'I Wish I'd Made You Angry Earlier'

Fifty years ago, the great un solved problem of biology seemed to be the structure of proteins. Bill Astbury, a physicist and X-ray crystallographer working for the Wool Research Association in Leeds (United Kingdom), discovered that the fibrous protein keratin, found in wool, horn, nails and muscle, gave a common X-ray diffraction pattern consisting of just two reflections, a meridionalone at 5.1 Å and an equatorial one at 9.8 Å. Astbury called this the a-keratin pattern. When these fibers were stretched under steam, a new pattern appeared with a meridional reflection at 3.4 Å and two equatorial ones at 4.5 and 9.7 Å. Astbury called this the b-keratin pattern. He concluded that it arose from the regular repeat of amino acid residues along straight polypeptide chains, whereas the chains in a-keratin must be folded or coiled such that several amino acid residues form a pattern repeating every 5.1 Å along the fiber axis. It appeared that the key to understanding the structure of proteins lay in the elucidation of this ubiquitous fold, but the meager information contained in the X-ray diffraction patterns did not provide enough clues to uncover it. By 1950, J.C. Kendrew and I had evidence that the same fold of the polypeptide chain occurred also in the two globular proteins myoglobin and hemoglobin. WL. Bragg, the pioneer of X-ray crystallography who was our professor at the Cavendish Laboratory in Cambridge, stimulated us to attack the problem by building molecular models. To start us off, he hammered nails representing amino acid residues into a broomstick in a helical pattern with an axial distance between successive turns (or pitch) of 5.1 centimeters. Kendrew and I had great difficulty in building real models of helical polypeptide chains with the right pitch; no matter whether we built them with two, three or four amino acid residues per turn, their bond angles were always strained. After some months we published our work jointly with Bragg in the Proceedings of the Royal Society, but without any firm conclusions about the correct fold. One Saturday morning shortly after our paper had appeared, I went to the Cavendish library, and in the latest issue of the Proceedings of the National Academy of Sciences of the United States of America found a series of papers by Linus Pauling, the famous American chemist, together with the crystallographer R.B. Corey. In their first paper they proposed an answer to the longstanding riddle of the structure of a-keratin, suggesting that it consisted of helical polypeptide chains with a non-integral repeat of 3.6 amino acid residues per turn. Their helix had a pitch not of 5.1 Å, as Astbury's X-ray picture seemed to demand, but of 5.4 Å, which was consistent with the repeat found in fibers of certain synthetic polypeptides by C.H. Bamford and A. Elliott and their colleagues at the Courtaulds Research Laboratories. I was thunderstruck by Pauling and Corey's paper. In contrast to Kendrew's and my helices, theirs was free of strain; all the amide groups were planar and every carboxyl group formed a perfect hydrogen bond with an imino group four residues further along the chain. The structure looked dead right. How could I have missed it? Why had I not kept the amide groups planar? Why had I stuck blindly to Astbury's 5.1 Å repeat? On the other hand, how could Pauling and Corey's helix be right, however nice it looked, if it had the wrong repeat? My mind was in a turmoil. I cycled home to lunch and ate it oblivious of my children's chatter and unresponsive to my wife's inquiries as to what the matter was with me today. Suddenly I had an idea. Pauling and Corey's a-helix was like a spiral staircase in which the amino acid residues formed the steps and the height of each step was 1.5 Å. According to diffraction theory, this regular repeat should give rise to a strong X-ray reflection of 1.5 Å spacing from planes perpendicular to the fiber axis. As far as I knew, such a reflection had never been reported, either from "natural" proteins like hair and muscle or from synthetic polypeptides. Hence, I concluded, the a-helix must be wrong. But wait! Suddenly I remembered a visit to Astbury's laboratory and realized that the geometry of his X-ray setup would have precluded observation of the 1.5 Å reflection because he oriented his fibers with their long axes perpendicular to the X-ray beam, while observation of the 1.5 Å reflection would have required inclining them at the Bragg angle of 31°. Furthermore, Astbury used a flat plate camera that was too narrow to record a reflection deflected from the incident X-ray beam by 2 x 31°. In mad excitement, I cycled back to the lab and looked for a horse hair that I had kept tucked away in a drawer. I stuck it on a goniometer head at an angle of 31° to the incident X-ray beam; instead of Astbury's flat plate camera I put a cylindrical film around it that would catch all reflection with Bragg angles of up to 85°. After a couple of hours, I developed the film, my heart in my mouth. As soon as I put the light on I found a strong reflection at 1.5 Å spacing, exactly as demanded by Pauling and Corey's a-helix. The reflection did not by itself prove anything, but it excluded all alternative models that had been put for-ward by ourselves and others and was consistent only with the a-helix. On Monday morning I stormed into Bragg's office to show him my X-ray diffraction picture. When he asked me what made me think of this crucial experiment, I told him that the idea was sparked off by my fury over having missed building that beautiful structure myself. Bragg's prompt reply was, "I wish I had made you angry earlier!" because discovery of the 1.5 Å reflection would have led us straight to the a-helix. The inconsistency between the 5.4 and 5.1 Å continued to worry me until one morning about two years later when Francis Crick arrived at the lab with two rubber tubes around which he had pinned corks with a helical repeat of 3.6 corks per turn and a pitch of 5.4 centimeters. He showed me that the two tubes could be wound around each other to make a double helix such that the corks neatly interlocked. This shortened the pitch of the individual chains, which projected onto the fiber axis, from 5.4 to 5.1 centimeters, as required by the X-ray pattern of a-keratin. Such a double helix was eventually found by my colleagues A.D. McLachlan and J. Karn in the muscle protein myosin. Perutz works at the Medical Research Council's Laboratory of Molecular Biology, Cambridge CB2 2QHQ, UK, with support from the U.S. National Institutes of Health and the U.S. National Science Foundation.

'I Wish I'd Made You Angry Earlier'

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