Sometimes I can’t believe how awesome people are…Dorothy Crowfoot Hodgkin (May 12, 1910 – July 29, 1994) was a true pioneer in x-ray crystallography – She wanted to get a look at biologically-important molecules. But the technology didn’t exist to do it – so she developed it. And used it to harness x-rays to solve the structure of increasingly complex molecules including the antibiotic penicillin in 1945, the most complex vitamin (vitamin B12) in 1955, and – her lifelong goal – the protein hormone insulin in 1969. She won the Nobel Prize in Chemistry in 1964 – the 3rd woman to do so – you might know that – but did you know that she also won the Soviet Union’s version of the Nobel Peace Prize? In addition to an incredible scientist, Hodgkin was active in opposing nuclear weapons and, in addition to advocating for women in science everywhere, she directly mentored several female scientists who went on to become influential crystallographers in their own right.
X-ray crystallography is a technique used to study the structure of molecules that are way too small to see with the naked eye, too small to even see with a light microscope. Instead of visible light (whose wavelengths are too long), it uses a much more energetic form of light – x-rays, which have much shorter wavelengths, better-suited for probing the small distances between a molecule’s linked atoms (individual carbons, hydrogens, oxygens, etc.). But that energy comes at a cost – x-rays can’t be focused with lenses like visible light can, so scientists have to work backwards from the “jumbled” x-rays that scatter when they hit a molecular crystal – and Dorothy Crowfoot Hodgkin largely made this possible!
Dororthy Crowfoot was born the eldest of four sisters on May 12, 1910 in Cairo, where her father worked for the Ministry of Education as an archaeologist and historian. She was raised in England and colonial North Africa and was fascinated by crystals from a young age, a fascination encouraged by her family and family friends – one such friend who was a soil scientist gave her a surveyor’s box of reagents and minerals she could explore with, and her mother gave her a book about X-ray crystallography by Henry Bragg when she was 16. She attended the Sir John Leman School in Beecles, England, where she and her friend Norah Pusey were the only 2 girls in their chemistry class – and they were only there because they had petitioned the school to take it instead of “domestic science”
Ever since Bragg’s book introduced her to x-ray crystallography, Dorothy was eager to try it out. Since she was only 16 at the time, she’d have to wait. But, at the age of 18, in 1928, she enrolled at Oxford University to study chemistry. As an undergraduate, she had her first experience trying out x-ray crystallography, but not on biological molecules – that would have to wait until her doctoral studies, which she began at Cambridge with a great mentor, J. D. Bernal. In addition to teaching her crystallography, provided a welcoming and supportive environment for female scientists, helping make crystallography one of the only fields hiring many women at the time.
She almost didn’t get to work with biochemicals here either – Bernal’s early work focused on metals – but thankfully for us, Bernal took an interest in biochemicals around the time Crowfoot joined his lab, switching focus to study the structure of sterols. Together with Bernal, Dorothy studied the x-ray crystallographic patterns of overs 100 steroids – at this point they weren’t trying to distinguish individual atoms, but rather to get a better sense of their overall dimensions and qualities. But, developing techniques and methodologies as they went, they began to take on larger, more complex, molecules, and higher and higher resolution.
In 1933, she received a research fellowship from Somerville that would support her work for one year at Cambridge and a second year at Oxford. So, in 1934, she returned to Oxford, where she would remain for the rest of her career – as an Official Fellow and Tutor in Natural Science, then a University lecturer and demonstrator (1946), a University Reader in X-ray Crystallography (1956), and a Wolfson Research Professor of the Royal Society (1960). Among other honors, she was elected a foreign member of the American Academy of Arts and Sciences (AAAS) in 1958 and a Fellow of the Royal Society in 1947. I don’t know much about how the British academic system is set up, but sounds impressive. And her work was impressive. Really impressive. As a pioneer of macromolecular crystallography, she developed techniques to “look” at biochemical molecules like vitamins, hormones, proteins, and pharmaceutical drugs – things much smaller than had ever been looked at before – at the “atomic level.”
You see, it’s really hard to look at really small things – likes atoms separated by tiny distances. You can’t use visible light microscopes because the wavelengths of visible light are thousands of times longer than bond lengths and light’s only useful for resolving (telling apart) things that are separated by distances about half a wavelength or more. X-rays have short enough wavelengths, but short wavelengths come from having high energy – and this makes it so that we can’t focus them with lenses. So x-ray crystallography, while able to provide incredibly valuable information, gets tricky. Really tricky. And it’s only possible to solve the structures of large proteins today because of contributions by Dorothy Crowfoot Hodgkin and other early pioneers.
Much more complicated situation grossly simplified, x-ray crystallography works like this: you get molecules to crystallize (arrange themselves into an orderly 3D lattice) -> beam x-rays at them -> x-rays get scattered by the molecules -> scattered x-rays interfere with one another, some “cancelling out” while others strengthen one another depending on their relative phases (where in their peak-trough-peak-trough… cycle they are) -> these “diffracted” x-rays hit a detector, leaving a pattern of spots called a diffraction pattern -> you work backwards from those spots to figure out where they scattered from.
Even for expert crystallographers these days, it isn’t easy. Even if they have really powerful computer software helping them out. But in the early days of x-ray crystallography, they were working with pen and paper (and wire and corks – they built physical models). Dorothy even had to collect money to get an X-ray apparatus at Oxford (which she had to climb staircase-like steps to work with, which must have been really painful given her arthritis). And, lacking her own laboratory in the early years, she and her students worked in various rooms of the University museum.
In 1934, Bernal got an x-ray diffraction pattern from a crystal of the protein pepsin – Hodgkin helped with the prep & analysis (but was at a doctor’s appointment for what would be diagnosed as rheumatoid arthritis during the actual “photo shoot”) and she’s on the paper announcing this momentous feat. Part of the prep was keeping protein crystals wet – figuring out dry protein crystals diffract poorly but if you keep them wet they’re happier was a major advancement. But what made this “picture” so important? They couldn’t even figure out much about pepsin from it because the technology and analytic methods required for that were still years off. But by obtaining a diffraction pattern they were able to show that proteins – even non-fibrous ones – can have orderly structures. And it opened the door wide for protein crystallography.
As they end their paper, “At this stage, such ideas are merely speculative, but now that a crystalline protein has been made to give X-ray photographs, it is clear that we have the means of checking them and, by examining the structure of all crystalline proteins, arriving at far more detailed conclusions about protein structure than previous physical or chemical methods have been able to give.” https://www.nature.com/articles/133794b0.pdf
But there was still a lot of work to do to be able to achieve the “atomic resolution” we routinely see from crystal structures these days.
So she turned to what she thought should be a much “simpler” protein – the hormone insulin – an attractive target given that it’s small and biomedically important. It has the role of telling cells in your body to let in and use glucose (blood sugar). But, for patients with diabetes, they either don’t make enough insulin (type I) or their cells have reduced sensitivity to it (type II) – so insulin can be used as a treatment.
She thought it wouldn’t be “too bad” in terms of complexity – it was certainly a lot smaller than the pepsin! Insulin is made as a single longer chain that gets folded up & then clipped up to form 2 chains but the chains stick together because they’re connected through disulfide crosslinks – so you get an A chain of 21 amino acids & a B chain of 30 amino acids. So each mature insulin “monomer” is 51 amino acids. But pairs of them formed dimers. And then 3 of those pairs joined up to form a hexamer. At least in the presence of zinc, like they’d used in their crystal prep. And these hexamers work for storing insulin in your pancreas and releasing it into the bloodstream to fall apart into the monomers which bind cell receptors to tell them to let in glucose.
But turns out that the hexamer-izing can be a problem if you want to use it as a drug to treat diabetes. Because, while the monomers and dimers can easily diffuse into the bloodstream, those big ole hexamers have a harder time. Thankfully the structure offered later scientists a key to preventing this – The structure showed that the dimers where forming because of hydrogen-bonding between the C-termini of the monomers. Knowing this, once recombinant protein expression became a thing (being able to stick protein instructions into cells to have them make it for you) pharma companies could change insulin’s spelling in a way that didn’t affect its receptor binding but did prevent dimerization – for example, insulin lispro swaps the lys & pro to prevent this H-bonding
But this was years off. Even the structure would prove to be years off – 34 years, actually from when she took the first X-ray diffraction photos of insulin in 1935 (and was so excited she panicked herself into thinking the diffraction pattern was coming from a salt or some contaminant or something instead of insulin that she rushed in the following morning to check!) to when, in 1969, she reported the structure of 2 Zn insulin at a resolution of 2.8A.
But, while working on and off on the insulin project, her “bucket list protein” over the decades, she was also able to determine some other really really important crystal structures – and pick up a Nobel Prize while she was at it…
It “only” took her 4 years to determine the structure of the first molecule for which she is famous – the antibiotic penicillin. In 1945, she solved its structure with the help of her graduate student, Barbara Low. Its discovery came at a time it was greatly needed. It was in the midst of World War II, when battlefield wounds and infections were rampant, and knowing the structure helped chemists develop modified versions of penicillin to treat a wider range of infections (and Hodgkin figured out the structure of many of those modified versions as well) http://bit.ly/2QLTvT1
And it took her 8 years to determine the structure of the next molecule to bring her fame (she wasn’t getting slower, she was just dealing with a molecule that was way more complex!). Instead of “just” 17 (non-hydrogen) atoms, vitamin B12 had a whopping 90!
Solving the structure of vitamin B12 required some creative thinking… One of Dorothy’s lab’s members, Galen Lenhert showed that one of vitamin B12’s carbons was bound to cobalt. Which was weird – because cobalt is a metal, and scientists weren’t used to finding it in “organic” (carbon/hydrogen backboned) molecules. In fact, the discovery made vitamin B12 the first known naturally occurring biologically significant organometallic compound.
Dorothy realized that this wasn’t just “cool” – for structure determination, it could be really useful! As the “heavy” in “heavy metals” hints at, metals like cobalt have a lot of electrons. And, electrons are what scatter x-rays when crystals get hit – when x-rays hit crystals, they get scattered from the electrons of the atoms because they kinda “ring” the electron clouds, getting the electrons to send off their own waves of the same wavelength (kinda like billiard balls getting tossed in a pool and generating rippling waves rather than billiard balls bouncing off pool table walls). The waves add together through wave interference, sometimes canceling out, other times strengthening each other, leading to kinda “megawaves.” You can capture the signals from these megawaves on the detector and deconstruct the megawave to the miniwaves using a math thing called a Fourier transform. But it requires you to know the “phases” – was a wave peaking or troughing when it hit? And you lose this information in crystallography – you only measure the amplitudes (how strong was the wave when it hit). And this leads to the “phase problem”
Carbon and oxygen and hydrogen and all those “usual players” in biochemical molecules don’t vary very much in terms of their electron stock (hydrogen has 1, carbon 4, and your “big guy” oxygen has 8). Cobalt? In its neutral form it has 27. So it’s kinda like having 27 hydrogens in one spot – the cobalts will scatter more strongly than the other atoms in the structure. And this kinda “puts a pin in the map” – if you know what to look for, it “sticks out” in the diffraction pattern, allowing scientists to orient themselves in the signal.
So, to solve the structure, Dorothy pioneered the use of heavy atoms for phasing. Cobalt isn’t *that* heavy, so she also used a few other tricks, including making a version where she added selenocyanate to complex with the cobalt – this introduces another heavy atom, selenium (34 electrons in neutral form), so you get a second pin in the map.
So she ended up working with crystals of 4 forms to solve the structure – “wet” & “dry” versions of the normal, a selenocyanate derivative, and a partly degraded, simpler version, a hexacarboxylic acid derivative, that allowed them to really nail down the central core region – that never-before-seen “corrin ring”
The work involved several collaborations and 2500 x-ray photos. Their initial work used a punch card machine like they used for penicillin, but later they finally got some much-needed computer help. The culmination of 8 years of work, solving this structure in 1955 was a tremendous achievement – both for its size (it was by far the largest & most complex structure solved to date) and its biomedical relevance – vitamin B12 is an essential nutrient that can be used to treat pernicious anemia, a disease caused when people’s bodies have problems absorbing it. And seeing what it looked like helped chemists figure out ways to synthesize it so they could produce the vast quantities needed to treat patients whose bodies needed it but weren’t good at taking it if you give it to them.
Stockholm folks agreed that it was a tremendous achievement, awarding her the Nobel Prize in chemistry in 1964 for “her determination by X-ray techniques of the structures of biologically important molecules.” Scientists clearly saw the value of her work, but some in the general public seemed to be more fascinated in her gender – with tabloids reading things like “Oxford Housewife Wins Nobel Prize” & “Nobel Prize for British Wife,” and reporters asking about how she managed to do all that scienceing with a busy domestic life.
Dorothy did lead a busy family life – she married Thomas Hodgkin in 1937, a historian who focused on African and Arab history and politics and directed the Institute of African Studies at the University of Chana. They had three children (and Dorothy’s lab notes include measurements of her babies’ growth rates). Their eldest son became a mathematician, their younger son, a botanist/agriculturalist, and their daughter a historian.
Dorothy also lead an active activist life – in addition to fostering her love of science, her mother, who had lost all her brothers in WWI, influenced Dorothy’s political and philosophical leanings. A vocal advocate for world peace, Dorothy campaigned against the Vietnam War and spoke out against nuclear weapons, even serving as president of the Pugwash Conferences on Science and World Affairs, an organization formed to oppose the proliferation of weapons of mass destruction. She received the Soviet’s equivalent of the Nobel Peace Prize, the Order of Lenin, in 1987. She also believed that international conflicts should not impede science – instead, she urged international scientific cooperation, helping found the International Union of Crystallography and insisting that, even during the tense times of the Cold War, Soviet & Chinese scientists be included.
Speaking of differences in political opinions, while working at Oxford in the 1940s, Crowfoot served as a tutor to the future, conservative, Prime Minister Margaret Thatcher – despite their different political leanings, they maintained a strong relationship, with Thatcher inspired by Hodgkin’s position as a powerful woman. As Prime Minister, Thatcher displayed a photo of Hodgkins, and a play (titled “The Chemistry Between Them”) was even written about their relationship by Adam Ganz.
Dorothy Crowfoot Hodgkin retired in 1977 and died July 29, 1994, but her legacy lives on, including through the work of prominent female crystallographers whom she mentored, including Barbara Low and Jenny Glusker. Dorothy worked at a time when women lost fellowships and research grants when they married, with their funders claiming the women were now “living at home” with their spouses providing for them – Dorothy spoke out against these ludicrous policies, portioning for individual affected women when the issue arose time and time again.
more on x-ray crystallography: http://bit.ly/2QASc8h