The Story of Insulin Crystallography

HISTORICAL NOTES The story of insulin crystallography M. Vijayan Early efforts was stopped by a policeman. I said I was vector density (vector set) into electron just going back to college and slowly density (the structure). If there are n The discovery of insulin by Banting and turned west. Next morning I woke up atoms in the structure, the vector set Best1 in the early twenties of this century with a horrid fear – perhaps the objects I would contain n2–n non-origin peaks. In was a major event in the history of thera- photographed were not insulin? I hurried the absence of non-random features like peutics. Insulin has since been involved straight to the laboratory and tested my heavy atoms, deciphering the positions in many landmarks in the development of crystals for protein in the xantho protein of the n atoms from the n2–n peaks is an biology. For instance, insulin was the reaction – yellow with nitric acid, brown impossible task, even for the structure of first protein to be sequenced2 and also with ammonia – it worked. I went gladly a moderately-sized organic molecule. chemically synthesized3–7. Insulin has back to college and breakfast’ (Figure 1). The task is utterly hopeless when the also been involved, long before these During the next couple of years, Doro- structure contains hundreds or thousands developments, in the initiation of macro- thy carried out detailed investigations, as of atoms, as in a protein. Yet Dorothy molecular crystallographic studies. In detailed as could be done in those very undertook to calculate a Patterson fact, it was the second protein to be sub- early days of protein crystallography, on map manually using Beevers–Lipson jected to preliminary X-ray crystallo- the crystals of insulin and the diffraction strips18,19, the magic weapon in the graphic studies. Pepsin was the first. The patterns from them13–15. She painstak- armoury of crystallographers for comput- history of biological macromolecular ingly measured the density of the crys- ing Fourier series in the pre-computer crystallography began in 1934 at Cam- tals. They belonged to the rhombohedral days. At the end of this stupendous ef- bridge when J. D. Bernal and his young system, a crystallographer’s nightmare in fort, the Patterson function did not reveal Ph D student Dorothy Crowfoot (subse- the pre-computer days. Yet, she success- any meaningful pattern that Dorothy quently Hodgkin) studied the diffraction fully indexed all the diffraction spots might have hoped to see. However, the pattern from the crystals of the digestive recorded using a primitive oscillation preliminary X-ray work, as a whole, enzyme, pepsin8. Bernal observed that camera and measured their intensities showed the molecular weight of the pro- the crystals lost birefringence when using eye estimation. By then Patterson tein in the unit cell to be 37,600 Da, a removed from the solution of crystalliza- had discovered the synthesis named after value remarkably close to the weight of tion and dry crystals did not give a dif- him16,17. He showed that a Fourier syn- an insulin hexamer as we know it today. fraction pattern. Therefore, he mounted thesis computed with the intensities as The work also demonstrated that the unit crystals, along with the mother liquor, in coefficients would give a representation cell with this weight had trigonal Lindemann capillaries, a practice fol- of the interatomic vectors in the struc- symmetry14. lowed since then, except when the ture. A Patterson function would obvi- Pepsin and insulin were not the only recently introduced technique of flash ously correspond to objective reality. proteins to be examined using X-ray freezing is used. The problem is the deconvolution of the crystallography in the thirties. Dorothy On her return to Oxford, twenty-five- took up the study of lactoglobulin along year-old Dorothy took up the X-ray with Riley20. Max Perutz had also started analysis of insulin as the first problem in his monumental work on the X-ray her independent research career. Bovine analysis of haemoglobin21. These were insulin had earlier been crystallized by heroic efforts of the pioneers, with no Abel in 1926 (ref. 9). The method of reasonable prospects of success. That crystallization in the presence of salts of was a time when one did not even know zinc and other metals, was standardized what proteins were. Furthermore, it was by Scott in 1934 (ref. 10). It is this a time when the solution of the crystal method that Dorothy used for growing structure of even a small organic mole- crystals. She could then record a series of cule involved formidable intellectual X-ray diffraction photographs from these challenge. Yet, it was these heroic efforts crystals11. The excitement of seeing the that laid the foundations of macro- first diffraction photograph is best cap- molecular crystallography, which now tured in her own words12: ‘The moment forms an indispensable component of late that evening about 10 pm – when I modern biology. developed the photograph and saw the central pattern of minute reflections was probably the most exciting in my life. I Gathering momentum waited while the film was washed, hung it up to dry, and wandered out, too Dorothy herself drifted away from insu- excited to return to college immediately. Figure 1. Dorothy Crowfoot in the late lin in the late thirties to other problems. I wandered South to the Broad and there thirties. She had already been involved in the 1598 CURRENT SCIENCE, VOL. 83, NO. 12, 25 DECEMBER 2002 HISTORICAL NOTES structure analysis of cholesterol and tallographic work on insulin. The rhom- fold axis appeared to be about 1 Å from other sterols. During the next couple of bohedral forms, particularly 2Zn insulin, the threefold axis. decades, she solved the structures of received special attention. The rotation and translation functions many important molecules, including In the meantime, Rossmann and enable the delineation of the basic 26,27 penicillin and vitamin B12. The structure Blow had developed their rotation framework of the quaternary structure. solution of vitamin B12, which even and translation functions, which formed But heavy atom derivatives were neces- today shines through as a great intellec- the basis of the molecular replacement sary, the situation still substantially con- tual achievement and exhibition of sheer method; currently perhaps the most tinues to be so, to determine the atomic crystallographic prowess, fetched a widely used method of structure solution positions in a protein of totally unknown Nobel Prize. Yet insulin remained the of proteins. Rotation and translation structure. Perutz was the first to demon- problem closest to her heart. Come back functions seek to establish the geometric strate that the presence of one or a few she did to insulin in the fifties, while relation between chemically identical or heavy atoms such as mercury, uranium Perutz and Kendrew had been demon- similar, but crystallographically non- or lead per protein molecule in a crystal, strating through their X-ray analysis of equivalent protein molecules in the same would lead to measurable changes in the haemoglobin and myoglobin, that protein crystal or different crystals. This essen- intensities of X-ray diffraction spots29. structures could be solved using the iso- tially involves rotation and translation of These differences can then be used to morphous replacement method. vector sets as visualized by Patterson calculate the phase angles of the ampli- The last primary publication during maps. Application of the two functions to tudes which can be readily obtained from the early phase of insulin crystallography 2Zn insulin and 4Zn insulin was the intensities. Once the amplitudes and appeared in 1939. The next crystallo- described in a second paper published in the phase angles are known, the electron graphic paper on insulin co-authored by 1966 (ref. 28). The unit cell – the basic density can be calculated, and hence the Dorothy Hodgkin was published in 1966 unit, the repetition of which in three- structure obtained, by computing a Fou- (ref. 22), a remarkable instance of Doro- dimensional space produces the crystal – rier synthesis. In principle, two inde- thy’s ability to pick up the threads of a of 2Zn insulin has an internal threefold pendent heavy-atom derivatives are problem after long years of comparative symmetry and contains an insulin necessary to solve a protein structure30. neglect. The primary structure of insulin hexamer and two zinc ions in addition to In practice, several are desirable. The had been determined by then2. Also, the solvent. The two ions are located on preparation of heavy-atom derivatives Schlichtkrull had systematically grown the threefold axis with a distance of involves the attachment of heavy atoms four different crystal forms of pig insu- about 17 Å between them. The three or groups containing them to the protein lin; one cubic, another monoclinic and dimers that constitute the hexamer are molecules in a coherent manner, without two rhombohedral23. One of the rhombo- related to one another by the crystallo- altering the structure of the molecules hedral forms corresponded to the crystals graphic threefold axis. The rotation and and their arrangement in the crystal. The originally photographed by Dorothy translation function studies showed that derivatives so produced are called iso- (Figure 2). The rhombohedral forms the two monomers in the dimer are morphous heavy-atom derivatives and were called 2Zn insulin and 4Zn insulin related to each other by a non-cry- the method of solving structures using as they contained two and four zinc ions stallographic twofold axis perpendicular them is called the multiple isomorphous respectively, per insulin hexamer. It is to and passing through the crystallo- replacement method. Heavy-atom deri- the preliminary X-ray studies on these graphic threefold axis.

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