A Very Short History of Crystallography

You might possibly have been intrigued to read in my last post that 2014 has been designated as the International Year of Crystallography. This year was chosen to celebrate the fact that this discipline - the study of atomic and molecular structure through their crystal forms - is now almost exactly a hundred years old. Admittedly, the first paper in the discipline, rather charmingly titled just "The diffraction of short electro-magnetic waves by a crystal" was published in 1913, and the Nobel Prize awarded two years later, but 2014 is at least a good compromise.

It can be said, perhaps simplistically, that crystallography was invented by the father-and-son team of William Henry and William Lawrence (known as Lawrence) Bragg, at the Universities of Leeds and Cambridge in the UK. The Braggs, however, did not aim to found a new discipline or even to investigate the atomic properties of matter. They were more interested in solving a problem that had been puzzling the cleverest physicists in the world for almost two decades. X-rays had been discovered by Wilhelm Rőntgen in Germany in 1895, but their very name (the unknown X) suggests reveals their controversial nature. Were they particles or waves?

The older Bragg, William, was convinced that X-rays were particles, and set out to prove this to his son (who favoured the wave theory) by exploiting the discovery of another German physicist, von Laue, that X-rays shone at a crystal were scattered and could produce a pattern on a film. Lawrence was the first to realise that these patterns could be explained by the theory that the X-rays were reflected from planes of atoms in the crystal and interfered with each other.

Lawrence presented these results to the Cambridge Philosophical Society late in 1912 and published them in the paper mentioned above the following year. This paper also included the first formulation of one of the best known of all laws of physics: Bragg's Law. This relates the wavelength of incoming X-rays and the angles that they are scattered (diffracted) to the spacing between planes of atoms in a crystal, enabling scientists to determine the geometry of atomic crystal lattices.

The Braggs worked together in Leeds and published their first structures, including that of sodium chloride (common salt) before Lawrence was sent to France to fight in the First World War. He was in the trenches when he heard that he and his father had been awarded the 1915 Nobel Prize for Physics; that news reached him shortly after that of the death of his brother Robert. At only 25, he was (and still remains) the youngest ever recipient of a Nobel Prize.

Technical advances between the wars enabled scientists working in this new discipline to solve the structures of rather more complex molecules. Kathleen Lansdale, one many women who began their research careers as the Braggs' students, solved the structures of benzene derivatives and was the first to see that aromatic rings were flat. And two later developments paved the way for the explosion in structural science that characterised the later twentieth century. In 1934, John Desmond (J.D.) Bernal, who later became the first head of the School of Crystallography at Birkbeck (the predecessor department of our Biological Sciences) and his student Dorothy Crowfoot (later Hodgkin) obtained the first X-ray diffraction patterns from protein crystals. And in the following year Lindo Patterson developed a function that greatly simplified the mathematics involved in structure determination.

Even fifty years ago however, solving crystal structures was a long and at times tedious business. A typical crystallogaphy PhD thesis of the 1960s or 1970s would contain the structures of maybe three small or medium-sized molecules. It is now possible to generate as many in a few hours, so it is possible to see clearly how structures of molecules respond to changes in conditios such as temperature and pressure.

All these discoveries have been made possible by advances in technology, and particularly by the development of synchrotron radiation as a source of powerful beams of X-rays. Synchrotron radiation is produced when charged particles are accelerated radially, and synchrotrons built primarily as X-ray sources were first built in the 1980s. The UK's synchrotron, Diamond at Harwell in Oxfordshire, is currently the fifth largest in the world. It has 23 separate "beamlines", each providing a beam of X-rays with properties that have been optimised for a particular experimental technique.

Synchrotrons provide facilities for solving structures from single crystals of large and small molecules, including, of course, proteins, and from micro-crystalline samples (the latter technique is known as powder diffraction). Although structural biology attracts much of the attention (see almost all the other posts on this blog) structures of smaller molecules can still provide important insights. Sandy Blake, a crystallographer at the University of Nottingham, is using Diamond beamlines to solve the structures of novel materials called metal-organic frameworks or MOFs that are able to store gases including hydrogen (which is a potential fuel source) and greenhouse gases.

At 100, crystallography is still a young discipline but it has radically transformed many other areas of science and, through them, the world we inhabit today. This has been reflected in decisions made by the Nobel committees over the decades. The International Union of Crystallography maintains a list of Nobels awarded for ‘achievements directly related to, or involving the use of, crystallography’. There are now 29 of these, and the latest year with no crystallography-related Nobel was 2008. Even the 2013 Chemistry prize, awarded to Martin Karplus, Michael Levitt and Arieh Warshel, appears on the list: their discipline of computational chemistry would be impossible without structural knowledge obtained through crystallography.

And it almost goes without saying that protein structure, and structural biology more generally - the disciplines taught in this course and its associated MSc - owe their existence to the development of X-ray crystallography.

This blog post is based on an article I wrote for the Royal Society of Chemistry's membership journal, Chemistry World. It will be published in the January 2014 issue of the journal.

Welcome to PPS Students 2013-14!

This post is very like one I wrote at exactly this time last year. This is because what I have to say now is very, very similar...

I would like to offer 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 2013 is Molecular Mechanisms of Intracellular Trafficking: an important topic that relates quite closely to some of the material we cover in the later sections of the course. Other posts may be reports from conferences or summaries of recently published papers in protein structure, protein bioinformatics and allied areas.

And one topic that you are bound to hear more of on this blog, particularly after the New Year, is the history of structural science, particularly X-ray crystallography. Crystallography was the first method to be developed for solving the structure of biological macromolecules, and it is still the most important. The United Nations has designated 2014 as the International Year of Crystallography - the date is between the centenaries of the publication of the first papers on X-ray diffraction and the award of the 1915 Nobel Prize for Physics to the father-and-son team of William and Lawrence Bragg who made the principal discoveries.

Some of the posts on this blog are 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 of the earlier blog posts 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 2013-14 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

GroEL, giving misfolded polypeptides a second chance

Saibil, H.R., Fenton, W.A., Clare, D.K., Horwich, A.L. (2013) Structure and Allostery of the Chaperonin GroEL. J Mol Biol. 2013 May 13;425(9):1476-87

A recent paper, by Professor Helen Saibil’s team at Birkbeck, reviews the current understanding of chaperonin GroEL.  Chaperonins attract unfortunate proteins which are incompletely or incorrectly folded and provide them with an isolated chamber in which to bind and release until they achieve their native functional state.  GroEL and its partner GroES are profiled in PPS in Section 7 (symmetry) and Section 8 (action as a chaperone).  

GroEL is remarkable in its construction.  It consists of 14 identical protomers arranged in two back-to back rings, each of the two rings with seven subunits.  This forms a barrel with a 7-fold rotational symmetry axis through its centre and, perpendicular to this, seven 2-fold axes of symmetry, giving an overall symmetry of 72.

Each subunit comprises two main domains linked by an intermediate domain (see figure (c)).  The largest domain is equatorial at the centre of the barrel.  This contains the ATP binding site and is in contact with its two neighbours in the ring as well as the equatorial domains of its partner ring.  These domains form a stable platform from which the other two domains undergo large movements orchestrated by the cycle of ATP binding, hydrolysis and release.  

The apical domains are exposed at the outer ends of the GroEL barrel.  They are smaller and include a hydrophobic surface which is the binding site for many different nonnative polypeptides.  The intermediate domain has a hinge at the junction with each of the two main domains, such that it can mediate large movements of the domains as rigid bodies.  This can be seen by comparing figures (c) and (f).

 

X-ray crystal structures of GroEL and GroEL-GroES complexes.  (a) Longitudinal cross-section of GroEL (PDB 1OEL). (b) Top view of the GroEL barrel. (c) A protomer of GroEL, aligned approximately as the top left protomer in (a).  (d-f) Show the same set of views with GroES (d-e) and ATP (f) bound (PDB 1SVT).  Example helices have been coloured to demonstrate the extent of the rotation angles.  The red and orange helices of the apical domains can be seen to undergo a significant rotation.  Compare this with the relatively minor movements of the green helices in the intermediate domains and the violet helices of the equatorial domains.

The operations of the GroEL chaperone are initiated by rapid binding of ATP to the equatorial domain of one of the rings.  This is followed by the binding of the unstructured, partly folded or misfolded polypeptide.

Natively folded proteins have their hydrophobic residues buried in the stabilising core whilst those which have lost their way have exposed hydrophobic patches.  These patches bind to the hydrophobic surfaces of the apical domains.

The final ligand is GroES, a ring of seven homo-oligomers, which forms a lid for the GroEL barrel.  Each GroES monomer has a flexible hydrophobic loop which binds to the hydrophobic regions of the apical domain alongside the substrate polypeptide.  This loop is visible in figure (d). 

The apical domains undergo significant concerted rotations together as one movement, with the domains being held as rigid bodies (compare figures (a) and (d)).   These rotations replace the hydrophobic polypeptide binding surface with hydrophilic residues, so propelling the nonnative protein into the central lidded cavity, where it is isolated to refold.

As ATP binding stimulates positive cooperative movements within the cis ring, that is the ring binding the nucleotide, it is also responsible for negative cooperation between the rings.  This means that while the movements are coordinated to bind GroES and promote protein folding in the ATP bound cis ring, in the trans or partner ring the opposite rotation prompts the release of GroES, more than 100Å away, and the expulsion of the now native protein.  

Mutation studies have revealed that salt bridges, which are studied in PPS Section 9, hold the rings steady until full ATP occupancy is achieved and are probably involved in the positive cooperativity whilst the negative cooperativity is thought to be triggered by a pivoting of the equatorial domains.  This interferes with the staggered contacts between each equatorial domain and two of its partner equatorial domains on the opposite ring.

Recent work using single particle cryo-electron microscopy techniques, which is studied in the TSMB course, has captured images of the intermediate states between ATP binding and the active chaperone state where GroES is fully bound.  

Once ATP binds, the intermediate and apical domains tilt 35˚ sideways from the lower hinge.  This brings the intermediate domain towards the ATP binding pocket where the residue ASP398 forms several hydrogen bonds.  This action causes the breakage of salt bridges between the intermediate and apical domains of neighbouring protomers and between neighbouring apical domains with new salt bridges forming which support the new tilted architecture.

Following this, the apical domains lift and separate, to use an old advertising slogan, causing further breakage of salt bridges between apical domains.  This separation could help to unfold the misfolded polypeptide before it is released into the chaperone chamber and also positions the hydrophobic binding areas for docking of the GroES binding loops.

Once GroES is bound, the apical domains lift even further outwards and undergo a 100˚ twist to create the active folding chaperone with the GroES lid in a domed position and the polypeptide is released into the cavity to complete its folding.

The next stage is hydrolysis of the ATP, which triggers the release of the ligands on the cis ring, and the acceptance of ligands on the trans ring.   

The mechanism is believed to involve separation of a β sheet contact between equatorial domains of the trans ring.   The equatorial domains are primarily responsible for holding the rings together so that the ADP complex has reduced stability. Hydrolysis is followed by ATP binding to the equatorial domains of the trans ring.  This promotes the pivoting of the equatorial domains that defines negative cooperativity and the discharge of GroES, the native protein and ADP from the cis ring, although the exact movements which lead to the discharge are unknown.

It seems likely that the release is mediated through a reversal of the twist in the cis apical domains.  This is speculation, however, as this good Samaritan of nanomachines has not yet given up all of its trade secrets.  The progress made to date, however, in large part by Professor Saibil’s team, is a striking demonstration of the power of this recently developed method in structural biology.

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.