Protein Science (1994), 3:1625-1628. University Press. Printed in the USA. Copyright 0 1994 The Society

”. ” ” ~~ ”” . .. .. INVITED TRIBUTE, SPECIAL SECTION IN HONOR OF MAX PERUTZ Max Perutz’s achievements: How did he do it?

DAVID EISENBERG UCLA-DOE Laboratory of Structural and Molecular Medicine, Institute, and Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90024-1570

This issue of Protein Science honors the 80th birthday of Max sin, which Bernal and Dorothy Crowfoot Hodgkin had obtained Perutz, oneof the great contributorsto our science. Other great the year before. Professor Mark claimed he was so excited by contributors include EmilFischer, who showed that are these photos that he forgot aboutPerutz’s request to workwith polymerized peptides; James Sumner, who proved decisively Hopkins and instead arranged for him to work with Bernal. that enzymes are proteinsby crystallizing urease; Fred Sanger, When Perutz heard about this, he protested that he knew no who determined the amino acid sequence of insulin, and later . Professor Mark replied: “Never mind, my boy, along with Gilbert gave us accurate and rapidways to determine you will learn it.” DNA sequences fromwhich protein sequences can be derived; And so Perutz joined Bernal’s lab in September 1936. Perutz and , who came with up the basic secondary struc- (1980) recalls Bernal as “the mostbrilliant talker I have ever en- tures, the a-helix and 0-sheets. These were all great discover- countered . . . [and] was soon inspired by [Bernal’s] visionary ies. But Max Perutz gave us the method for determining the faith in the power of X-ray diffraction to solve the structures 3-dimensional structures of proteins and also provided the in- of molecules as large and complex as enzymes or viruses at a tellectual environment thatled to thediscovery of the structure time when the structure of ordinary sugar was still unsolved” of DNA. He hasbeen perhaps the greatest leaderin the merger (Perutz, 1985a). of chemistry with biology and is regarded by all as one of the But Ph.D. dissertations are not based on inspiration alone. principal protein scientists of the century. By the following summer, Perutz still had no thesis topic and was concerned about his future (Perutz,1980). When he returned to Austria for summer vacation, he remembered that onehis of The path to protein structure cousins in Prague had married a biochemist, Felix Haurowitz, How did Perutz comedevelop to a method to reveal the 3-dimen- and Perutzcalled on him for advice. Haurowitzsuggested as a sional structures ofproteins? It was through a series of accidents thesis the structure of . Apparently, like Perutz, he and then single-minded persistence.’ His father was an Austrian did notrealize that itwas then impossible to determine protein textile manufacturer who had spent his apprenticeship in En- structures: there was as yet no practical way to measure accu- gland (Perutz, 1980). As an ardent Anglophile, the father was rately so many intensities of diffraction; therewas no way to de- willing to underwrite the son’s graduate educationin Cambridge, termine the unknown phases of the diffracted beams; and there England. Max Perutz, as an undergraduate at theUniversity of were no computers, or even calculators, to compute electron in 1935, had learned of the work in Cambridge by Fred- density maps. Haurowitz showed that as enters hex- erick Gowland Hopkins on vitamins. When he heard that his agonal plates of horse deoxyhemoglobin, they disorder and physical chemistry professor from Vienna, Herman Mark,was monoclinicneedles of oxyhemoglobin form in their place going to attenda meeting in Cambridge, Perutz asked Mark to (Haurowitz, 1938). This must mean thatoxygen changes hemo- find a place for him in Hopkins’s laboratory. However, while globin’s shape, and Perutz wanted tosee how. He got down to in Cambridge, Professor Markvisited J.D. Bernal, whoshowed work, not knowing that it would take him 22 years, even to make him the first good X-ray diffraction pictures a of protein, pep- a start. At first Perutz tried to determine the phasesby scanning the ~ ~~~______.___.~ Reprint requests to: David Eisenberg, UCLA-DOE Laboratory of molecular transform,using the diffraction pattern of hemoglo- Structural Biology and Molecular Medicine, MolecularBiology Insti- bin crystals in different stages of swelling and shrinkage. Here- tute, and Departmentof Chemistry and Biochemistry, University of Gal- calls (Perutz, 1985a): “. . . with immense labor I mapped out the ifornia at LOSAngeles, Los Angeles, California 90024-1570; e-mail: nodes and antinodes of the molecular transform of hemoglo- [email protected]. bin, . . . but managed to determine thesigns of only a few low- ’ Fortunately, Max Perutz has written about his development as a order hol reflections. I also spent many years collecting and scientist and of his process of discovery. In this editorial, I have drawn from these sources, and 1 have freely quoted and paraphrased from visually measuring the intensities of the reflections needed for them. The most extensive sourceis Perutz’s 1989 book, Is Science Nee- a three-dimensional Patterson at 2.8 A resolution, hoping that essary?, which is recommended with enthusiasm to all protein scientists. some simplifying regular features of the molecular structure 1625 1626 D. Eisen berg would make that Patterson interpretable.I did in fact fool my- Success at last self into believing that I had solved the structure of hemoglo- bin, only to find my solution demolished by my newly arrived Nevertheless, from 1953 onward, there was steady progress: research student . . . . I was desperate that 16 years’ a projection map of hemoglobin in 1954 (Bragg & Perutz, 1954); toil had led me nowhere. . . .” a second heavy-atom derivative finally in 1958; a method by But just then, in 1953, renewed hope arrived in the form of (1960) (The Protein Society’s 1994 Stein and a reprint, from Austin Riggs at Harvard, showing that it was Moore Awardee) for determining the relative positions in the possible to bind mercury to hemoglobin. When Perutz exam- unit cell of the 2 heavy atoms; and at last,in 1960, a paper by ined X-ray pictures of hemoglobin with bound mercury, hesaw Perutz, Rossmann and othersdescribing the tertiary and quater- that therewere marked changes in the relative intensities of the nary structureof hemoglobin at 5.5 A resolution (Perutz et al., diffraction spots. He said that this,“. . . was the most exciting 1960). Meanwhile, Perutz’s colleague and former student, John moment in all my research career, because I realized at that mo- Kendrew, had pressed ahead to apply isomorphous replacement ment that now in principle, the protein problem was solved” to the smaller molecule. With myoglobin, but not at (Judson, 1979). In September 1953, he reported this finding at first with hemoglobin, heavy atoms could be bound between the Pasadena Conference on the Structure of Proteins, orga- molecules in the crystal lattice, and,by 1960, it was possible to nized by Linus Pauling. But the conferees took little notice of build an atomic model of the structure at2 A resolution (Ken- his talk. The attention instead went to Linus Pauling and his drew et al., 1960). “Today this lookslike child’s play,” recalled a-helix and to Watson and Crick for theirnew DNA structure. Perutz (1985a) about the 6-A-resolution myoglobin structure of This structure hadbeen worked out in Perutz’s lab, where Wat- 1959, “but remember that the phase angles had to be determined son had come as a postdoc to apply X-ray diffraction to the manually by superposition of circles for each reflection. . . . study of DNA, and where Crick was a graduate student. Nearly all the methods used had to be developed from scratch Even though the method of addingheavy atoms to proteins as the work progressed.” was the solutionin principle to the problem of protein structure, The photograph of the 1962 ceremony (Fig. I) Perutz found in practice that, “There were all kinds of dread- shows a beaming Perutz, who, alongwith Kendrew, received the ful complications. In retrospect, 6 years doesn’t seem such a ter- award in chemistry. Fortunately, neither awards norscientific ribly long time, but when you’re actually living six years, and fame quenched Perutz’s curiosity about hemoglobin. He and his trying, not very successfully, to apply this method which you coworkers extended the resolution of the hemoglobin structure knew could solve the structureof proteins, it seemed an eternity” to 2.8 A (Perutz et al., 1968) and then to 1.74 A (Fermi et al., (Judson, 1979). 1984). Perutz (1970) presented a detailed atomic model for the

Fig. 1. In 1962, Perutz (second from left) and Kendrew(on right) received the . At the same ceremony, \Watson (second from risht) and Crick (third from left)received the award in physiology and medicine, sharing it with from London (on left). The fourth principal in the DNA structure, , had died at age 37, 4 years before. At the center is , the literature awardee. (Photograph made available by UPI/Bettmann.) Max Perutz and protein science 1627 stereochemical basis of of oxygen, and he tein Society members know enough about the propertiesice of and coworkers explained the effects on hemoglobinphysiology to understand what this means for the volumeice of below the pH and of dozens of mutants species and differences. Onecol- water), and with a cruising range of 7,000 miles. Its displace- league recently heard Perutz speak on an unexpected mutation ment of 2.2 million tons was 26 times that of the QueenEliza- in high-altitude globins. It conflicted with his original interpre- beth, then the biggest ship afloat. The rudder was to be the tation. In pursuing the problem, Perutz explained,“I just had height of a 15-story building. Turbo-electric steam generators to know.” were to supply 33,000 horsepower to drive the bergship. The dual Nobelprizes for the structures of both proteins and Pyke’s mind raced ahead to work out new ways bergships DNA constituted tangible recognition for MaxPerutz’s second would win the war.Pyke’s idea was that bergships would force great achievement: the Medical Research Council Laboratory their way into enemy harbors, where defending troops would of Molecular Biology in Cambridge, England. This lab, with be frozen solid by sprays of supercooled water, supplied from Perutz as Director from 1947 to 1979, has members who have enormous tanks on board. Pyke did not concernhimself with made many discoveries, in additionto the 3-dimensional struc- details such as how the supercooledwater in the tanks wouldbe tures of proteins and DNA. Thereis the sequencing of proteins, prevented from crystallizing. Meanwhile, Perutz was assigned the sequencing of DNA, the sliding filament model of muscle, to find some location on earth for freezing 2 million tons of monoclonal antibodies, 3-dimensional reconstruction from mi- during the winter of 1943. crographs, electron crystallographyof membrane proteins, and However, pykrete and bergships fell from favor. A major rea- the pioneering work on molecular replacement. How couldall son was Pyke’s megalomania, but another was that a demon- of this, and much more, have happened under one roof? stration of pykrete to the Chiefs of Staff literally backfired. To have a chance of answering thisquestion, we need to know Perutz prepared large blocks of ice and pykrete, but as an Aus- more about Max Perutz, the person. We are helped in this by trian, he was not invited to make the demonstrationhimself. The Perutz’s graphic World WarI1 memoirs, which were published officer who carried out the demonstration fireda revolver into in the New Yorker (Perutz, 1985b), and reprinted in his book, the block of ice, which shattered. When he fired into theblock Is Science Necessary? (Perutz, 1989). In the following section, of pykrete, thebullet rebounded and hit theChief of the Impe- I paraphrase closely from these memoirs. rial General Staff in the shoulder. Pyke was disappointed about thedemise of the bergships but busied himself with new plans. One was to constructa gigantic World War I1 pneumatic tube to run under the Himalayas, to propel soldiers In May 1940, just several weeks after he received his Ph.D., and tanks from Burma to China with compressed air. At this Perutz was arrested in Cambridge as an enemy alien, interned, point, Perutz returned to Cambridge, “sad atfirst that [his] ea- and then deported to Canada, locked in the airless hold of a gerness to help in the war against Hitler had not founda more troop ship with 1,200 other deportees.A sister ship, crammed effective outlet, but later relieved to have worked ona project with interned Austrian and Germanyrefugees to England, was that never killed anyone-not even the chief of the Imperial torpedoed by a German U boat, drowning 615. Perutz was em- General staff” (Perutz, 198513). bittered, having first been rejected as a Jew by his native Aus- Perutz’s brilliant narration of his war experiences is both mov- tria, which he loved, and now finding himself rejected as a ing and hilarious andcarries serious messages. The centralmes- German by his adopted country. He was frustrated at having sage seems to be that war provides an excuse to suspend our to idle away time instead of helping in the war against Hitler. normal critical evaluations. The Pykes of this world take the To keep spirits up, Perutz organizeda university in the prison place of study sections, and then science and technology, funded camp, with courses on vectoranalysis, theoretical physics, and uncritically, go wild, as with plans for bergships. Society also of course, crystal structure analysis. suffers fromuncritical social judgments, such as the British de- At the start of1941, Perutz’s friends at homewere successful cision to deport thousands of innocent and loyal refugees. Of in getting him released to return to England and hemoglobin. course, we in the United States fell prey to similar bad judgment: But soon he was approached by a shadowy figure named Pyke, in 1942, we arrested and interned 120,313 Japanese-Americans, to help in a top-secret war project. Later Perutz learned that some 80,000 of whom were U.S. citizens by birth. Pyke’s career had been a string of failures, but “despitehis fail- ures Pyke remained unshakenin his faith that heknew how to Max Perutz as scientist and leader perform any job better than those whose profession that job happened to be, and as soon as the.. . war broke out he became To complete this editorial,I attempt to summarize thequalities intent on telling the soldiers how to win it.” that allowed Max Perutz tobuild a great lab, and to make great At first Perutz was told only that he shoulddiscover how to discoveries. Perutz hints at his own view in a book review he make ice strong. This he did,with the help of a report from his wrote of a biography of Lord Rutherford (Perutz,1984, 1989, former professor fromVienna, Herman Mark,by freezing it in page 164). Perutz says that, duringhis first year in Cambridge, the presence of wood pulp,in analogy to theway concrete is re- he became imbued with Rutherford’s scientific values. These inforced by steel bands. Perutzcalled this reinforcedice “pykrete,” came from the atmosphereof the lab and from Rutherford’s pa- in honor of Pyke, the head of the project. Only gradually did pers, which he still looks to as modelsof the way science should Perutz learn what Pyke had in mind for strong ice: it was to be done. Rutherford’s values are stated as “loyaltyto your lab- build immense aircraft carriers out ofice, called bergships. The oratory, extreme devotion to hard experimental work, and grandiose design of bergships kept growing until it called for strong aversion to speculation beyond what is justified by the a self-propelled, self-refrigerating ice ship, 2,000 feet long by experimental results.”But having said that, Perutzimmediately 200 feet wide, with a flight deck 50 feet above the water (Pro- adds 3 sentences that reveal that he has a wider view: He says, 1628 D. Eisenberg

“When Crick and Watsonlounged around, arguing about prob- ership by example, a deep interest in both ideas and people, and lems for which there existed as yet no firm experimental data an openness to varied research styles. But perhaps the most im- instead of getting down to the bench and doing experiments, portant factoris his driving need to know. Perutz simply needed I thought they were wasting their time. However, like Leonardo, to know how hemoglobin changes shape when it binds oxygen. they sometimes achieved most when they seemed to be work- What other thanthis could have kepthim going, through 16 years ing least, and their apparent idleness led them tosolve the great- with no positive results, on to the structure of proteins? est of all biological problems, the structureof DNA. There is more than oneway of doing good science.” References In 1985, I was privileged to spend an 8-monthsabbatical leave Bragg WL, Perutz MF. 1954. The structure of haemoglobin: Fourier pro- in the Cambridge laboratory that Perutz had built. Onmy first jections on the 010 plane. Proc R SOC Lond Ser A 225:315-329. morning, Perutz’s secretary sought me outwith a message from Fermi 0,Perutz MF, Shaanan B, Fourme R. 1984. The crystal structure of him. This in itself was surprising because I had made my sab- human deoxyhaemoglobin at 1.74 A resolution. JMolBiol175:159-174. Haurowitz F. 1938. Das Gleichgewicht zwischenHamoglobin und Sauerstoff. batical arrangements with his successor as Director, and I had Hoppe-Seyler 254:266-274. no reason to think that Perutz would take it upon himself to Judson HF. 1979. The eighth day of creation: Makers of the revolution in greet me. But themessage held another surprise: he apologized biology. New York: Simon & Schuster. for not being at the lab towelcome me becausehe had been up Kendrew JC, Dickerson RE, Strandberg BE, Hart RG, Davies DR, Phillips DC, Shore VC. 1%. Structure of myoglobin: A three-dimensional Fou- most of the night, trying to get his experiment going. He was rier synthesis at 2 A resolution. Nature 185:422-427. then in his 70th year. And his love of experiments continues: Perutz MF. 1970. Stereochemistry of cooperative effects in hemoglobin. Na- a recent issue of PNAS contains his article on polar zippers ture 373:726-739. Perutz MF. 1980. Origins of molecular biology. New Scientist 85(1192): (Perutz et al., 1994). 326-329. In the Cambridge lab,I failed to find the formality thatI had Perutz M. 1984. Review of Rutherford by David Wilson. London Review expected from my own graduate studentdays at Oxford.Every- of Books 6(7):7-8. [Also reprinted in Is Science Necessary?] one, from students andtechnicians to Max, was on a first-name Perutz M. 1985a. Early days of protein crystallography. Methods Enzymol 114:3-19. basis. Nearly the whole lab met in the top floor canteen for Perutz M. 1985b. That was the war: Enemy alien. The New Yorker Aug. morning coffee, lunch, and then afternoon tea. Inmy first few 12 1985. pp 35-54. [Also reprinted in Is Science Necessary?] days, I resented these intrusions into the work day. ButI soon Perutz M. 1989. Is science necessary? New York: E.P. Dutton. Perutz M, JohnsonT, Suzuki M, Finch JT. 1994. Glutamine repeats as po- caught on that there would always be someone at or tea coffee lar zippers: Their possible role in inherited neurodegenerative disease. who could help me conquermy current problem. I never went Proc Natl Acad Sci USA 91~5355-5358. more than 3 hours wrongin my research. I was able toprogress Perutz MF, Muirhead H, Cox JM,Goaman LC. 1868. Three-dimensional fourier synthesis of horse oxyhemoglobin at 2.8 A resolution: The atomic rapidly and, toward the end of my Cambridge stay, I wrote a model. Nature 219:131-139. paper on my findings. Perutz asked tosee the manuscript and Perutz MF, Rossmann MG, Cullis AF, Muirhead H, Will G, North ACT. returned itwith an encouraging note, including suggestions 1960. Structure of haemoglobin: A three-dimensional Fourier synthesis about science and the use of the English language. at 5.5 A resolution, obtained by X-ray analysis. Nature 1853416-422. Rossmann MG. 1960. Theaccurate determination of the position and shape To sum up, the factors thatI see in Perutz’s colossal achieve- ofheavy-atom replacement groups in proteins. AcraCrystallogr ments are a love of experiments, intense intellectual focus, lead- 13~221-226.