PERSPECTIVE

The discovery of the ␣- and ␤-sheet, the principal structural features of

David Eisenberg* Howard Hughes Medical Institute and University of California–Department of Energy Institute of Genomics and Proteomics, University of California, Los Angeles, CA 90095-1570

PNAS papers by , Robert Corey, and Herman Branson in the spring of 1951 proposed the ␣-helix and the ␤-sheet, now known to form the backbones of tens of thousands of proteins. They deduced these fundamental building blocks from properties of small molecules, known both from crystal structures and from Pauling’s resonance theory of chemical bonding that predicted planar peptide groups. Earlier attempts by others to build models for helices had failed both by including nonplanar peptides and by insisting on helices with an integral number of units per . In major respects, the Pauling–Corey–Branson models were astoundingly correct, including bond lengths that were not surpassed in accuracy for >40 years. However, they did not consider the hand of the helix or the possibility of bent sheets. They also proposed structures and functions that have not been found, including the ␥-helix.

decade before the structures these historic articles, and then mention that information about interatomic dis- of entire proteins were first some surprising omissions from them. tances, bond angles, and other configu- revealed by x-ray crystallogra- The most revolutionary of these arti- rational parameters might be obtained phy, Linus Pauling and Robert cles is the first, submitted to PNAS on that would permit the reliable prediction CoreyA of the California Institute of Pauling’s 50th birthday, February 28th, of reasonable configurations of the Technology (Fig. 1) deduced the two 1951. It is The Structure of Proteins: Two polypeptide chain.’’ In other words, the main structural features of proteins: the Hydrogen-Bonded Helical Configurations structural Pauling believed that ␣-helix and ␤-sheet, now known to form of the Polypeptide Chain (1), in which with an accurate parts list for proteins the backbones of tens of thousands of Pauling and Corey are joined by a third in hand he would be able to infer major proteins. Their deductions, triumphs in coauthor, H. R. Branson, an African- aspects of their overall architecture, and building models of large molecules American physicist, then on leave from this proved to be so. based on features of smaller molecules, his faculty position at Howard Univer- The next two paragraphs concisely set were published in a series of eight arti- sity (Fig. 1). In the opening paragraph, out the method: ‘‘The problem we have cles, communicated to PNAS in Febru- the authors state that ‘‘we have been set ourselves is that of finding all hydro- ary and March 1951. Their work had a attacking the problem of the structure gen-bonded structures for a single significance for proteins comparable to of proteins in several ways. One of these polypeptide chain, in which the residues that 2 years later of the Watson–Crick ways is the complete and accurate deter- are equivalent (except for the differ- paper for DNA, which adopted the mination of the crystal structure of ences in the side chain R).’’ That is, the Pauling–Corey model-building approach. amino acids, peptides, and other simple authors sought all possible repeating Here I summarize the main points of substances related to proteins, in order structures (helices) in which the car- bonyl CAO group of each residue accepts an NOH from another residue. Why did they be- lieve that there would be only a small number of types of helices? This was because of the constraints on structure imposed by the precise bond lengths and bond angles they had found from their past studies of crystal structures of amino acids and peptides, the compo- nents from which proteins are built up. These constraints are summarized in the third paragraph of their paper, which specifies to three significant figures the bond lengths and bond angles that they had found.† The most important con- straint was that all six of the (or peptide) group, which joins each amino acid residue to the next in the protein chain, lie in a single plane.

Fig. 1. Linus Pauling and Robert Corey (A) and Herman Branson (B). Pauling’s deep understanding of chemical structure and bonding, his retentive memory for details, and his creative flair were all factors in This perspective is published as part of a series highlighting in the discovery of the ␣-helix. Robert Corey was a dignified and shy x-ray crystallographer with the landmark papers published in PNAS. Read more about this classic PNAS article online at www.pnas.org͞misc͞ know-how and patience to work out difficult structures, providing Pauling with the fundamental classics.shtml. information he needed. Herman Branson was a physicist on leave at the California Institute of Technology, who was directed by Pauling to find all helices consistent with the rules of structural chemistry that he and *E-mail: [email protected]. Corey had determined. The wooden helix between Pauling and Corey has a scale of 1 inch per Å, an †The bond lengths are all within 1 standard deviation of enlargement of 254,000,000 times. (A) Courtesy of the Archives, California Institute of Technology. (B) those determined 40 years later (15). Courtesy of the Lincoln University of Pennsylvania Archives. © 2003 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.2034522100 PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11207–11210 Downloaded by guest on September 30, 2021 istry is important in determining which dled them with the notion of integral helices are possible, and integral symme- numbers of units per unit cell. They also try has no role whatever. missed the necessity of planar peptide Today, we accept without a second groups. Working in the physics depart- thought that helices do not need to have ment at University (Cam- Scheme 1. an integral number of monomer units bridge, U.K.), they were unaware of per turn. But in 1950, the crystallo- conjugation with nearby double bonds. graphic backgrounds of Bragg, Kendrew, The professor of organic chemistry at Pauling had predicted planar peptide and Perutz, three of the greatest struc- Cambridge at that time was Alexander groups because of resonance of elec- tural scientists of the 20th century, sad- Todd, who worked across the courtyard trons between the double bond of the carbonyl group and the amide CON bond of the peptide group (Scheme 1). In fact, such planar peptide groups had been observed in the crystal struc- tures of N-acetylglycine and ␤-glycylgly- cine. As the authors put it: ‘‘This struc- tural feature has been verified for each of the that we have studied. Moreover, the resonance theory is now so well grounded and its experimental substantiation so extensive that there can be no doubt whatever about its ap- plication to the amide group.’’ When Pauling, Corey, and Branson constructed helices with planar amide groups, with the precise bond dimen- sions they had observed in crystal struc- tures, and with linear hydrogen bonds of length 2.72 Å, they found there were only two possibilities. These two they called the helix with 3.7 residues per turn and the helix with 5.1 residues per turn (Fig. 2), soon to be called the ␣- helix and the ␥-helix. Much of the rest of this short, bril- liant paper is taken up with a compari- son of these two helices with helices proposed earlier by others, most notably Bragg, Kendrew, and Perutz (2) in a paper the year before, that attempted to enumerate all possible protein helices, but missed these two. In their ␣-helix paper, Pauling et al. take a tone of tri- umph: ‘‘None of these authors propose either our 3.7-residue helix or our 5.1- residue helix. On the other hand, we would eliminate by our basic postulates all of the structures proposed by them. The reason for the difference in results obtained by other investigators and by us through essentially similar arguments is that both Bragg and his collaborators . . . discussed in detail only helical struc- tures with an integral number of resi- dues per turn, and moreover assume only a rough approximation to the re- quirements about interatomic distances bond angles, and planarity of the conju- gated amide group, as given by our in- vestigations of simpler substances. We contend that these stereochemical fea- tures must be very closely retained in stable configurations of polypeptide chains in proteins, and that there is no Fig. 2. The ␣-helix (Left) and the ␥-helix (Right), as depicted in the 1951 paper by Pauling, Corey, and special stability associated with an inte- Branson (1). Biochemists will note that the CAO groups of the ␣-helix point in the direction of its gral number of residues per turn in the C terminus, whereas those of the ␥-helix point toward its N terminus, and, further, that the ␣-helix shown helical molecule.’’ In short, stereochem- is left-handed and made up of D-amino acids. (Reproduced with permission from Linda Pauling Kamb.)

11208 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2034522100 Eisenberg Downloaded by guest on September 30, 2021 from Bragg and his team. Todd recalled other. The wider excursion of the ␣- the light on I found a strong reflection (3) that ‘‘despite the proximity, Bragg helix in the coiled-coil reduces its repeat at 1.5-Å spacing, exactly as demanded never, to my knowledge, set foot in the distance to 5.1 Å. This knack of know- by Pauling and Corey’s ␣-helix.’’ chemical laboratory...until one ing which contradictory fact to ignore On Monday morning, Perutz showed day...hecame to my room in a some- was one of Pauling’s great abilities as a his x-ray diffraction picture to Bragg. what agitated state of mind, bearing a creative scientist. ‘‘When he asked me what made me bunch of papers in his hand,’’ including think of this crucial experiment, I told ␤ the Pauling–Corey–Branson paper and The -Sheets him that the idea was sparked off by my his own on helices. Bragg asked Todd The second paper of the series appeared fury over having missed building that whether he preferred the ␣-helix over as one of a group of seven in a single beautiful structure myself. Bragg’s the helices that Bragg and his coworkers issue of PNAS. It was: The Pleated prompt reply was, ‘I wish I had made had invented. Todd responded, ‘‘I think Sheet, A New Layer Configuration of you angry earlier!’ because discovery of that, given the evidence, any organic Polypeptide Chains (6). In this article, the 1.5-Å reflection would have led us chemist would accept Pauling’s view. Pauling and Corey report that they have straight to the ␣-helix.’’ Perutz also Indeed, if at any time since I have been discovered a hydrogen-bonded layer found the 1.5-Å reflection in diffraction in Cambridge you had come over to the configuration of polypeptide chains, in from . He wrote to Pauling chemical laboratory, I...would have which the planar peptide groups lie in (8), ‘‘The fulfillment of this prediction told you that.’’ the plane of the sheet, and successive and, finally, the discovery of this reflec- The idea of the nonintegral ␣-helix protein chains can run in opposite direc- tion in hemoglobin has been the most had come to Pauling 3 years before, tions, giving an antiparallel sheet, as thrilling discovery of my life.’’ Perutz, when he was visiting professor at Oxford well as a parallel sheet. In both, linear along with his coworkers Dickerson, University. He caught cold in the damp H-bonds are again formed, but between Kendrew, Strandberg, and Davies, was weather and spent several days in bed. protein chains rather than within a sin- to make even more thrilling discoveries He recalled (4) that he was soon bored gle chain. This results in protein chains later, including seeing direct pictures of with detective novels and ‘‘I didn’t have that are not fully extended: the rise per ␣-helices in and hemoglobin. any molecular models with me in Ox- residue is 3.3 Å, a spacing seen in x-ray ␤-Sheets and single-stranded ␤-rib- ford but I took a sheet of paper and diffraction patterns of ␤-keratin, rather bons were first seen in globular proteins sketched the atoms with the bonds be- than 3.6 Å, expected for a fully ex- as in the structure of egg white ly- tween them and then folded the paper tended protein chain. sozyme in 1965 (9). An initial surprise to bend one bond at the right angle, was that both the strands and the sheets ␣ what I thought it should be relative to Confirmation of the -Helical and are twisted, unlike the straight strands ␤ the other, and kept doing this, making a -Sheet Models and pleated sheets of Pauling and Co- helix, until I could form hydrogen bonds Confirmation of the ␣-helix came from rey. In 1989 Pauling recalled that as between one turn of the helix and the , one of the three authors of soon as he saw the structure of ly- next turn of the helix, and it only took a the 1950 article that had enumerated sozyme with its twisted sheet he realized few hours of doing that to discover the the wrong helices. One Saturday morn- he should have incorporated the twist in ␣-helix.’’ ing in spring 1951, he came across the the original model. More recently there Why did Pauling delay 3 years in pub- PNAS paper (7). ‘‘I was thunderstruck have been thorough analyses of twist lishing this finding that came to him in by Pauling and Corey’s paper. In con- and shear in ␤-structures (10, 11). only a few hours? He gave the answer in trast to Kendrew’s and my helices, theirs his banquet address at the third sympo- was free of strain; all of the amide Some Surprising Omissions from the sium of the Protein Society in Seattle in groups were planar and every carbonyl 1951 Papers 1989. He was uneasy that the diffraction group formed a perfect hydrogen bond who take a careful look at the pattern of ␣-keratin shows as its princi- with an imino group four residues fur- ␣-helix of Fig. 2 will notice two surpris- pal meridional feature a strong reflec- ther along the chain. The structure ing features: (i) It is a left-handed helix, tion at 5.15-Å resolution, whereas the looked dead right. How could I have unlike ␣-helices of biological proteins, ␣-helix repeat calculated from his mod- missed it ?...Icycled home to lunch which are now known to be right- els with Corey was at 5.4 Å. As he says and ate it oblivious of my children’s handed. That is, if your left thumb in his fourth paper of the PNAS series chatter and unresponsive to my wife’s points along the helix axis, the helix with Corey: ‘‘The 5.15-Å arc seems on inquiries as to what the matter was with turns in the direction of the fingers of first consideration to rule out the ␣- me today.’’ your left hand. (ii) The configuration of helix, for which the c-axis period must Suddenly Perutz had an idea: ‘‘Paul- chemical groups around each ␣-carbon be a multiple of the axis distance per ing and Corey’s ␣-helix was like a have the D-configuration, rather turn...’’ But then came the paper in staircase in which the amino acid resi- than the naturally occurring L-configura- 1950 by Bragg, Kendrew, and Perutz dues formed the steps and the height of tion of amino acid residues in proteins. enumerating potential protein helices. each step was 1.5 Å. According to dif- That is, this model of Pauling et al. is Pauling told his audience in 1989: ‘‘I fraction theory, this regular repeat the mirror image of an ␣-helix in a nat- knew that if they could come up with all should give rise to a strong x-ray reflec- ural protein. In contrast, the ␥-helix in of the wrong helices, they would soon tion of 1.5 Å spacing from planes per- Fig. 2 is a right-handed helix made up come up with the one right one, so I pendicular to the fiber axis...Inmad of D-amino acid residues. Why did the felt the need to publish it.’’ excitement, I cycled back to the lab and authors choose to draw the ␣-helix as The origin of the discrepancy between looked for a horse hair that I had kept left-handed, with D-amino acids? the repeat of the ␣-helix and the x-ray tucked away in a drawer...’’ and put it The basis for this choice has recently reflection of ␣-keratin was hit on a year in the x-ray beam at an angle of 31° been analyzed by Dunitz (12), who had later by (5), then a gradu- to the beam to bring the 1.5-Å repeat been a postdoctoral fellow at the Cali- ate student with Perutz, and also by into the reflecting position. ‘‘After a fornia Institute of Technology at the Pauling. It is that keratin is a coiled- couple of hours, I developed the film, time of the Pauling–Corey research. In coil, with ␣-helices winding around each my heart in my mouth. As soon as I put fact, it was Dunitz who persuaded Paul-

Eisenberg PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11209 Downloaded by guest on September 30, 2021 ing to change his terminology from ‘‘spi- more stringent than those used today that muscle contraction is a transition ral’’ to ‘‘helix’’ in describing the new (13), now that we know accepts from extended ␤-strands to compact ␣ protein structures. In his analysis, the . -helices. Nevertheless, the breathtaking Dunitz notes that 1951, the year of the One other omission from the set of correctness of the ␣-helix and ␤-sheets ␣-helix, was also the year in which J. M. 1951 papers is the Ramachandran dia- and the bold approach of modeling bio- Bijvoet established the absolute configu- gram. This is a 2D plot of the allowed logical structures from chemical princi- ration of molecules by the anomalous values of rotation about the NOC␣ and ples overshadow the rest. scattering of x-rays. After recalling dis- C␣OCAO bonds in the protein back- These papers are all the more re- cussions of handedness at the California bone, introduced by Ramachandran and markable when we consider the political Institute of Technology in that year, others in 1964 (14). This diagram shows context in which they were written. Dur- Dunitz concludes: ‘‘Either Pauling was that most values of rotation about these ing this period, Pauling was also heavily unaware of these developments when he two bonds are forbidden by collisions of involved in defending academics, includ- wrote the ␣-helix paper, or he knew protein atoms. Only two major regions ing himself, against charges of disloyalty about them but was uninterested. . . of the diagram are allowed: one corre- to the United States, brought about by I tend to believe that when they wrote sponds to the ␣-helix, and one to the the pressures of the Cold War and what the paper, or quite possibly even when nearly extended chains of the ␤-sheets. became known as McCarthyism. He was they made the models, Pauling (or his Today the Ramachandran diagram is subpoenaed to appear before various colleague Robert B. Corey) simply taught in all classes on protein structure anticommunist investigating committees, picked one of the two amino acid con- and is featured in every textbook to give he received hate mail for his work on figurations (as it happened, the wrong insight into the forces that determine liberal causes, and he faced cancellation one) to illustrate the helical structures the structures of proteins. But there is of his major consulting contract and and did not give the problem of abso- nothing in this diagram beyond what coolness from some California Institute lute configuration much thought. . . Pauling and Corey knew well: they built of Technology colleagues. On the day Problems of absolute configuration re- models of their proposed structures that after Pauling and Corey submitted their ceived little or no attention because embodied all features of the Ramachan- seven protein papers for publication, the House Un-American Activities Commit- there seemed to be no need for them dran diagram. Apparently they under- tee named Pauling one of the foremost then. Perhaps they were even regarded stood the principles so well that they felt Americans involved in a ‘‘Campaign to as a distraction from the task at hand. no need to explain them by a diagram Disarm and Defeat the United States’’ Sometimes one can focus more clearly of this sort. Another factor may have (8). The press release read, ‘‘His whole by closing one eye.’’ been that Pauling and Corey focused record. . . indicates that Dr. Linus Paul- Also missing from the first paper is more on the stability provided by hydro- ing is primarily engrossed in placing his anything more than passing mention of gen bonds and less on the restrictions on scientific attainments at the service of a the 310 helix, a component of globular possible structures dictated by collisions host of organizations which have in proteins found rarely in short segments, between nonbonded atoms. common their complete subservience to but more common than the Pauling– the Communist Party of the USA, and Corey–Branson ␥-helix, which is virtually The Other Six PNAS Articles by Pauling and Corey and the Wider Context the Soviet Union.’’ Somehow, even in never seen. The H-bonds of the 310 helix the face of such false invective and mul- are somewhat too long and bent to have The remaining six articles in PNAS give tiple distractions, Pauling could main- been acceptable by the stringent thresh- the atomic coordinates of the models tain his focus as a top creative scientist. olds set by the authors. Their intuition and interpret the diffraction patterns of about bent and long hydrogen bonds fibrous proteins in terms of the models. I thank David R. Davies, Richard E. Dicker- destabilizing structures was basically cor- There is much in these papers than has son, Jack Dunitz, Richard E. Marsh, and rect, but the thresholds they set are not been borne out, including a proposal Doug Rees for discussion.

1. Pauling, L., Corey, R. B. & Branson, H. R. (1951) 6. Pauling, L. & Corey, R. B. (1951) Proc. Natl. Acad. 10. Chothia, C. (1973) J. Mol. Biol. 75, 295–302. Proc. Natl. Acad. Sci. USA 37, 205–211. Sci. USA 37, 251–256. 11. Bosco, K. H. & Curmi, P. M. G. (2002) J. Mol. Biol. 2. Bragg, L., Kendrew, J. C. & Perutz, M. F. (1950) 7. Perutz, M. (1998) I Wish I’d Made You Angry 317, 291–308. Proc. R. Soc. London Ser. A 203, 321–357. Earlier (Cold Spring Harbor Lab. Press, Plainview, 12. Dunitz, J. D. (2001) Angew. Chem. Int. Ed. 40, 3. Todd, Lord Alexander (1990) in The Legacy of NY), pp. 173–175. 4167–4173. Sir , eds. Thomas, J. M. & Phil- 8. Hager, T. (1995) Force of Nature: The Life of 13. Kortemme, T., Morozov, A. V. & Baker, D. (2003) lips, D. (Science Reviews Limited, Northwood, Linus Pauling (Simon & Schuster, New York), J. Mol. Biol. 326, 1239–1259. U.K.), p. 95. pp. 379–380. 14. Ramachandran, G. N., Sasisekharan, B. & 4. Serafini, A. (1989) Linus Pauling: A Man and His 9. Blake, C. C. F., Koenig, G. A., Mair, G. A., North, Ramakrishnan, C. (1963) J. Mol. Biol. 7, 95–99. Science (Paragon House, New York), p. 131. A. C. T., Phillips, D. C. & Sarma, V. R. (1965) 15. Engh, R. A. & Huber, R. (1991) Acta Crystallogr. 5. Crick, F. (1952) Nature 170, 882–883. Nature 206, 759–761. A 47, 392–400.

11210 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2034522100 Eisenberg Downloaded by guest on September 30, 2021