What Mad Pursuit (6 page)

Even before I left the Admiralty there had been some quite unexpected evidence pointing to DNA as near the center of the mystery. In 1944 Avery, MacLeod, and McCarty, who worked at the Rockefeller Institute in New York, had published a paper claiming that the “transforming factor” of pneumococcus consisted of pure DNA. The transforming factor was a chemical extracted from a strain of bacteria having a smooth coat. When added to a related strain lacking such a coat it “transformed” it, so that some of the recipient bacteria acquired the smooth coat. More important, all the descendants of such cells had the same smooth coat. In the paper the authors were rather cautious in interpreting their result, but in a now-famous letter to his brother Avery expressed himself more freely. “Sounds like a virus—may be a gene,” he wrote.

This conclusion was not immediately accepted. An influential biochemist, Alfred Mirsky, also at the Rockefeller, was convinced that it was an impurity of the DNA that was causing the transformation. Subsequently more careful work by Rollin Hotchkiss at the Rockefeller showed that this was highly unlikely. It was argued that Avery, MacLeod, and McCarty’s evidence was flimsy, in that only one character had been transformed. Hotchkiss showed that another character could also be transformed. The fact that these transformations were often unreliable, tricky to perform, and only altered a minority of cells did not help matters. Another objection was that the process had been shown to occur just in these particular bacteria. Moreover, at that time no bacterium of any sort had been shown to have genes, though this was discovered not long afterward by Joshua Lederberg and Ed Tatum. In short, it was feared that transformation might be a freak case and misleading as far as higher organisms were concerned. This was not a wholly unreasonable point of view. A single isolated bit of evidence, however striking, is always open to doubt. It is the accumulation of several different lines of evidence that is compelling.

It is sometimes claimed that the work of Avery and his colleagues was ignored and neglected. Naturally there was a mixed spectrum of reactions to their results, but one can hardly say no one knew about it. For example, that august and somewhat conservative body, the Royal Society of London, awarded the Copley Medal to Avery in 1945, specifically citing his work on the transforming factor. I would dearly love to know who wrote the citation for them.

Nevertheless, even if all the objections and reservations are brushed aside, the fact that the transforming factor was pure DNA does not in itself prove that DNA alone is the genetic material in pneumococcus. One could quite logically claim that a gene there was made of DNA
and
protein, each carrying part of the genetic information, and it was just an accident of the system that in transformation the altered DNA part was carrying the information to change the polysaccharide coat. Perhaps in another experiment a protein component might be found that would also produce a heritable change in the coat or in other cell properties.

Whatever the interpretation, because of this experiment and because of the increased knowledge of the chemistry of DNA, it was now possible that genes might be made of DNA alone. Meanwhile the main interest of the group at the Cavendish was in the three-dimensional structure of proteins such as hemoglobin and myoglobin.

L
ET US NOW return to my own career. I still had to make contact with Max Perutz. One day in the late 1940s, I was returning to Cambridge from a visit to London, having arranged to call on Perutz at the physics laboratory where he worked. The train journey from London was uneventful. I watched the countryside slide past but my thoughts were elsewhere, focused mainly on my impending visit to the Cavendish Laboratory. For a British physicist the Cavendish had a unique glamour. It had been named after the eighteenth-century physicist Henry Cavendish, a recluse and an experimenter of genius. The first professor had been the Scottish theoretical physicist James Clerk Maxwell, of Maxwell’s equations. While the laboratory was being built he did experiments in his kitchen at home, his wife raising the room temperature for him by boiling pans of water.

It was at the Cavendish that J. J. Thomson had “discovered” the electron by making measurements of both its mass and its charge. Thompson was an interesting case of an experimenter who was so clumsy that his associates tried to keep him away from his own apparatus, for fear of his breaking it. Ernest Rutherford, fresh from New Zealand, had started his main research career there and later returned to succeed J.J. as Cavendish Professor. There, under his direction Cockroft and Walton had first “smashed the atom”—that is, had produced the first artificial atomic disintegration. Their original accelerator was still there. And in the early 1930s James Chadwick (whom I knew later as Master of Caius College) had in a few short weeks discovered the neutron. At that time the Cavendish was in the very forefront of research in fundamental physics.

The current Cavendish Professor was Sir Lawrence Bragg (known to his close friends as Willie), the formulator of Bragg’s law for X-ray diffraction. He was the youngest Nobel Prize winner ever, having been only twenty-five when he shared it with his father, Sir William Bragg. It was no wonder that I was in awe of such a world-famous institution and excited at the prospect of visiting it.

At the station I decided to take a taxi. After settling my bags, I leaned back in my seat. “Take me,” I said, “to the Cavendish Laboratory.”

The driver turned his head to look at me over his shoulder. “Where’s that?” he asked.

I realized, not for the first time, that not everyone was as deeply interested in fundamental science as I was. After fumbling in my papers I found the address.

“It’s in Free School Lane,” I said, “wherever that is.”

“Not far from the Market Square,” said the cabby, and off we went.

Max Perutz, whom I was to visit, was Austrian by birth. He had obtained his first degree, in chemistry, at the University of Vienna. He had wanted to go to Cambridge to work under Gowland Hopkins, the founder of the Cambridge School of Biochemistry. Perutz had asked Herman Mark, the polymer specialist, to try to arrange this for him when Mark went on a short visit to Cambridge. Instead Mark ran into J. D. Bernal (known to his close friends as “Sage,” because he appeared to know everything). Bernal said he would be happy to have Perutz work with him and so Max became a crystallographer. This was all before the Second World War.

By the time of my visit Perutz was working, under the loose supervision of Bragg, on the three-dimensional structures of proteins. As I explained in the last chapter, proteins belong to one of the key families of biological macromolecules. How each protein acts depends on its exact three-dimensional structure. It is therefore crucially important to discover such structures experimentally. At that time the largest organic molecule whose three-dimensional structure had been determined by X-ray diffraction was two orders of magnitude smaller than a typical protein. A determination of the three-dimensional structure of a protein seemed, to most crystallographers, almost impossible or, at best, very far away. Bernal had always been enthusiastic about it, but then he was a visionary. However, it also had a great appeal for the hard-headed Bragg, since it represented a challenge. Having started his career unraveling the very simple structure of crystals of sodium chloride (common table salt), Bragg hoped he might crown his achievements by solving one of the
largest
possible molecular structures.

Before the war, Bernal had founded the study of the X-ray diffraction of protein crystals. One day, he was observing the optical properties of a protein crystal, using the light microscope (actually a polarizing microscope). The crystal was sitting on an open glass slide, with a little bit of the mother liquor of the crystal (the solution in which the protein crystal had been grown) attached to it. Slowly the water in the mother liquor evaporated into the air till eventually the crystal became dry. As it did so Bernal saw the optical properties deteriorate, since the dry jumbled crystal transmitted the light in a more confused way than before. Bernal immediately realized that it was important to keep protein crystals wet and proceeded to mount a crystal in a small silica tube, sealed with a special wax at each end. Fortunately the silica interfered very little with the X rays being diffracted from the crystal. All previous attempts to get X-ray diffraction photos from protein crystals had produced only a few smudges on the photographic plate since the crystals used had dried in the air. Great was the excitement in Bernal’s lab when the wet crystal produced many beautiful spots. The study of protein structure had taken a decisive first step.

Before I first visited Max Perutz at the Cavendish I read the two papers he had recently published in the
Proceedings of the Royal Society
about his X-ray diffraction studies on crystals of a variety of hemoglobin. Hemoglobin is the protein that carries oxygen in our blood and makes red blood cells red, though the variety Perutz had studied came from a horse, as horse hemoglobin happens to form crystals that are especially convenient for X-ray studies. We now know that each hemoglobin molecule is made up of four rather similar subunits, each of which contains about 2, 500 atoms, arranged in a precise three-dimensional structure.

Since one cannot easily focus X rays, it is impossible to make X-ray photographs in the way one uses a lens to make photographs using visible light or by focusing electrons in the electron microscope. However, the wavelength of convenient X rays is about the same distance as the distance between close atoms in an organic molecule. For this reason the pattern of X rays that molecules scatter can, under optimal circumstances, contain enough information for the experimenter to determine the positions of all the atoms in the molecule. More correctly, such a picture shows the density of the electrons that surround each atom and that, since they have very little mass, scatter the X rays more effectively than the heavier atomic nuclei. A crystal is used because the X rays scattered from a single molecule would be too feeble. If long exposures were used to try to overcome this difficulty, the heavy dose of X rays would damage the molecule far too much and effectively cook it before enough X rays had been scattered to be useful.

In those days the X rays were registered by special photographic film, developed in much the same way that ordinary photographic negatives are developed. Nowadays the X rays are caught and measured by counters. A special camera had to move the crystal in the beam, and the X-ray film with it, in order to record a particular portion of the diffraction data at a time.

Although I must have learned all this when I took my B.Sc. in physics, I had forgotten most of it by this time, so that I could get only a rough idea of what Perutz had been doing. I learned that protein crystals usually had a lot of water in them, tucked away in the interstices in the crystal between one large molecule and its neighbors. In a drier atmosphere a crystal could shrink somewhat, as the protein molecules packed more closely together, and it was these shrinkage stages that Perutz had been studying. If the atmosphere were
too
dry, the packing of the molecules would become jumbled, as the bulky molecules vainly tried to get as near together as possible. The nice X-ray diffraction pattern, with many sharp discrete spots, would then deteriorate to a few smudges on the X-ray film. In diffraction,
regular
three-dimensional structures produce a whole series of discrete spots, as Bragg had explained many years before.

I also knew about the major problem of X-ray crystallography. Even if the strength of all the many X-ray spots were measured (in those days a tremendous undertaking) and even if the atoms in the crystals were so regular that those X-ray spots corresponding to fine details were also recorded, the mathematics showed clearly that the spots contained just half the information to reveal the three-dimensional structure. [In technical terms the spots gave the intensities of all the many Fourier components but not their phases.] If by some magic the position of each atom were known, then it was possible (though in those days very laborious) to calculate exactly what the X-ray diffraction pattern would look like, and also to calculate the missing information—the phases. But given only the spots, the theory showed that very, very many possible three-dimensional arrangements of electron density could give exactly the same spots, and there was no easy way to decide which was the correct one.

In recent years it has been shown, mainly by the work of Jerome Karle and Herbert Hauptman, how to do this for small molecules by putting various rather natural constraints into the mathematics. For this work they were awarded the Nobel Prize for Chemistry in 1985. But even today such methods cannot, by themselves, be used for large molecules of the size of most proteins.

Thus it was not surprising that in the late 1940s Perutz had not progressed very far. I listened carefully to his explanation of his work and even ventured a few comments. This must have made me appear more perceptive and quicker on the uptake than I really was. In any event, I impressed Perutz sufficiently for him to welcome the idea of my joining him, provided the MRC would support me.

In 1949 Odile and I got married. We had first met during the war when she was a naval officer—strictly speaking a WREN officer (the British equivalent of the WAVES, the women’s naval service). Toward the latter part of the war she worked at the Admiralty headquarters in Whitehall (the main government street in London), translating captured German documents. After the war she became an art student again, this time at St. Martin’s School of Art, in Charing Cross Road, not far from Whitehall. I was then working in Whitehall myself, in Naval Intelligence, so it was easy for us to meet. In 1947 Doreen and I were divorced. Odile had transferred to a new course in fashion design at the Royal College of Art, but after the first year she decided she preferred marriage to further study.

We spent our honeymoon in Italy. Only after we returned did I discover that the First International Congress of Biochemistry had taken place in Cambridge while we were away. In those days there were nothing like as many scientific meetings as there are now. As a beginner in research, still almost an amateur, I was not especially aware of even those meetings that did take place. I think at the back of my mind was the idea that science was an occupation for gentlemen (even if somewhat impoverished gentlemen). Incredible as it may seem, I had not realized that for many it was a highly competitive career.

The Perutzes had lived for some time in a tiny furnished apartment very conveniently located near the center of Cambridge and only a few minutes’ walk from the Cavendish. They now planned to move into a suburban house, to have more room, and suggested to us that we might take their place. We were delighted with the idea and moved into The Green Door, as it was called, a set of two and one-half rooms and a small kitchen, at the top of The Old Vicarage, next to St. Clement’s church on Bridge Street, between the top of Portugal Place and Thompson’s Lane. The owner, a tobacconist, and his wife lived in the main body of the house while we occupied the attic. The actual Green Door was on the ground floor, at the back, leading to a narrow staircase that went up to our set of rooms. The washbasin and lavatory were halfway up these stairs and the bath, covered by a hinged board, was tucked into the small kitchen. It was often necessary to move a miscellaneous collection of saucepans and dishes if one wanted to have a bath. One room served as a living room, the other as a bedroom, while the smallest room was used as a bedroom for my son Michael, when he came home for the holidays from his boarding school.

Odile and I had our leisurely breakfasts by the attic window in the little living room, looking out over the graveyard to Bridge Street and beyond that to the chapel of St. John’s College. There was much less motor traffic in those days, though many bicycles. Sometimes in the evening we would hear an owl hooting from one of the trees that bordered the college. We had only a small income but fortunately the rent was also very small, even though the apartment was rented furnished. The landlord apologized profusely when he felt compelled to raise our rent from thirty shillings a week to thirty shillings and sixpence. Odile luxuriated in her newly found leisure, read French novels in front of the small gas fire, and attended, informally, a few lectures on French literature, while I reveled in the romance of doing real scientific research and in the fascination of my new subject.

The first thing I had to do was to teach myself X-ray crystallography, both the theory and the practice. Perutz advised me which textbooks to read and I was shown the elements of mounting crystals and taking X-ray pictures. Simple inspection of parts of the X-ray diffraction pattern usually gave, in a fairly straightforward manner, not only the physical dimensions of the unit cell—the spatial repeat unit—but also revealed something about its symmetry. Because biological molecules often have a “handedness"—their mirror image is not usually found in living things—certain symmetry elements [inversion through a center, reflection, and the related glide planes] cannot occur in protein crystals. This limitation reduces drastically the possible number of symmetry combinations, or space groups, as they are called.