on average big structures are less well determined than small structures, but this is because they are lower resolution. Structural genomics groups are no better or worse than targetted labs at determining structures.

From a PPS point of view probably the most interesting talk was by Chris Tate from MRC in Cambridge who said that the retracted structure had to be wrong because it was incompatible with the biochemical data. Although structure is powerful, it has to be compatible with the biology. The system he worked on (EmrE) has another peculiarity in that half the protein inserts into the membrane in one direction (N terminus in) and half in the other (N terminal out) and the active molecule consists of one of each of these protein chains.

Tim Hunt's Lecture

2008-01-11T07:44:00.001-08:00

The Institute of Structural Molecular Biology, based at Birkbeck and University College, hosted a star performer as its first seminar speaker of 2008: Tim Hunt of the Cancer Research UK London Research Institute. Tim was awarded the Nobel Prize for Physiology or Medicine in 2001, with Leland Hartwell and Paul Nurse, for his discovery of cyclins - proteins that control the expression of the cyclin-dependent kinases (CDKs) which control a cell's passage through the cell cycle. So cyclins can be described as regulators of the regulators of the cell cycle.

Maybe only a Nobel Laureate could do it. Tim started his lecture with a quick tour through several hundred years of Physics, inspired by his small daughter's (unanswered) question "Why is the sky opaque?" He introduced (or re-introduced) his audience to Schrodinger's equation and Maxwell's idea of a "field" before confessing that he didn't understand quantum mechanics: no one should ever be ashamed of admitting as much.

The cell cycle, and its control, is, like quantum mechanics, "very interesting and very complicated". Tim's critical observation, which he made studying frog oocytes, was that cell division is controlled by the concentrations of the proteins that we now know as cyclins. They were given this name because their concentration in cells goes up and down according to where those cells are in the cell cycle - whether they are growing, replicating their DNA, undergoing mitosis...

Cyclins control the progress of cells through cell division by regulating the function of cyclin dependent kinases (CDKs). By binding to CDKs, cyclins control their activation state, and active CDKs drive the cells through the cell cycle. The press release for the 2001 Nobel Prize succinctly described CDKs as the cells' "motors" and cyclins as the gear boxes that control whether cells will be in idle or overdrive.

Cyclins are all-alpha proteins (link is to the PPS material) with 5-helical cores. PDB entry 1H1S shows human cyclin A bound to CDK2.

It is impossible to do justice to such a complex topic, and lecture, in a few paragraphs. To learn more, try the resources on the Nobel website would be a good first port of call.

Happy New Year!

2008-01-11T07:31:00.000-08:00

It must have been the way the holidays fell in the middle of the week, this year, but that was certainly a long break! But we are now back in earnest.

And I hope that none of you have forgotten what you have been learning over the long holiday. Section 5 of the course will be released on Monday 14th Jan.

And there have already been a few interesting scientific events of 2008...

British Crystallographic Association Winter Meeting

2007-12-19T08:45:00.000-08:00

The British Crystallographic Association is - as its name implies - the principal UK based scientific society for crystallographers. It is organised into several sections, one of which, the Biological Structures Group, is concerned with protein structure (as studied by X-ray diffraction).

The BSG, as it is known, holds its main yearly meeting (known as the Winter Meeting) in mid-December, often, but not always, in London. The 2007 meeting was held yesterday (18 Dec.) at the London School of Pharmacy. The overall theme of a wide-ranging programme was "Structural Investigations of Gene Regulation", covering an enormous variety of proteins that interact with DNA and control how and when genes are expressed.

Overall, the programme was divided into three sections: The Histone Code, Upstream Regulation, and Transcription, Translation and Recombination. And Birkbeck was represented in all but the first section. Tracey Barrett, who is course director for our Protein Crystallography course, presented her new structure of a kinase complexed to a protein from the virus that causes Kaposi's sarcoma (the most common cancer found in people with AIDS). Our new bioinformatics lecturer, Alona Sosinsky, described a model for DNA sequence recognition based on subtle differences in DNA structure rather than specific chemical groups.

More later, perhaps...

And Merry Christmas!

Welcome!

2007-12-19T08:33:00.000-08:00

Dear PPS People...

Welcome to the Principles of Protein Structure blog.

This is a new way for students and tutors on the PPS course to communicate with each other. We, the course tutors, will post accounts of conferences and seminars that we have attended, summaries of new papers, book reviews and anything else we can think of related to protein structure. Please comment on these!

Very soon you will also be asked to join in - please accept the request (note that you will need a Google account to do so) and write your own posts. There is only one rule, which is that all posts have to be related to protein structure - however peripherally...

This is an experiment - it is the first time that blogs have been used in our Internet based courses. It is up to all of us what we make of it. Enjoy!