Munc18: No Munc-eying around with our molecular transport machinery!

What is it?

The transport of chemicals or proteins across the membranes of cells is a vital part of life. Chemicals may need to be released in order to send a signal to the cell next door, or sometimes, a protein must be relocate from inside the cell to sit on the cell membrane so it can do its job.

For this reason, the Sec/Munc18 (SM) family of proteins—involved in fusion between two membranes—are an essential for this molecular transport and play a key part of healthy cell function. These SM proteins are involved in neurotransmitter release in the brain and in the maintenance of glucose levels (i.e. by relocating the glucose transporter to the cell surface so it can pump glucose into the cell).

Within our cells, chemicals or molecules can be isolated from the rest of the cell contents by containing them within small membrane encapsulated vesicles. But the molecular mechanisms behind how proteins, including SM, coordinate the fusion of these vesicle membrane to the outer membrane of the cell, so that neurotransmitters or the glucose transporter can do their thing, is not understood.

Crystal structures of SM proteins alone, and in complex with their partner proteins are helping us to decode the complex mechanisms of the membrane fusion event. And the importance of research in this area is essential for the development of new treatments for neurological diseases or diabetes. Indeed, Randy W. Schekman, Thomas C. Südhof, and James E. Rothman were awarded the 2013 Nobel Prize in Physiology or Medicine for their discoveries on the protein machinery that regulates vesicle traffic, including the SM proteins! You can read more about their Nobel Prize and their research here.

What does it look like?

SM proteins, such as Munc18-1 (or Munc18a) shown below, consist of three separate domains that fold into a U-shaped or arch-shaped architecture. Image generated by Pymol (using the coordinates from the protein data bank (accession code: 3PUJ).

SM proteins, such as Munc18-1 (or Munc18a) shown below, consist of three separate domains that fold into a U-shaped or arch-shaped architecture. Image generated by Pymol (using the coordinates from the protein data bank (accession code: 3PUJ).

Where did the structure come from?

This is the structure of rat Munc18-1 that was solved in complex with a portion of its partner protein, syntaxin (Sx). The structure was published in 2011 PNAS by a team of researchers from the Institute for Molecular Bioscience, UQ, Australia.

A total of 12 SM structures have been determined, either alone or complexed to a truncated protein partner or short peptide sequence. Click here for a review on the current structural information of SM proteins.

From crystal to structure in 84 years

What is it?

In 1926 Jack Bean urease was the first protein to be crystallised 1, earning a place in the history books for this humble plant protein and a Nobel Prize for James B. Sumner (see yesterday’s post for more). Besides establishing that proteins could be crystallised, Sumner’s work was remarkable for demonstrating that proteins could be isolated and purified whilst retaining enzymatic activity. Despite this landmark advance in protein biochemistry and crystallography, it was a further 84 years before the crystal structure of Jack Bean Urease was determined, in what must surely be the longest running crystallography endeavour in history2.

Where did the structure come from?

Urease is an enzyme in plants, bacteria and fungi that helps to break urea into ammonia and carbon dioxide. Whilst other urease structures were known – for example Helicobacter pylori urease, the structure of which helped to explain how this bacterium can survive in the acid environment of the human stomach3 – the structure of Jack Bean urease was only determined in 2010.

What does it look like?

Urease

Plant ureases are single chain proteins. Jack Bean urease is a T-shaped molecule of four domains (coloured here in red, blue, yellow and pink.) In common with other ureases, Jack Bean Ureases binds two nickel ions (blue spheres) in its active site; these are required for enzymatic breakdown of urea.

This structure is Protein Data Bank ID 3LA4

References

  1. Sumner, 1926: J. Biol. Chem. 69:435-441
  2. Balasubramanian & Ponnuraj. 2010 J. Mol. Biol. 400:274-283
  3. Ha et al., 2001. Nature Structural Biology 8: 505-509

Crystallising enzymes from beans – celebrating Sumner

What does it look like?

4GY7 jack bean urease deposited in the PDB in 2012. From the paper Crystallographic structure analysis of urease from Jack bean (Canavalia ensiformis) at 1.49 Å resolution deposited by Begum, A.,  Banumathi, S.,  Choudhary, M.I., & Betzel, C

4GY7 jack bean urease deposited in the PDB in 2012. From the paper Crystallographic structure analysis of urease from Jack bean (Canavalia ensiformis) at 1.49 Å resolution deposited by Begum, A., Banumathi, S., Choudhary, M.I., & Betzel, C

What is it and where did the structure come from?

This urease, extracted from a jack bean, was the first enzyme ever to be crystallised meaning that the arrangement of atoms that make it up could then be studied and understood.  The man that achieved this, Jame Batcheller Sumner, was later to win a Noble prize in 1946 for this achievement.  Sunmer, who’s birthday it would be today, actually achieved this feat in 1926 – but it took a number of years to convince the skeptical scientific community that he had isolated an enzyme in his crystals .

James Sumner and his first crystals of an enzyeme

James Sumner and his first crystals of an enzyme

The Nobel Prize in Chemistry 1946 was divided, one half awarded to James Batcheller Sumner “for his discovery that enzymes can be crystallized”, the other half jointly to John Howard Northrop and Wendell Meredith Stanley “for their preparation of enzymes and virus proteins in a pure form”.

Sunmer’s biography on the Nobel Prize website, gives a great insight into how this achievement gradually gained acceptance:

“Gradually, recognition came. In 1937, he was given a Guggenheim Fellowship; he went to Uppsala and worked in the laboratory of Professor The Svedberg for five months. He was awarded the Scheele Medal in Stockholm in the same year. When Northrop, of the Rockefeller Institute, obtained crystalline pepsin, and subsequently other enzymes, it became clear that Sumner had devised a general crystallization method for enzymes. The opponents gradually admitted Sumner’s and Northrop’s claims – Willstätter last of all – and the crowning recognition came in 1946 when the Nobel Prize was awarded to Sumner and Northrop. In 1948, Sumner was elected to the National Academy of Sciences (USA).”

In Sumner’s Nobel acceptance lecture he said:- “Why was it difficult to isolate an enzyme? …i.e. in pure condition. It does not seem difficult to isolate and crystallize an enzyme now, but it was difficult 20 years ago….When I succeeded I then telephoned to my wife ‘I have crystallized the first enzyme.

Tomorrow we’ll have a post about the urease structure in detail, and why it took 85 years from when Sumner first grew the crystals to determining the structure.

References

James B. Sumner The chemical nature of enzymes Nobel Lecture, December 12, 1946

“James B. Sumner – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 13 Nov 2014. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1946/sumner-bio.html

See also:-

P A Karplus, M A Pearson and R P Hausinger 70 Years of Crystaline Urease: What Have We Learned? Acc Chem Res 1997 30, 330-337.

N E Chayen, J R Helliwell and E H Snell “Macromolecular Crystallization and Crystal Perfection” (International Union of Crystallography Monographs on Crystallography” Oxford University Press International Union of Crystallography Monographs on Crystallography (2010).

 

 

A Nobel Explosive – Trinitroglycerin

After a bit of a break Dr Dave T is back with an explosive post!

What does it look like?

Picture1What is it?

With the upcoming release of a new AC/DC album, I found myself raising a cacophony in the shower (note the deliberate avoidance of the word ‘singing’). The lyrics “I’m TNT, I’m dynamite” got stuck in my head and made me realise that they’re actually wrong (science fact not getting in the way of a good rock song). TNT (trinitrotoluene) is not dynamite, which is composite based on trinitroglycerin. The timing of this revelation is even more significant, as the Nobel prizes have just been awarded and few people realise that Alfred Nobel, who bequeathed his estate to a trust to fund the prizes (including for peace) which bear his name, was the inventor of dynamite.

Nobel came from a family that was rooted in science and engineering, and Alfred’s particular interest was in explosives, particularly nitroglycerin (1,2,3-trinitroxypropane). His research was in part aimed at making the compound more stable, particularly after an accident at his factory that killed 5 people including his younger brother. He invented dynamite, a composite of nitroglycerin with diatomaceous earth as an absorbent, which has increased stability and could therefore be handled in industrial uses (particularly mining). Military dynamite is not nitroglycerin-based.

The molecular structure of trinitrogylycerin is quite simple, being propane with three nitro groups (one at each carbon). The presence of nitro groups in small molecules is generally a theme of explosive compounds, and seeing too many in one molecule causes concern amongst synthetic chemists. The packing of the molecules in the crystal structure lacks any traditional ‘strong’ interactions such as hydrogen bonding. Instead the crystal lattice is held together by van der Waals interactions and dipole-dipole interactions between the nitro groups.

Where did the structure come from?

The structure was determined and published in Acta Crystallographica C in 1984 by Espenbetov and co-workers (Acta Cryst., 1984, C40, 2096-2098). These are braver crystallographers than me, given that mishandling the material would lead to a bad case of, as Jazzy Jeff and the Fresh Prince said, ‘Boom Shake the Room’ (although I’m fairly certain the song is not about explosives).

Sodium hydride, simple yet historic.

What is it?

Though it looks pretty simple, this structure has a special place in history as it was one of the first structures to be discovered with neutron diffraction.  Though the positions of the sodium atoms in sodium hydride were known for some time (from x-ray diffraction), there was still debate if the hydrogen atoms were at the ½, ½, ½ point in the unit cell (a zinc blende structure) or at the ½, 0, 0 point in the unit cell (a rock salt structure).

In the 1940’s a number of researchers realised that instead of using x-rays to diffract off a material, they could use some different radiation – a beam of neutrons.  This was only possible with the development of nuclear reactors, which could provide a steady source of neutrons.

Neutrons interact with matter in a very different way to x-rays.  With  x-rays, the amount they scatter from an atom is proportional to the number of electrons they have in their outer shell – for instance sodium atoms (with 11 electrons each) scatters much better than hydrogen atoms (with only one electron for each atom).  Instead of interacting with the ‘cloud’ of electrons, neutrons diffract by interacting with the nucleus of the atom itself, and are not affected by it’s size, and so have a much more complex trend as you move through the elements of the periodic table.    For instance heavy hydrogen (deuterium) has a scattering cross length from neutrons that is twice that of sodium.

The effect that is to x-rays the structure of sodium hydride looks like this:

The structure of sodium hydride showing the relative scattering of the sodium (yellow) and hydrogen (pink) atoms.

The structure of sodium hydride showing the relative scattering of the sodium (yellow) and hydrogen (pink) atoms.

and to neutrons it looks like this!

And this shows the relative scattering of the heavy hydrogen (pink) and sodium atoms (yellow) to neutron diffraction.

And this shows the relative scattering of the heavy hydrogen (pink) and sodium atoms (yellow) to neutron diffraction.

This meant that the first researchers using neutron diffraction could work out that the structure of sodium hydride was, in fact, the rock salt structure.  This paved the way for many other structures to be solved with neutron diffraction, locating atoms that would be difficult to see with x-rays.
Shull, one of the authors of this work on sodium hydride, was later to share the Nobel Prize in Physics for his development of neutron diffraction.

Not going anywhere – low expansion alloys.

What does it look like?

Details of the crystal structure of a low thermal expansion alloy (Manganese Nickel) investigated by Yokoyama et al at the National Institute for Materials Science in Japan http://www.ims.ac.jp/english/topics/2012/130206.html.  The purple atoms are the manganese and the silver atoms are nickel.

Details of the crystal structure of a low thermal expansion alloy (Manganese Nickel) investigated by Yokoyama et al at the National Institute for Materials Science in Japan http://www.ims.ac.jp/english/topics/2012/130206.html. The purple atoms are the manganese and the silver atoms are nickel.

What is it?

One of the most fundamental things we can observe is that when we heat a material up it expands. This is a consequence of the heat transferring energy to the atoms within the material, and them moving about a bit more.

Metals often expand quite a bit, more than glass for instance (handy way to get a lid off a jar when it’s stuck is to run the lid under a hot tap – it will expand more than the glass of the jar and should pop off a bit easier!). But there is a family of metal alloys that this wouldn’t work for, low expansion alloys.

The most famous of these is INVAR, which is an Iron Nickel alloy. The crystal structure is a simple face centred cubic structure, the left image in the picture, like gold for instance. INVAR is usually about 64 % iron, and 36 % nickel with these atoms existing in a solid solution, they are mixed up through the structure. The combination of these two elements in this ratio give a thermal expansion coefficient which is tiny around 0.5 parts per million for every 1°C in heat increase.  It’s still not totally understood why this effect occurs.

There are actually a range of alloys in this family that have thermal expansion coefficient tuned to different materials. For instance, Kovar has the same expansion rate as glass so was used widely in the early electronics industry.

 Where did these structure come from?

The discovery of low expansion alloys occurred around 1890’s was deemed so import that the discoverer Guillame received the Nobel prize the year before Einstein! Being able to build a scientific instrument (particularly chronometers) that are inherently unaffected by temperature led to huge improvements in what could be done.

DNA: Life’s blueprint.

Today is the birthday of Rosalind Franklin, whose pioneering work on X-ray diffraction of DNA was crucial in determining its structure.

Rosalind Franklin.  Picture sourced from Jewish Chronicle Archive/Heritage-Images

Rosalind Franklin. Picture sourced from Jewish Chronicle Archive/Heritage-Images

What is it?

DNA is the pieces of code we receive from our parents that make us all unique, life’s master plan or blueprint, the molecules that contain information necessary to make up a living being…

Where did the structure come from?

The original double helical model so widely known today was published by Watson and Crick in 1953.  Like most scientific breakthroughs, their discovery was based on nearly a century of incremental advances in DNA research. Importantly, Rosalind Franklin’s beautiful X-ray diffraction images, collected while she was working at King’s College, London, were evidence of the helical structure.

Picture1

On the left, the famous ‘photo 51’, a diffraction image of DNA fibres produced under Rosalind Franklin’s supervision, which was shown to Watson and Crick before they published the structure. On the right, Rosalind Franklin.

What does it look like?

The famous double helix structure of DNA is now well known and can be widely seen in various stylised ways in everyday life.

DNA, as drawn in the 1953 paper by Watson and Crick.

DNA, as drawn in the 1953 paper by Watson and Crick.

What about water?

Rosalind Franklin also realised the importance of water in the structure of DNA, and showed that different forms of DNA were produced depending on water content… which brings me to an unavoidable plug for neutron scattering. The neutron fibre diffraction work on DNA is a beautiful example of how neutrons can be used to localise water molecules in a structure, as shown below.

Water in DNA: the yellow sticks show the DNA helix, while blue shows where water sits in the structure. From [1]

Water in DNA: the yellow sticks show the DNA helix, while blue shows where water sits in the structure. From [1]

[1] Fuller W, Forsyth T, Mahendrasingam A, Phil Trans R Soc Lond B (2004) 359 1237-1248.

A mineral of history – Fluorite

William Henry Bragg's portrait on winning the Nobel Prize, image from the Noble Foundation

William Henry Bragg’s portrait on winning the Nobel Prize, image from the Noble Foundation

Today would have been the 152nd birthday of one of the founders of crystallography, William Henry Bragg. Together with his son WL Bragg, he won the 1915 Nobel Prize in physics. Between this work and the work of the previous year’s Nobel Prize won by Max von Laue, these researchers put down the rules for us to discover how atoms are arranged in solids.

After finishing his PhD with J.J. Thomson (the man who discovered the electron) W.H. Bragg journeyed to Australia, where at the age of 23 he became a professor of physics at Adelaide University. Here he also married Gwendoline Todd the daughter of Charles and Alice Todd (who Alice Springs is named after), and their first son William Lawrence was born.

The family moved to the UK in 1909, W.H. Bragg to take up a position at the university of Leeds, and W.L. Bragg to continue his university studies at Cambridge. It was during this time that the both worked on the equation that was to bear there name, and the research that would earn them a Noble Prize.

We’ve already covered the mineral, Braggite, named after WH and WL Bragg – then instead today we thought we’d cover one of the first crystal structures they worked out, Fluorite.

What does it look like?

So far this year we come across many ways to represent crystal structures. This may be the most original yet!

What is it?

Calcium Floruride, CaF2 or Flourite, is a colourful mineral that is found all over the world. It comes in a large range of colours, from green through to dark purple, all dependent on the small amount of element impurities contained within it.  Given the right impurities, it will also fluorescence under UV light – the name of this property actually takes its name from fluorite.

In working out the structure of fluorite, the Bragg’s noted the similarity of its diffraction to that of diamond. From this they could work out that the atoms would be similarly arranged but with gaps as there isn’t a 1 to 1 ratio of fluorine atoms to calcium atoms.

Where did the structure come from?

Like the rock salt structure, you can read about how this structure was discovered straight from the book ‘X-rays and crystal structure’ by W.H. and W.L. Bragg.

Haemoglobin: Bloody protein!

Today to celebrate what would have been Max Pertuz’s 100th birthday, we examine the structure of haemoglobin for which Perutz shared – with John Kendrew – the 1962 Nobel Prize for Chemistry.

What is it?

Haemoglobin is an iron-containing oxygen carrier protein that transports oxygen from the lungs to elsewhere in the body and which when associated with that oxygen gives our blood its characteristic bright red colour.

Where did the structure come from?

To examine the structure of haemoglobin, Perutz and colleagues isolated the protein from horse blood which is abundant in haemoglobin1; horses typically have a haemoglobin concentration of ~150 g per litre of blood. By comparison in the modern day lab scientists producing recombinant protein ( via a process of co-opting bacteria or other cells to produce large amounts of proteins that they don’t normally produce) usually have to work very hard, growing tens of litres of culture to produce just milligrams (i.e. one thousandth of a gram) of protein for experiments. Hence, in these early days of protein science, horse blood provided a ready source for Perutz and co. to study.

What does it look like?

haemoglobin-finalExtensive investigation of the structure of haemoglobin has revealed much about the intricacies of oxygen binding and release. Haemogoblin is made up of four subunits, each of which contains an iron-containing heme group. Remarkably, binding of oxygen to iron in one subunit directly increases the propensity of another of the subunits to bind oxygen, meaning that as the protein binds oxygen it becomes easier to bind more oxygen. This so-called co-operative binding is pivotal to the efficiency with which haemoglobin can be loaded with oxygen and was the first time such an allosteric phenomenon was observed in proteins.

This structure is of human deoxyhaemoglobin 2 i.e. haemoglobin without oxygen bound. Each of the four subunits are coloured differently, with the oxygen binding heme groups show in black. This structure is Protein Data Bank ID 2HHB

References

1. Perutz, Rossmann, Cullis, Muirhead, Will & North. 1960 Nature 185: 416-422

2. Fermi, Perutz, Shaana and Fourme. 1984 J. Mol. Bio. 175: 159-174

Dorothy Crowfoot Hodgkin and the structure of Vitamin B12

Today is the birthday of Dorothy Crowfoot Hodgkin O.M. FRS who was awarded the 1964 Nobel Prize for her determinations by X-ray techniques of the structures of important biochemical substances

DHodgkin

What does it look like?

A space filling model of Vitamin B12 (cyano-cobalamin) (top)  - and a “capped stick representation of the same molecular structure (red= oxygen, grey= carbon, white = hydrogen, blue = cobalt, mauve = nitrogen, orange = phosphorus)  both structures depict the molecule in the same orientation – note the ease of access to the CN group bound at the top of the corrin ring – the reactive position of the molecule.  Isolated red crosses or spheres are water of crystallization. (coordinates used from DOI: 10.1039/c003378b)

A space filling model of Vitamin B12 (cyano-cobalamin) (top) – and a “capped stick representation of the same molecular structure (red= oxygen, grey= carbon, white = hydrogen, blue = cobalt, mauve = nitrogen, orange = phosphorus) both structures depict the molecule in the same orientation – note the ease of access to the CN group bound at the top of the corrin ring – the reactive position of the molecule. Isolated red crosses or spheres are water of crystallization. (coordinates used from DOI: 10.1039/c003378b)

What is it and how was the structure found?

This is Vitamin B-12.

A vitamin is an organic compound vital to the nutrition of an organism. For any particular organism if the substance cannot be synthesized in sufficient (but very small) amounts, the vitamin must then be obtained from the diet.  For instance, Vitamin D is a vitamin in humans because in certain circumstances, particularly low exposure to UV radiation, the normal synthesis of the substance does not occur and it must be consumed from dietary sources to meet metabolic requirements.

Vitamin B12 is chemically the most complex vitamin with a group of closely related substances functioning in the organism to provide the necessary source of cobalamin to vital enzymatic pathways of the metabolism.   The recommended dietary intake in humans varies but is in the range of 1-3 micrograms (millionths of a gram) per day. Vitamin B12 deficiency can result either from an inadequate dietary intake, or from conditions in which the ability to absorb the nutrient is diminished. Biosynthesis of vitamin B12 only occurs in bacteria and archaea and passes into the food chain via due to bacterial symbiosis.

The common features of the Vitamin B12 group of compounds are (i) the corrin ring (similar to the porphyrin http://en.wikipedia.org/wiki/Porphyrin ring which binds to the metals present in haem, chlorophyll and cytochrome) which has (ii) a cobalt atom bound in its centre. (iii) One of the carbon atoms on the “outside” of the corrin ring carries to a long chain of atoms ending in a dimethylbenzimidazole group which joins on to the cobalt through one of its nitrogen atoms on one side of the corrin ring and on the other side of the corrin, a sixth substituent bonds to the cobalt atom – this is the site of the biological reactivity of Vitamin B12 and can be occupied by hydroxyl (-OH), methyl (-CH3), cyano (-CN) or 5’-deoxyadenosyl (via its C5’ atom). All of these compounds form crystals which are deep red in colour.

The complete structure of vitamin B12 was determined by Dorothy Hodgkin and her collaborators by means of X-ray crystallographic methods. Using structures from fragments and combining knowledge of the chemistry and with access to early computer based fourier methods, the structure was fully revealed. It is interesting to note the sharing of information which went on between the crystallographic groups and the chemists who were engaged in studying the structure. The documents demonstrate that the two different approaches were to be published simultaneously and Dorothy maintained an inclusive and generous approach to publication of this important structure. Dorothy learned that Alexander Todd (Nobel Prize for Chemistry 1957) had agreed to present a talk about “some new nitrogen-containing compounds” at a meeting of the Chemical Society which he had not mentioned to Dorothy when he was in Oxford a day or two prior to this for the purpose of discussing the publication of the structure. When Dorothy got wind of this talk, she attended and finding that he was announcing the structure, at the end of the talk Dorothy stood up and explained how it had been done. After this occurrence, Dorothy insisted that members of her crystallographic group went wherever Todd was speaking so that they could give a comment at the end.

A study designed to isolate the substance in liver which cured anaemia in dogs (iron), along the way revealed the presence of a different substance which cured pernicious anaemia in humans. For several years sufferers of pernicious anaemia were required to consume large amounts of raw liver or drink “liver juice” to treat their condition. The 1934 Nobel Prize in Physiology or medicine was awarded to Whipple, Minot and Murphy for their work in pointing the way to a treatment for this condition. In 1928 Edwin Cohn prepared an extract that was 50-100 times more effective in the treatment of the disease than raw liver products and together these discoveries led to the identification of the group of compounds known as Vitamin B12