Molecules that Walk

This post was contributed by Dr Clare Sansom, senior associate lecturer in the Department of Biological Sciences. Dr Sansom attended Dr Anthony Roberts’s lecture during Birkbeck Science Week 2016.

Molecules

The Department of Biological Science’s contribution to Science week 2016 kicked off on 11 April with a lecture by Dr Anthony Roberts, a young Principal Investigator who arrived at Birkbeck in 2014. Anthony received his B.Sc. from Imperial College in London and his Ph.D. from the University of Leeds, and spent four years as a postdoc at Harvard in the USA before moving here to start his own research group as a Sir Henry Dale Fellow of the Wellcome Trust and Royal Society.

Walking molecules and their importance for human health

Anthony began his lecture by explaining that he was going to talk about molecules that have the capacity to produce directed movement – or to ‘walk’ – and their importance for human health. These molecules are all proteins, and the context in which they move is the interior of living cells. Both the proteins he studies, kinesin and dynein, ‘walk’ on a network of highways conceptually not unlike the transport system that we use to move around London. These cellular highways are filaments called microtubules, which, unlike our roads and railway tracks, are able to self-assemble and also to self-destruct.

The ability to move is one of the fundamental properties of life, and scientists and philosophers have been studying it for millennia. Muscles were identified as the organs of movement in antiquity, but it was not until the mid-twentieth century that the molecules involved in muscle contraction could be identified. The Hungarian physiologist Albert Szent-Györgyi discovered the muscle proteins now named actin and myosin using very simple equipment during the Second World War.

These proteins have similarities with kinesin and dynein, although historically they have been easier to study due to their abundance in muscle; actin forms fibrils and the enzyme myosin binds to and ‘walks’ along these filaments. This process, like all movement, requires energy, and this is obtained from the cell’s power source, the small molecule adenosine triphosphate (ATP). The part of the myosin molecule that binds to actin, which is called its head, breaks a phosphate bond in this molecule to liberate energy and power the walking motion; many of these ‘power strokes’ together cause the muscle fibre to contract.

Snapshots

Dr Anthony RobertsIdeally, we would want to watch this, or any other form of molecular motion, in real time, but this is impossible because molecules are far too small: smaller than the wavelength of light, so they cannot be viewed in a light microscope. Studies of molecular structure require techniques like X-ray crystallography and electron microscopy, both of which have been used to study motor molecules.

However, neither of these techniques can do more than generate still images. Movement can only be inferred by taking lots of snapshots of the molecules at different points during the movement cycle, rather like the earliest movies. We have now built up a complete picture of actin and myosin that is detailed enough for the positions of individual atoms to be seen clearly.

Not all movement in nature, however, uses muscles. Single-celled organisms – the ‘animalcules’ observed by pioneer microscopist Antonie van Leeuwenhoek in the 1670s – have directed movement, as do bacteria, and these have neither muscles nor nervous systems. And directed movement also occurs inside cells. A good example of this is the division of replicated DNA between daughter cells during cell division.

The interior of all cells is a viscous mixture, crowded with molecules; it is possible for small molecules to move from one part of a cell to another through diffusion, but this process would be impossibly slow for larger ones. Motor proteins, on the other hand, can carry ‘cargo’ molecules across cells remarkably quickly and efficiently. Motor proteins can traverse a distance of 0.1 mm – the length of a large animal cell – in two minutes, which in terms of lengths per second is approximately three times faster than a car.

Both the motor proteins studied in Anthony’s lab, kinesin and dynein, ‘walk’ along microtubules inside cells. These filaments typically form with one end towards the centre of the cell, and its nucleus, and the other towards the cell periphery, and the motor proteins move in opposite directions: dynein towards the nucleus, and kinesin towards the cell edge.

Any kind of directed movement by molecules is challenging for several reasons. Motor molecules have no equivalent of our nervous systems for controlling movement, and they are far too small to be held on their tracks by gravity; instead, they grip the microtubules using chemical forces. They experience negligible inertia, and are constantly buffeted by other molecules in the cell. It would therefore be catastrophic for the whole of a walking molecule to leave its path at once.

A single leg with two feet

The structure and function of conventional kinesin are now fairly well understood. It consists of two identical protein chains, and each chain has two major domains separated by a short linker. The larger domain of each chain coils together to form a single long stalk; the smaller domain is globular and attaches to the microtubule, so the molecule looks rather like a single leg with two feet. Each of the feet is an enzyme that generates the energy for the motion by breaking down ATP to form ADP and release a phosphate group, and it cycles between ATP-bound, ADP-bound and empty states.

The step between ADP-bound and empty is a bottleneck that can be relieved when the foot attaches to the microtubule in a particular position, ensuring that the whole molecule moves in the correct direction. The trailing foot is released from the microtubule and the cycle begins again once ATP has bound to the front foot, triggering a conformational change in the whole molecule.

The core of kinesin is similar in structure to myosin, suggesting that these two proteins have a common ancestor. The other microtubule-bound motor protein, dynein, has a different origin. Although we still know comparatively little about it, it was actually the first of the microtubule-bound motor proteins to be discovered: this was in the 1960s, when it was found as the protein that generates the force that allows protozoa and sperm cells to swim. Anthony’s group, however, has been studying how it functions inside cells to move ‘cargo’ – often nucleic acids or other proteins – from the edges of the cell towards its interior. It also helps to pull the duplicated genetic material between the two halves of the cell during cell division.

The structure of Dynein

Dynein

Dynein

Dynein is a much larger and more complex molecule than the other motor proteins. Its structure, like those other proteins, has several components: in this case, a stalk, a ring and a tail, with a linker between the stalk and the ring. Much of what we know about this large structure has come from electron microscopy, and more recently X-ray crystallography.

Anthony’s group and others have developed a model in which the main mechanical element is the linker, which bends and straightens to displace the cargo-bound end of the structure along the microtubule in the direction of travel. The image shown here is a still from an animated model of how dynein generates movement, which remains speculative in places and is helping to stimulate new experiments in these areas. It is also incomplete, as it only shows one half of the molecule: we do know that dynein, like kinesin, is a biped, but exactly how its ‘feet’ are coordinated remains at the frontier of our knowledge.

Anthony ended his talk by discussing some actual and potential medical applications of studies of walking molecules. Some commonly used anti-cancer drugs, including taxol, work by stabilising microtubules to prevent motion and therefore stop cancer cells from dividing. Molecules that interact with motor proteins are also being studied as potential treatments for neurodegenerative diseases and for some types of heart disease. One such compound is a myosin activator, omecamtiv mecarbil, which is showing promise as a treatment for heart failure. And we are likely to discover further applications as we learn more about these fascinating walking molecules.

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Computational Modelling in Structural Biology

This post was contributed by Dr Clare Sansom, senior associate lecturer in the Department of Biological Sciences. Dr Sansom attended Dr Maya Topf’s lecture on Computational Modelling during Birkbeck Science Week 2016.

Brain computer (copyright Marcos Fernandez via Flickr. Image cropped)

Brain computer (copyright Marcos Fernandez via Flickr. Image cropped)

The Wednesday of Birkbeck Science Week – 13 April – was set aside to celebrate women in science, and it included a talk by Maya Topf of the Department of Biological Sciences. Maya, who was educated in Israel and Oxford, came to Birkbeck on an MRC fellowship after a post-doc at UCSF and has rapidly worked up the academic ladder to the position of reader in computational biology. She will be appointed as a full Professor in October this year.

Maya began by explaining that her research involves making models: specifically, three-dimensional models of biological molecules. Models have enabled scientists to make sense of biological processes since Watson and Crick’s double helical model of DNA showed how this molecule could both replicate itself and act as a template for the synthesis of proteins. This model, celebrated in the film Life Story that was shown earlier in Science Week, would not have been possible without the X-ray photographs of DNA fibres obtained by Rosalind Franklin, then working at King’s College.

The purpose of computational modelling

And the main purpose of modelling molecules is the same now as it was in the 1950s: to discover how they function, and specifically how they function in the environment of the cell. We still have no means of observing what protein molecules – the tiny ‘machines’ that drive all cellular processes – look like when they are at work; all we have is models that may be more or less precise. The very first protein structures to be determined were of the oxygen-carrying proteins myoglobin and haemoglobin, and the first of these, published in 1960, were very imprecise: it was possible to see the shape of the chain but no individual atom positions. These, and all early protein structures, were obtained by X-ray crystallography; ten years later the same group used the same technique to determine a structure in which all atoms except hydrogens could be seen.

DNAThese two proteins have now also been studied using two other structural biology techniques, nuclear magnetic resonance and, most recently, electron microscopy. This last technique is best suited for studying large proteins and complexes of many protein chains, and therefore not suitable for studying most forms of haemoglobin, a small, simple protein. Haemoglobin in earthworms, however, functions as a complex of many individual molecules. Electron microscopy gave a low-resolution picture of the overall shape of these molecules, much like those first haemoglobin structures, and a more precise picture was built up by ‘docking’ atomic-resolution X-ray structures of a single haemoglobin molecule into the shape of the fold.

During the last half-century these three techniques have generated structures for a wide range of proteins, leading to insights in many areas of biochemistry: how the body’s catalysts, the enzymes, work; how drugs bind to their receptors; and how a ‘large’ molecular complex, the ribosome, can synthesise all the proteins that a cell needs from RNA templates. The first atomic structures of this ‘molecular machine’ were obtained in the early 2000s and have transformed our view of protein translation since then (see these videos from the Howard Hughes Medical Institute in the US: basic and more advanced versions).

But, as real proteins are too small to be visible with even the best light microscopes, we need to realise that even these experimental structures are models. Each of the three techniques has its own advantages and limitations. X-ray crystallography needs protein crystals, which can be difficult or even impossible to obtain for particular proteins; electron microscopy cannot be used to study small proteins, but NMR works best with these. All three techniques are complex, time-consuming and expensive, and therefore proteins with known structures are greatly outnumbered by those without structures. There are probably about 43,000 known structures of ‘distinctly different’ proteins known compared to over half a million well-characterised protein sequences.

Bridging the sequence-structure gap

Maya explained that much of her group’s work concerns trying to bridge this ‘sequence-structure gap’ by using computers to model unknown protein structures. There are several ways of doing this; if the computers are powerful enough and the molecule is small enough (and the smallest proteins can be) it is possible to generate a model structure ‘from first principles’ using physics. These techniques assume that the molecules are likely to occupy conformations in which their energy is low. The best results simulate protein folding to produce model structures that can be very close to the experimentally-determined ones, but these require an enormous amount of computational power. Less expensive computer modelling methods tend to rely more on experimental data; Maya collaborates with Helen Saibil in Biological Sciences to fit atomic structures of individual proteins to lower-resolution maps of protein complexes that were generated by electron microscopy. Proteins studied in this way include GroEL, a ‘molecular chaperone’ that forms a chamber that isolates unstructured proteins so that they can fold.

Dr Maya Topf

Dr Maya Topf

Another method of modelling protein structures uses evolution, and relies on the fact that there are remarkably few different basic protein structures – each of the 43,000 known protein structures takes up one of only about 1,000 different folds. Just as all birds have the same basic pattern, with two legs and two wings, all proteins with a particular function will usually have a similar fold. It is therefore possible to model the structure of a protein based on one or more of its evolutionary relatives, in a technique called ‘homology modelling’. In some cases, it is possible to produce a usable model from the structure of a related protein from a very different type of organism. It was more than a decade after the publication of the first bacterial ribosome structures before similar structures could be obtained from mammalian ribosomes, but many useful results were obtained during that time by modelling mammalian ribosome sequences using the bacterial structures and low-resolution electron microscopy data.

Maya ended her talk by stressing that structural biology is a science of model-building. It requires experimental data complemented by physics and by evolution, and, almost above all, it requires powerful computers. Generally, the more sources of information can be combined into a model, the nearer the ‘correct’ structure that model will be: and to quote the statistician George Box, ‘all models are wrong, but some are useful’.

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Crystallography: From Chocolate to Drug Discovery

This post was contributed by Dr Clare Sansom, senior associate lecturer in the Department of Biological Sciences. Dr Sansom attended inaugural Rosalind Franklin Lecture during Birkbeck Science Week 2016.

Rosalind Franklin - slide

Birkbeck has already established lecture series in honour of some of its most distinguished alumni. Until 2016, however, Rosalind Franklin – co-discoverer of the DNA structure and perhaps the most widely recognisable of its ‘famous names’ – was missing from the list of honourees. This gap has now been filled; the annual Rosalind Franklin lecture forms part of the college’s Athena SWAN programme and will always be given by a distinguished woman scientist.

And fittingly, the inaugural lecture, which was part of Science Week 2016, was devoted to Rosalind Franklin’s own discipline, crystallography. Elspeth Garman, Professor of Molecular Biophysics at Oxford University, gave an entertaining and illuminating lecture to a large audience that included Rosalind’s sister, the author Jenifer Glynn.

Exploring crystals

Garman began her lecture by showing a short video that she had produced for OxfordSparks.net that used a ‘little green man’ to illustrate the method of X-ray crystallography that is used to obtain molecular structures from crystals. The rest of the lecture, she said, would simply go through that process more slowly. She started by showing some beautiful examples of crystals. All crystals are formed from ordered arrays of molecules. They can be enormous, such as crystals of the mineral selenite in a cave in Mexico that measure over 30’ long or too small to be visible with the naked eye.

In the early decades of crystallography, structures could only be obtained from crystals of the smallest, simplest molecules: the first structure of all, published in 1913 by the father-and-son team of W.H. and W.L. Bragg, was of table salt. When they were jointly awarded the Nobel Prize for Physics in 1915, the younger Bragg was a 25-year-old officer in the trenches on the Western Front. His record as the youngest Nobel Laureate was unbroken until Malala Yousafzai’s Peace Prize in 2014.

The Braggs’ discoveries paved the way for studies of the structures of many, many substances: including the chocolate of the lecture title. Few of the audience can have known that chocolate exists in six different crystal forms, or that only one of these (Form V) is good to eat. The process of ‘tempering’ – a series of heating and cooling steps – is used to ensure that it solidifies in the correct form.

Professor Nick Keep and Professor Elspeth Garman at the inaugural Rosalind Franklin lecture

Professor Nick Keep and Professor Elspeth Garman at the inaugural Rosalind Franklin lecture

Protein crystallography

Garman then moved on to talk about her own field of protein crystallography. Proteins are the ‘active’ molecules in physiology, and they are formed from long, linear strings of 20 different ‘beads’ (actually, small organic molecules known as amino acids). Chemists can quite easily find out the sequence of these beads in a protein, but it is impossible to work out from this the way that the string will fold up into a definite structure ‘like a piece of wet spaghetti’. And it is this structure that places different units with different chemical properties on the surface or in the interior of the protein, or near each other, and that therefore determines what the protein will do.

Protein crystallography only became technically possible in the mid-twentieth century, and even then it was a painfully slow and complex process that could only be used to study the smallest, simplest proteins. Dorothy Hodgkin, also a professor at Oxford, won her Nobel Prize in Chemistry in 1964 for the structures of two biologically important but fairly small molecules: penicillin, with 25 non-hydrogen atoms and vitamin B12, with 80. She is perhaps better known for solving the structure of insulin, the protein that is missing or malfunctioning in diabetics. This has 829 non-hydrogen atoms; in contrast, the 2009 Chemistry Nobel Prize was awarded for the structure of the ribosome, the large (by molecular standards) ‘molecular machine’ that synthesises proteins from a nucleic acid template. The bacterial ribosome used for the Nobel-winning structural studies is well over 300 times larger than insulin, with over a quarter of a million atoms.

Real world applications

Dr Rosalind Franklin

Dr Rosalind Franklin

Protein structures are not only beautiful to look at and fascinating to study, but they can be useful, particularly for drug discovery. Many useful drugs have already been designed at least partly by looking at a protein structure and working out the kinds of molecule that would bind tightly to it, perhaps blocking its activity. Some viral proteins have been particularly amenable to this approach.

Rosalind Franklin did some of the first research into virus structure when she was based at Birkbeck, towards the end of her tragically short life, and her student Aaron Klug cited her inspiration in his own Nobel lecture in 1982. X-ray crystal structures were used in the design of the anti-flu drugs Relenza™ and Tamiflu™ and of HIV protease inhibitors, and more recently still structures of the foot and mouth virus are helping scientists develop new vaccines for tackling this potentially devastating animal disease. The foot-and-mouth virus structure even made the front page of the Daily Express.

The equipment that Dorothy Hodgkin and her contemporaries used to solve protein structures in the 1960s and 1970s looks primitive today. Now, almost every step of protein crystallography has been automated. Powerful beams of X-rays generated by synchrotron radiation sources, such as the UK’s Diamond Light Source in Oxfordshire, allow structures to be determined quickly from the smallest crystals. It is even possible to control some of these machines remotely; Garman has operated the one at Grenoble from her sitting room. Yet there is one step that has changed remarkably little. It is still almost as difficult to get proteins to crystallise as it was in the early decades. Researchers have to select which of a large number of combinations of conditions (temperature, pH and many others) will persuade a protein to form viable crystals. Guesswork still plays a large part and some researchers seem to be ‘better’ at this than others: Garman adds the acronym ‘GMN’ or ‘Grandmother’s maiden name’ to her list of conditions to reflect this.

Yet, with every step other than crystallisation speeded up and automated beyond recognition, the trickle of new structures in the 70s and even 80s has become a torrent. Publicly available structures are stored online in the Protein Data Bank, which started in 1976 with about a dozen structures: it now (May 2016) holds over 118,000. Protein crystallography as a discipline is thriving, but there are many challenges ahead. We are only now beginning to tackle the 70% or so of human proteins that are only stable when embedded in fatty cell membranes and are therefore insoluble in water. It is possible to imagine a time when it is possible to solve the structure of a single molecule, with no more need for time-consuming crystallisation. And, hopefully, women scientists will play at least as important a role in the second century of crystallography as they – from Quaker Kathleen Lonsdale, who developed important equations while jailed for conscientious objection during World War II, through Franklin and Hodgkin to Garman and her contemporaries – have in the first.

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Microtubules and Microscopes: Exploring the Cytoskeleton

This post was contributed by Clare Sansom, Senior Associate Lecturer at the Department of Biological Sciences

"The binding site for End Binding protein 1 (highlighted in green) on the microtubule lattice at the corner of four tubulin dimers, visualised using cryo-electron microscopy" (Credit Cell by Maurer et al (2012))

“The binding site for End Binding protein 1 (highlighted in green) on the microtubule lattice at the corner of four tubulin dimers, visualised using cryo-electron microscopy” (Credit Cell by Maurer et al (2012))

Electron microscopist Carolyn Moores, the most recently appointed professor in the Department of Biological Sciences at Birkbeck, gave her inaugural lecture at the college on June 1.

Moores arrived at Birkbeck in 2004 to start her research group and has risen rapidly and steadily up the academic ladder ever since. Introducing the lecture, the Master of Birkbeck, David Latchman, explained that Moores’ CV stood out in every way; she was clearly as gifted a teacher and administrator as she was a researcher. Furthermore, as she has won several awards for science communication, he predicted that the audience would be in for a treat. We were not disappointed.

Educational journey

Moores began her lecture by saying that she would talk about three different things: her own career development; her group’s research into the structure and function of microtubules; and the advancement of women in science, a cause that is close to her heart.

She remembered that she had wanted to work as a scientist as soon as she knew what a laboratory was, and she started young, as an intern in a research lab at Middlesex Hospital while still in the sixth form. School was followed by a BSc in Biochemistry at Oxford and a PhD in John Kendrick-Jones’ lab at the world-famous Laboratory for Molecular Biology (LMB) in Cambridge. She then moved to work as a post-doc with Ron Milligan at the Scripps Research Institute in La Jolla, California, USA, and it was there that she began her studies on microtubules.

Coming to Birkbeck

The award of a David Phillips research fellowship in 2004 gave her the opportunity to return to the UK as an independent researcher. She explained that there were three reasons – or more accurately three people – that led her to choose to come to Birkbeck. Working in electron microscopy, she was inspired by the work of Helen Saibil, one of the UK’s principal exponents of that technique; she had known Nicholas Keep, then a lecturer in Biological Sciences, as a friend since her time at the LMB; and she knew that she would value the interdisciplinary working environment of the Institute for Structural Molecular Biology under the ‘inspired’ leadership of Gabriel Waksman.

Research into microtubules

Moores then moved on to discuss the main topic of her group’s research: the three-dimensional structure, function and role in disease of tiny cylindrical structures known as microtubules. These are one of the building blocks of the cytoskeleton, which forms a framework for our cells in the same way that our skeletons form a framework for our bodies. They are about 25nm in diameter, which puts them firmly into the ‘nano-scale’ of biology that is easily studied using electron microscopy.

There is a cytoskeleton in every living cell, and it, and the microtubules that form it, are involved in many important cellular processes including shape definition, movement and cell division. Diseases as diverse as cancer, epilepsy, neurodegeneration and kidney disease have been linked to microtubule defects. Understanding their fundamental structure and function, as Moores’ group aims to, should help in understanding these disease processes and perhaps also in developing effective treatments.

Microtubules are built up from many copies of a small protein called tubulin, which, in turn, is a dimer of two similar proteins called alpha and beta tubulin. These tubulin dimers make contacts with each other both head-to-tail and side-to-side to create the cylindrical microtubule wall, fuelled by energy derived from the molecule GTP. Each tubulin unit has a definite “top” and a “bottom” and, as the units are oriented in parallel, so has the complete microtubule.

Microtubules are dynamic structures; they continue to grow by the addition of tubulin units to one end as long as GTP is available, and then begin to unravel and shrink. This dynamism, which allows them to respond to the changing needs of the cell, is essential for their function in healthy cells. In particular, microtubules organise chromosome structures during cell division and are therefore necessary for cell proliferation. As cancer is a disease of uncontrolled cell proliferation, it is possible to imagine that a molecule that could specifically block microtubule growth and assembly in the nucleus might be useful as an anti-cancer drug.

Moores and her group are aiming to understand the process of microtubule growth at as high resolution as possible, using electron microscopy. Unfortunately, however, the most detailed images can only be obtained if the specimen is at very low temperatures (in so-called cryo-elecron microscopy) and using this means that the dynamics of the specimens must be “frozen” into a still image. While it is now possible to see the individual tubulin subunits in the static microtubule images, many details of their structure can only be inferred from computational analysis.

Understanding growth

Moores went on to describe one project in her lab in a little more detail. This was an investigation of the structure and role of proteins that bind only to growing microtubule ends, falling off when the growth stops. It is possible to obtain low-resolution images of microtubules in which these molecules have been made to fluoresce, so only growing microtubules are tracked.

In order to understand the growth process in detail, the group developed an analogue of the GTP “fuel” molecule which can bind to the tip of a microtubule that is extending but not break down to release its energy, so the microtubule does not in fact grow. This forms a static analogue of a growing microtubule that retains all the characteristics of the dynamic structure but that can be studied at low temperatures.

Images of this structure have shown that the end binding proteins bind at the corner of four of the tubulin units. They have explained a lot of the properties of growing microtubules, but there is still more to learn. A full understanding will need structures that are at even higher resolution, where the positions of individual atoms can be made out. Following many years of technical development, today’s most powerful electron microscopes are now making this possible.

Women in science

In the last section of the lecture, Moores left the topic of research to talk briefly about another of her passions: the promotion of women in science. She explained that although 65% of under-graduates in the biological sciences are now women, the proportion of women drops to 40% at any academic grade and 25% for full professors.

A study cited by the European Molecular Biology Organisation has suggested that the barriers for women scientists to progress are set so high that at the current rate of progress full equality would never be achieved. Birkbeck has signed up to the Athena SWAN Charter, set up to encourage higher education institutions to transform their culture and promote gender equality. She described her work with the Athena SWAN team that has so far resulted in the college gaining a bronze award as being as exciting as, but also as challenging as, her studies of microtubules.

Nicholas Keep, Dean of the Faculty of Science and, as Moores had stated, a personal friend, gave the vote of thanks after the lecture. He paid tribute in particular to her value as a colleague, her administrative skills, and the importance of her contribution to the college’s application for the Athena SWAN award.

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