Welcome to new PPS students - and a few more links...

This post is very like those I have written at the beginning of the academic year for the past few years; this is because what I have to say now is also very similar...

I would like to offer a warm welcome to the Principles of Protein Structure blog to all students who have recently started studying Birkbeck's Principles of Protein Structure (PPS) course, and a welcome back to any who have taken a break in studies and intend to complete the course this year. Welcome too if you are thinking that you might want to study with us in the future, or if you are just interested in learning more about a fascinating and fast-moving area of research in molecular biology.

I run this occasional 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. 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 programme for Autumn 2018 has the intriguing title of 'Mischievous Microbes'; its themes of microbiology and infectious disease biology have links to some of the later sections of the course. Other posts may be reports from conferences (such as this one at Imperial College, London in December) or summaries of recently published papers in protein structure and allied areas/

Some earlier posts were written by "guest blogger" Jill Faircloth, who took the MSc in Structural Molecular Biology a few years ago and is now working as a freelance science communicator. She introduces herself in this post written in March 2012, in which she also describes how she found the later part of the PPS course and her thoughts on the two choices available for the second year of the MSc.

Do, if you get a chance, look through some blog posts from earlier years 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!

I also work part-time as a freelance science writer, and sometimes I even have a chance to write about structural biology. You might like to follow a set of blog posts I wrote from the International Union of Crystallography's triennial meeting in Hyderabad, India last summer (posts from 22 August - 6 September). The first entry, featuring a talk by Sir Tom Blundell, a former head of the Department of Crystallography at Birkbeck (now part of the Department of Biological Sciences) is perhaps most relevant to PPS. Sir Tom was involved in solving the structure of HIV protease, target of some of the most successful drugs for AIDS, and he went on to found a drug discovery company, Astex. This year I reported on a meeting much nearer at hand (in Liverpool) and, specifically, on one of the most exciting advances in structural biology by X-ray crystallography for some years: X-ray free electron lasers.

Finally, the best of luck to new students for the 2017-18 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
Tutor, Principles of Protein Structure

From Crystallography to Light (according to the United Nations)

Those of you who started the PPS course in 2014 may not have realised that they began their studies of structural biology at an auspicious time for this subject. Last year was designated the International Year of Crystallography to mark the centenaries of both the discovery of X-ray diffraction by crystals (first published in 2013) and the award, in 2015, of the Nobel Prize to the father-and-son team of William and Lawrence Bragg who made the discovery. I have blogged about this in much more detail over the previous year, with a post in December 2013 on "a very short history of crystallography" and one in July reporting on lectures by two of our distinguished emeritus professors, Paul Barnes and David Moss. Professor Moss took the subject up to date with a race through the history of structural biology, from the publication of the first protein structures - myoglobin and haemoglobin - in 1958 to the 100,000th structure that entered the Protein Data Bank in 2014. If you want to read more about the numerous events and publications around the world that honoured and publicised crystallographers during 2014, the best place to start is probably the official website of IYCr2014. And if you have access to Chemistry World, the membership magazine of the Royal Society of Chemistry, you can read two more pieces by me: "Crystal Clear" in the January 2014 issue and "Life in the Freeze Frame", featuring structural biology, in September. (Unfortunately, it is not available in the Birkbeck e-library.)

2014 was designated as the International Year of Crystallography by the United Nations, as part of its programme of using anniversaries to highlight topics and issues that it sees as important. There have been many international years on science-based topics, some broader than others: 2005, for example, was the International Year of Physics. And in this new year the focus moves on to another physical science topic. 2015 is the International Year of Light: or more precisely that "... of Light and Light-based Technologies".

And optics, the science of light, is very closely related to crystallography. Both light and X-rays are parts of the electro-magnetic spectrum and as such are both particles and waves; the differences in their frequencies and wavelengths lead to their very different properties. The first X-ray diffraction experiments were conducted mainly to find out whether the hypothesis that X-rays were electromagnetic waves with a wavelength of about 1 Angstrom was correct. The utility of this technique as a probe for molecular structure was initially a side effect of the similarity between this wavelength and the length of a covalent bond. (So-called "copper K-alpha" radiation, which is often used in crystallography, has a wavelength of 1.54Å, which is exactly equivalent to the length of a single C-C bond.) And the basic equation that defines X-ray diffraction - Bragg's Law, or nλ = 2d sin θ - applies to all electromagnetic radiation, not just to X-rays.

Advances in optical technology have been responsible for some important advances in crystallography. The discovery, in the mid- twentieth century, that charged particles emit electromagnetic radiation in the form of X-rays when accelerated radially was initially viewed as a problem of energy loss. This "lost" radiation, which is orders of magnitude more intense than the X-rays used in the first diffractometers, was soon exploited in synchrotron radiation sources and most non-routine protein structures are now solved using synchrotron radiation. The UK's synchrotron source, a doughnut-shaped structure half a kilometre across located at Harwell in Oxfordshire, is even known officially as "Diamond Light Source". And recent advances in optics are gradually reducing the size of intense X-ray sources so that it will become possible to do complex structural biology that now needs a synchrotron using a lab-based machine.

The next big technical advance to influence X-ray crystallography is the free electron laser, which can generate pulses of X-rays that are much more intense than those produced by even the largest synchrotrons. This is a beam of electrons that is accelerated to almost the speed of light through a side-to-side magnetic field to produce pulses of extremely intense electromagnetic radiation. These pulses are exceptionally short, each lasting only a few femtoseconds (1 fs = 10^-15s). This is a feature of the technology that leads to two advantages: the crystals remain intact, even though they are exposed to extremely intense radiation, and it becomes possible to take "snapshot" structures very close together in time study the dynamic behaviour of molecules. Free electron laser radiation also enables precise structures to be obtained from much smaller crystals, which makes large complexes and membrane-bound proteins more tractable to crystallography. And it may one day be possible, using this technique, to determine a structure from the smallest "crystal" of all - a single molecule - and thus liberate protein crystallography from one of its most important bottlenecks: the need to grow protein crystals.

Acknowledgement:This post owes much to this one on the "Crystallography 365" blog (author unknown)

Science Week 2013: Structures of Sodium Channels

Since 2010, Birkbeck College has held a week of lectures, most often in the spring, to highlight some of the research carried out in the School of Science. This year’s speakers included Professor Bonnie Wallace from the Department of Biological Sciences, who presented a fascinating and accessible lecture on the structures of sodium channels, and what these new structures are already teaching us human health, and particularly about some rare neurological diseases.

Professor Nicholas Keep, Dean of the Faculty of Science (and director of the MSc in Structural Molecular Biology and the second-year option TSMB) introduced Professor Wallace. She has been at Birkbeck for about twenty years and now directs the department’s impressive research work on the structural biology of membrane ion channels. You will learn a lot about membrane proteins in general in section 11 of PPS; they are ubiquitous, are responsible for the transport of both chemicals and signals into and out of cells, and form some of the most important drug targets. They are also, as Wallace made very clear in her talk, some of the most challenging of all proteins for structural biologists to work with.

All cell membranes are semi-permeable, which means that some substances can pass across them easily while others are excluded. Ions, which are charged, are generally excluded by the hydrophobic (“water hating”) membranes. This could be something of a problem, as ion transport into and out of cells is an essential physiological process. Ion channels are evolution’s solution to this problem: proteins embedded in membranes that allow ions to selectively enter and leave cells.

Much of Wallace’ work over the last ten years has focused on the structures of voltage gated sodium channels. These open to allow sodium ions to enter cells, and close to prevent them from doing so, in response to changes in potential across the membrane, and they are found throughout nature. Small molecules can bind to these channels, holding them either open or closed; some of these are severely toxic, but others are important drugs for cardiac arrhythmias, epilepsy, and pain.

Human voltage gated sodium channels are composed of a single protein chain, divided into four similar domains. Each of these domains has six transmembrane helices, four of which (labelled S1-S4) act as a voltage sensor while the other two (S5 and S6) fold together to form an eight-helix pore. This protein has so far proved impossible to crystallise, and the breakthrough involved a bacterial protein. Similar proteins are found in the membranes of some species of bacteria, enabling them to live in “extreme” environments that are rich in salt. Their structures are similar to those of the human protein, but in this case the channel is built up from a complex of four identical proteins, each of which is homologous to a single domain of the human channel.

Although this simpler bacterial protein proved easier to work with than the human protein, it was still not at all easy. It took over ten years for Professor Wallace and her group to isolate the gene, clone and purify the protein, obtain crystals and finally solve the structure of the pore. The structure was finally solved using the powerful X-rays generated at Diamond, the UK’s only synchrotron radiation source located near Harwell in Oxfordshire.

These channels exist in three different structural forms: “open”, “closed” and “inactivated”. Many years before the detailed structures were solved Wallace and her group had used a biophysical technique, circular dichroism (CD) spectroscopy, to examine the conformational changes that occurred when mammalian and bacterial channels switched from one state to the other. As always, however, the full atomic-crystal structures yielded very much more information.

The first of these structures to be solved was a slightly strange one: the pore was held in the “closed” conformation that prevents sodium ions from entering the cell, although the voltage sensor was in the structure associated with the “active” state (PDB code 3RVY). The “top” part of this structure, towards the extracellular membrane surface, has a hydrophobic surface, and the pore in this part of the membrane acts as a selectivity filter to allow sodium ions in while keeping others, including potassium and calcium ions, out. Wallace and her group were the first to solve the structure of a fully open channel and showed that the upper portion of the channel containing the selectivity filter was virtually unchanged. The conformational change associated with opening and closing the channel occurs at the internal or cytoplasmic side of the protein (PDB 4F4L). When the pore closes, a small turning motion of the “bottom” part of the helical bundle causes the diameter of the pore to shrink, in a motion rather like the closure of a camera lens; the resulting channel is too small for sodium ions to pass through, so any inside the pore become trapped there.

Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure

All voltage gated sodium channels have a domain at the C-terminal end of the molecule that is necessary for channel activity but that was not visible in any of the crystal structures. Wallace and her group looked at this part of the molecule in the bacterial protein using a particularly powerful form of CD spectroscopy called synchrotron radiation CD spectroscopy that she had pioneered, and showed that each subunit had an extremely flexible protein chain separating the pore from a C-terminal helix. Using this information, the group have proposed a novel mechanism for channel opening in which the conformational change in the pore is enabled by these helices oscillating up and down.

Two subunits of the bacterial sodium channel pore in the “open” conformation, shown as a ribbon structure

The final part of Wallace’ talk was devoted to the role of sodium channels in health and disease, and as a drug target. A few unfortunate individuals have mutations in a type of channel that is involved in the response to painful stimuli. If this channel is jammed open, patients experience a constant, burning pain termed erythromelalgia, most commonly in their hands and feet. Wallace showed that an equivalent mutation from phenylalanine to valine at the base of one of the bacterial protein subunits caused the channel to open just enough for ions to pass through. There are also people in whom these channels are jammed in the closed position, and they feel no pain, even if they walk on hot coals. It may one day be possible for drugs based on our knowledge of these structures to be designed to ease both these conditions.

Twenty Years of Structural Biology in Drug Discovery

Approximately once a term, scientists working in structural biology and related areas in and near London, meet under the auspices of the London Structural Biology Club to discuss their recent research. These Club meetings generally involve four or five lectures followed by an informal discussion over beer and pizza, generously provided by a sponsoring company.

Most speakers at LSBC meetings are academic researchers and a typical presentation will highlight a novel structure or two, emphasising, for a specialist audience, some of the trickier aspects of how they were solved. The most recent meeting, however – held on 3 July 2012 in Birkbeck’s Clore Management Centre, and sponsored by specialist light scattering company Avid Nano – included one rather unusual talk. Dave Brown recently left the pharmaceutical giant Pfizer after sixteen years in its structural biology group. He now combines an academic post at the University of Kent at Canterbury with work in a new biotech company, Cangenix Molecular Solutions. During his time at Pfizer, Brown contributed his expertise in structural biology to drug discovery programmes for a wide range of cardiovascular, inflammatory and infectious diseases. The topic of his LSBC presentation was the evolution of structural biology in the pharmaceutical industry as he had experienced it during the last two decades.

The history of structural biology in drug discovery goes back a little longer than that, to the use of NMR by companies such as Abbott and Agouron to determine structure-activity relationships for compound series. The first drugs to be largely designed based on structural principles were the HIV protease inhibitors, which are covered in some depth in both section 5 and section 10 of PPS. The first protease inhibitor to be licensed for treating AIDS was Saquinavir (Invirase™), which entered the clinic in 1995, only ten years after the protease gene was first detected in the newly sequenced HIV genome.

By the mid-1990s, most major pharma companies had specialist structural biology groups. The most important developments since then have been driven by technical improvements in molecular biology, gene cloning and protein purification, and in diffraction technology. Synchrotrons such as the UK’s Diamond Light Source make their facilities available for commercial use at a price that large companies, at least, have no trouble affording. X-ray data collection is now extremely fast and can largely be automated so it has become accessible to non-expert users. Although dedicated structural biology groups in industry are no larger than they were in the 1990s, there are many more researchers there – medicinal chemists and molecular biologists – who spend some of their working life doing structural biology. Arguably, a reasonable knowledge of protein structure is essential for all scientists working in drug design.

But what, exactly, does structural biology contribute to the complex process of drug discovery? Once a likely protein target for a new discovery programme has been identified – an enzyme to be targeted by an inhibitor, for example – knowing its three-dimensional structure can help both in the selection of likely starting molecules or “hits” and in their development into “lead” compounds that are potent enough inhibitors to go forward to the later stages of drug discovery. In the related technique of fragment-based drug discovery, a huge number of very small compounds or fragments are screened against a target and those that bind to different parts of the protein’s active site are selected and linked together to form a larger and theoretically more potent compound. Typically, individual fragments bind to their target so weakly that they can only be identified experimentally rather than through computer modelling. Structural experiments can also be used to probe unexpected ligand-binding interactions, perhaps even identifying previously unknown functional binding sites, and to build selectivity for one member of a large protein family into a drug molecule. Selectivity is essential for the development of safe, non-toxic drugs that bind proteins such as kinases and G-protein coupled receptors (see section 11) that have hundreds of homologs in the human proteome.

Brown then went on to describe some of the work that he had been involved with during his career at Pfizer. Phosphodiesterases are enzymes that catalyse the breaking of phosphodiester bonds; this function is responsible for regulating the concentration of the essential cyclic nucleotide monophosphates in cells, and enzymes in this class are drug targets for a range of diseases. Cyclic GMP-specific phosphodiesterase type 5 or PDE5 is the target of Pfizer’s most famous drug, sildefanil, which is known worldwide as Viagra™ (link is to the Wikipedia entry). Researchers at Pfizer have used structure-based techniques to modify the basic structure of this compound into drugs with the potential to treat very different diseases, including Reynaud’s syndrome (in which the blood supply to the extremities is reduced in cold temperatures) and stroke recovery. The recent structure-led discovery of the mechanism by which another protein in this family, PDE4, regulates cyclic AMP may even lead to the design of drugs for a range of devastating neurological conditions including Alzheimer’s disease and schizophrenia.

Brown summed up his lecture with a series of recommendations for structural biologists working in industry (and by extension, for industrial researchers doing structural biology there). He advised them to get involved in drug discovery programmes at the earliest stage, to use structure to understand mechanisms of action rather than simply to design inhibitors and to collaborate between disciplines and sometimes even across companies. And, overall (and more prosaically), to work as quickly and cheaply as reasonable. These are not bad recommendations for academic researchers, either.