Linus Pauling and the Structure of Proteins: A Documentary History

Today is Linus Pauling’s birthday – he would have been 112 years old.  Every year on February 28th we try to do something special and this time around we’re pleased to announce a project about which we’re all very excited: the sixth in our series of Pauling documentary history websites.

Launched today, Linus Pauling and the Structure of Proteins is the both latest in the documentary history series and our first since 2010′s The Scientific War Work of Linus C. Pauling. (we’ve been a little busy these past few years)  Like Pauling’s program of proteins research, the new website is sprawling and multi-faceted.  It features well over 200 letters and manuscripts, as well as the usual array of photographs, papers, audio and video that users of our sites have come to expect.  A total of more than 400 primary source materials illustrate and provide depth to the site’s 45-page Narrative, which was written by Pauling biographer Thomas Hager.

Warren Weaver, 1967.

That narrative tells a remarkable story that was central to many of the twentieth century’s great breakthroughs in molecular biology.  Readers will, for example, learn much of Pauling’s many interactions with Warren Weaver and the Rockefeller Foundation, the organization whose interest in the “science of life” helped prompt Pauling away from his early successes on the structure of crystals in favor of investigations into biological topics.

So too will users learn about Pauling’s sometimes caustic confrontations with Dorothy Wrinch, whose cyclol theory of protein structure was a source of intense objection for Pauling and his colleague, Carl Niemann.  Speaking of colleagues, the website also delves into the fruitful collaboration enjoyed between Pauling and his Caltech co-worker, Robert Corey.  The controversy surrounding Pauling’s interactions with another associate, Herman Branson, are also explored on the proteins website.

Linus Pauling shaking hands with Peter Lehman in front of two models of the alpha-helix. 1950s.

Much is known about Pauling’s famously lost “race for DNA,” contested with Jim Watson, Francis Crick and a handful of others in the UK.  Less storied is the long running competition between Pauling’s laboratory and an array of British proteins researchers, waged several years before Watson and Crick’s breakthrough.  That triumph, the double helix, was inspired by Pauling’s alpha helix, discovered one day when Linus lay sick in bed, bored and restless as he fought off a cold. (This was before the vitamin C days, of course.)

Illustration of the antibody-antigen framework, 1948.

Many more discoveries lie in waiting for those interested in the history of molecular biology: the invention of the ultracentrifuge by The Svedberg; Pauling’s long dalliance with a theory of antibodies; his hugely important concept of biological specificity; and the contested notion of coiled-coils, an episode that once again pit Pauling versus Francis Crick.

Linus Pauling and the Structure of Proteins constitutes a major addition to the Pauling canon. It is an enormously rich resource that will suit the needs of many types of researchers, students and educators. It is, in short, a fitting birthday present for history’s only recipient of two unshared Nobel Prizes.

Happy birthday, Dr. Pauling!

On the Formation of Antibodies

By the 1940s, Linus Pauling’s research interests had expanded to include many subjects generally outside the purview of a typical chemist. In particular, immunology was rapidly becoming a fascination of his – one that would come to devour more and more of his time both in and out of the lab. For Pauling, much of the human body could be viewed as a gigantic set of very complicated chemistry problems, and he derived great joy from being able to solve some of these problems. Among his most important immunological discoveries were his elucidation of the role that hemoglobin plays in sickle cell anemia and his theory of antibody formation.  The latter is the topic of today’s post.

In 1940 Pauling published a paper – now his ninth most cited – in the Journal of the American Chemical Society on the subject of antibodies and antigens. This manuscript, titled “A Theory of the Structure and Process of Formation of Antibodies” is fundamentally based around one important assumption that Pauling made about antibody structure, “…that all antibody molecules contain the same polypeptide chains as normal globulin, and differ only in the configuration of the chain; that is, in the way that the chain is coiled in the molecule.”

Antibodies are protein molecules that play an extremely important role in the human body. Their main function is to identify and neutralize foreign objects, called antigens, that have been taken up by the body. Antigens come in many varieties, including high-molecular-weight carbohydrates, lipids, pollen and bacterial cells. It is important to note too that only antigens marked by the body’s systems as “foreign” will set off an antibody response; antigens marked as “self” are tolerated by the body.

Pauling with Dan Campbell, a primary colleague in Pauling's antibody work.

Antigens play an important role in Pauling’s theory, which argues that antigens alone determine the configuration of a specific portion of the antibody molecule. For example, without the presence of an antigen, a normal globulin protein will be synthesized. In the presence of an antigen however, a specific antibody will be produced, a portion of which will be complementary in structure to the antigen that in question. In describing this process, Pauling’s paper first details the four steps that occur in the formation of a normal globulin molecule, which are summarized below.

1. The polypeptide chain is synthesized. The two ends of the chain are free, but the center of the chain is still attached at the site of synthesis.
2. The ends coil into either their most stable, or another very stable, configuration. Hydrogen bonds and other weak forces between amino acids in the polypeptide chain stabilize the two ends.
3. The center of the chain is freed from the site of synthesis.
4. The center coils into its most stable configuration, and the globulin molecule is completed.

Because antibodies are simply modified globulin molecules, the process for their formation is closely related to that of globulin. Summarized below then, are Pauling’s six steps of antibody formation.

1. An antigen is held in place at the site of antibody production and the antibody is synthesized around the antigen molecule.
2. The ends of the newly synthesized antibody coil into a configuration complementary to groups on the antigen and attach to these complementary groups.
3. The center of the chain is freed from the site of synthesis, causing one of two things to happen. If the forces between the ends of the chain are sufficiently strong, both ends will continue to be attached to the antigen, and the antibody will never be completed.
4. If they forces between the ends of the chain and the antigen are weak, one end will dissociate from the antigen.
5. Assuming one end of the chain dissociates from the antigen, the center of the chain coils into its most stable configuration, making a complete antibody.
6. Eventually, the antibody will dissociate from the antigen and float away.

To summarize, Pauling’s theory of antibody formation argues that every antibody has the same configuration in the center of its polypeptide chain, and that the configuration of the ends of the chain are dependent on which antigen is present at the time of the antibody’s synthesis.  In present day, even with our better understanding of antibody synthesis, the core principles of Pauling’s theory – most prominently the idea that each antibody shares a common structure – remain sound.

The entirety of Pauling’s manuscript is available here.  In it, he discusses other topics related to his theory, including the formation of different structures based on antibody to antigen ratios, the characteristics that define a molecule or substance as an antigen, and the compatibility of his theory with experimental results. For more information on Pauling’s immunological work, visit It’s in the Blood: A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia.

The Alpha Helix

Space-filling model of the alpha helix.

[The Paulings in England: Part 5 of 5]

It has been said that sometimes blessings come in disguise, and so it may be that we have the damp English spring to thank for the elucidation of the alpha-helix structure of alpha-keratin – a fundamental and ubiquitous secondary structure pattern found in many proteins.

Linus Pauling was plagued by sinusitis for much of his time in England, and for three days in March 1948 it had become severe enough to put him in bed (as he was fond of saying over the years, this was before his vitamin C days). After a day spent devouring mystery novels, Pauling asked Ava Helen if she would bring him some paper and his slide rule, at which point he started trying to figure out how polypeptide chains might fold up into a satisfactory protein structure.

Pauling’s canvas was just an ordinary 8 1/2 by 11 inch sheet of paper. His first step was to draw the correct bond angles and distances onto the sheet, as determined from previous x-ray crystallographic work on polypeptides. Next he folded the sheet along parallel lines into a sort of squared-off tube. Doing so allowed him to add in representations of hydrogen bonds, which the impromptu model suggested would form between amino acid residues and, as a result, hold the turns of the polypeptide together.

The model made sense and pretty quickly it was clear that Pauling had discovered something important.  As he later wrote, his folded creation “turned out to be the structure of hair and horn and fingernail, and also present in myoglobin and hemoglobin and other globular proteins, a structure called the alpha-helix .”

Reconstruction of the alpha-helix paper model. Drawn and folded by Linus Pauling, 1982.

Pauling kept this idea to himself until his return to the United States because something didn’t match up quite right with the current laboratory data. Specifically, the turns of Pauling’s helix didn’t mirror the 5.1 angstrom repeat found in all of William T. Astbury‘s x-ray patterns. Pauling’s structure came close, but made a turn every 5.4 angstroms, or every 3.7 amino acid residues.

After his return home, with the assistance of colleagues Robert Corey and Herman Branson, Pauling continued refining his alpha helix structure and developing others, including the beta sheet. Simultaneously, the Caltech group’s chief British rivals at the Cavendish Laboratory published a paper titled “Polypeptide Chain Configurations in Crystalline Proteins.” The paper promised more than it delivered though, and while it listed many possible structures, Pauling found none of them to be likely. The competition was still on.

Pauling was finally convinced to publish when he received word that a British chemical firm called Courtaulds had created a synthetic polypeptide chain that showed no sign of Astbury’s 5.1 angstrom reflection in x-ray diffraction images. This was enough evidence for Pauling to decide that the 5.1 angstrom repeat was, perhaps, not a vital component of all polypeptide chains.  And so it was that in April 1951 Pauling, Corey and Branson published “The structure of proteins: Two hydrogen-bonded helical configurations of the polypeptide chain,” in the Proceedings of the National Academy of Sciences.

After devouring the Pauling group’s results shortly after their publication, Max Perutz headed to the Cavendish lab at Cambridge to check the data himself. Having confirmed the structure in images of horsehair, porcupine quill, synthetic polypeptides, hemoglobin and, for good measure, some old protein films that had been tucked away, Perutz wrote to Pauling, “The fulfillment of this prediction and, finally, the discovery of this reflection in hemoglobin has been the most thrilling discovery of my life.” He then published an analysis of his own data, concluding, “The spacing at which this reflexion appears excludes all models except the 3.7 residue helix of Pauling, Corey and Branson, with which it is in complete accord.”

Video Link: Pauling Recounts His Discovery of the Alpha Helix

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It wasn’t until a couple of years later that the mystery of Astbury’s 5.1 angstrom reflection was finally solved. In 1953, on a visit to the Cavendish, Pauling met Francis Crick, the then-graduate student who would go on to play a huge part in the discovery of the structure of DNA. The two maintained similar interests and during a taxi ride around Cambridge found themselves discussing the matter of the alpha helix. “Have you thought about the possibility,” Crick asked Pauling, “that alpha helixes are coiled around one another?” Whether Pauling had or had not considered this possibility remains a point of contention, but Pauling remembered replying that he had, because he had been considering a number of higher-level schemes for his helixes, including some which wound around each other.

Regardless, Pauling returned to Caltech and both he and Crick set to work on the problem. With help from Corey, Pauling discovered a means by which the alpha helixes could wrap around each other in a coiled-coil to produce the problematic 5.1 angstrom found in Astbury’s pictures of natural keratin.  Crick, in the meantime, was conducting a very similar study.  Pauling and Crick, independent of one another, ultimately submitted the solution to this puzzle for publication within days of each other, and at first there was a bit of grumbling as to whom the credit should be awarded. Though Crick’s note was published first, the Cavendish camp eventually conceded that Pauling’s paper included considerably more detail of consequence, and it was finally settled that both scientists had independently come to the same general conclusion.

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Pauling receiving his honorary degree from the University of Paris, 1948.

After Pauling’s two fruitful terms as Eastman Professor at Oxford were up in July, the family split their remaining time between travels in Amsterdam, Switzerland and Paris. Pauling rounded off the trip by receiving yet another honorary degree from the University of Paris, and on August 25, 1948, the Paulings set sail once more on the Queen Mary.

His eight months in Europe had been productive and enlightening, but Pauling was ready to return to Pasadena where he could share the myriad ideas he had generated and gathered during his time away from Caltech. As we have seen, he was especially eager to get back to work on proteins, writing shortly before his departure that “I have continued to work on my theory of metals, and have been doing nothing about proteins. However, I am looking forward to being back home, and to thinking about that subject again.”

An Era of Discovery in Protein Structure

Linus and Ava Helen Pauling, Oxford, 1948.

[The Paulings in England: Part 4 of 5]

Though metals were consuming a good portion of his time during his fellowship at Oxford, Linus Pauling’s other projects never strayed far from his thoughts.  High on the list were the mysteries of proteins, whose structures and functions were slowly starting to be unraveled.

Pauling’s interest in proteins was spurred in the mid-1930s when the Rockefeller Foundation began to look most favorably upon the chemistry of life when deciding where their grant money would go. Early on, Pauling set out to tackle hemoglobin and though his affair with the molecule lasted for the remainder of life, Pauling certainly didn’t limit himself to the study of just one protein.

At a time when most were looking at proteins from the top down, trying to sort out the complicated data produced by an x-ray diffraction photograph of an entire protein, Pauling was working from the bottom up, in the process determining the structures of individual amino acids – the building blocks of proteins.

A specific protein that kept coming back into view over the years was keratin. In the 1930s, the English scientist William Astbury had studied the structure of wool, which along with hair, horn, and fingernail is made up primarily of this enigmatic protein, keratin. Astbury proposed that the structure was akin to a flat, kinked ribbon, but Pauling disagreed. “I knew that what Astbury had said wasn’t right,” Pauling recalled, “because our studies of simple molecules had given us enough knowledge about bond lengths and bond angles and hydrogen-bond formation to show that what he said wasn’t right. But I didn’t know what was right.” Pauling attempted to construct a model at the time, but could not match his structure to the measurements dictated by Astbury’s blurry x-ray diffraction images. Pauling wrote the project off as a failure and continued pursuing other interests.

In 1945 Pauling found himself seated next to Harvard medical Professor William B. Castle on a railroad journey from Denver to Chicago. Castle was a physician working on the nature of sickle cell anemia and the conversation that he shared with Pauling planted a seed in Pauling’s mind about the cause of this debilitating disease.

In the bodies of those suffering from sickle cell anemia, red blood cells assume a sickled shape when they are in the deoxygenated venous system but retain their normal flattened disk shape in the oxygen-rich arterial system. Noting this, Pauling suggested that perhaps the source of the problem could be a defect in the oxygen-carrying protein itself: hemoglobin.

Amidst his travels in Europe, Pauling continued to act on this idea as maestro from afar, directing the scientists in his Caltech laboratory to continue searching for differences in the hemoglobin of normal and sickled cells. In the meantime, he sought out and communicated new ideas gleaned from meetings such as the Barcroft Memorial Conference on Hemoglobin, held at Cambridge in June 1948. Pauling’s research team, in particular Harvey Itano and S. Jonathan Singer, were able to show experimentally that his hunch had been right, and less than a year after his return to Pasadena a paper was published that established sickle cell anemia as the first illness to be revealed as a truly molecular disease.

Linus and Peter Pauling at the model Bourton-on-the-water, England. 1948.

While in England, Pauling had occasion to interact closely with a number of scientific greats.  Among these were his close friend Dorothy Crowfoot Hodgkin, who is credited as a pioneer in the development of protein crystallography and was the winner of the 1964 Nobel Prize for Chemistry.  Likewise, Pauling conversed with Max Perutz, a protege of Sir William Lawrence Bragg‘s at the Cavendish Laboratory at Cambridge, who would go on to discover the structure of hemoglobin and receive the Nobel Prize for Chemistry in 1962.  While fruitful in many respects, these interactions served to increase Pauling’s feelings of urgency as concerned the race to determine the structure of proteins.

Bragg shared the 1915 Nobel Prize in Physics with his father for their early development of X-ray crystallography, and though there existed a long-standing scientific rivalry between Pauling’s and Bragg’s laboratories, it wasn’t until Pauling saw, with his own eyes, the work that was being done that he admitted he was “beginning to feel a bit uncomfortable about the English competition.” As he wrote to his colleague Edward Hughes back at Caltech

It has been a good experience for me to look over the x-ray laboratory at Cambridge. They have about five times as great an outfit as ours, that is, with facilities for taking nearly 30 x-ray pictures at the same time. I think that we should expand our x-ray lab without delay.

This realization prompted Pauling to get researchers in his lab started on work with insulin – an arduous and complicated process that required sample purification and crystallization prior to x-ray investigation. In relaying research findings from English scientists working on insulin to his partners back in Pasadena, Pauling intimated that

It is clear that there is already considerable progress made on the job of a complete structure determination of insulin. However, there is still a very great deal of work that remains to be done, and I do not think that it is assured that the British school will finish the job. I believe that this is the problem that we should begin to work on, with as much vigor as possible, under our insulin project.

Little did Pauling know that, while laying in bed, using little more than a piece of paper, a pen and a slide rule, he would soon make a major breakthrough in protein chemistry on his own.

A Theory of the Denaturation of Proteins

Alfred E. Mirsky, 1960s

In 1935, as a result of being prompted toward the biological sciences in order to keep his Rockefeller Foundation funding, Linus Pauling began his research on proteins. Hemoglobin, the oxygen-binding agent in blood, was his first target; but as he became more aware of the complex nature and diversity of proteins, he began contemplating broader topics related to the subject – one of which was the theory of protein denaturation.

In the spring of 1935, Pauling traveled to the Rockefeller Institute in New York City, where he met Dr. Alfred Mirsky. Mirsky was a Rockefeller scientist who had previously conducted denaturation research, and because of his new interest in the subject, Pauling arranged for Mirsky to spend fifteen months working with him at Caltech. Although initially hesitant, Mirsky eventually agreed, and the pair began collaborating in the summer of 1935.

In July 1936, the duo’s paper, titled “On the Structure of Native, Denatured, and Coagulated Proteins” was published in the Proceedings of the National Academy of Sciences.  In this paper, the authors loosely describe protein denaturation as “the loss of certain highly specific properties by the native protein,” and provide examples of the types of changes that have been experimentally observed.

In so doing, Pauling and Mirsky point out that while many proteins in their native form have been crystallized, no denatured protein exist in this state. Likewise, in proteins that act as enzymes, denaturation causes a disappearance of the enzymatic activity.  And one fact that was of particular interest to Pauling was that the process of denaturation is occasionally reversible.

Early Pauling notes on the characteristics of protein denaturation, ca. 1935

As researchers are now aware, any given protein has a certain structure – or rather, four different structural levels – that needs to be maintained in order for the molecule to function correctly. Although this crucial bit of information was still unknown at the time of Pauling and Mirsky’s research, the authors essentially touch on this exact detail in their 1936 paper:

Our conception of a native protein molecule (showing specific properties) is the following. The molecule consists of one polypeptide chain [the amino acid sequence] which continues without interruption throughout the molecule (or in certain cases, of two or more such chains); this chain is folded into a uniquely defined configuration, in which it is held by hydrogen bonds…

The collaborators further posited that, as a result of this “structure equals function” characteristic of proteins, denaturation is “characterized by the absence of a uniquely defined configuration” and can be accomplished in a number of different ways, including heating, subjection to ultraviolet light, or an attack by certain reagents.

In presenting their theory of denaturation, Pauling and Mirsky associated both the heating of the protein and its treatment with certain reagents, as leading to the disruption or complete rupturing of hydrogen bonds.  From there they pointed out that ultraviolet light is not able to break a sufficient quantity of hydrogen bonds, and therefore must affect the molecule differently – an impact which they predicted to be an attack on the main polypeptide chain. Consequently, they suggested that denaturation caused by ultraviolet light was irreversible, while methods that disrupt the more easily re-formed hydrogen bonds would be reversible.

Although Pauling and Mirsky weren’t correct in every aspect of their denaturation theory (ultraviolet light does not disturb the polypeptide chain, and denaturation involves more than just the disruption of hydrogen bonds), it provided a strong start for further work.  The Pauling-Mirsky theory also touched on many details of the structure of proteins in their native forms, a field of inquiry that would not be completely elucidated for many years to come.

For more information on Linus Pauling, please visit the Linus Pauling Online portal. For more information on Alfred Mirsky, visit his key participants page within the It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia site.