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.

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The Importance of the Concept of Molecular Disease

The idea of Dr. Linus Pauling that an abnormal hemoglobin molecule might be responsible for the sickling process initiated the study of the hemoglobin molecule in hereditary anemias.
– Harvey Itano. “Clinical States Associated with Alterations of the Hemoglobin Molecule.” Archives of Internal Medicine, 96: 287-97, 295. 1955.

During his lengthy career, Linus Pauling maintained a long-running interest in the relationships between chemistry and the human body. In the mid-1930’s, he began to work extensively with the hemoglobin molecule. As we’ve seen in previous posts, this research would eventually lead to many important discoveries and his coining of the term “molecular disease.”

Sickle cell anemia was the first disease to be classified as a molecular disease. As was mentioned in this post, Pauling first learned of the disease in the spring of 1945 when Dr. William B. Castle, a physician and Professor of Medicine at Harvard University, described it at a meeting of the Medical Research Association. As Dr. Castle listed off the characteristics of the disease, Pauling, through the prism of his deep knowledge of the structural chemistry of hemoglobin, developed an almost-immediate formulation of sickle cell anemia as a disease of the hemoglobin molecule, rather than of the entire blood cell.

Listen: William Castle recounts his first meetings with Linus Pauling…

Listen: …and Pauling responds in kind

A few months later, Pauling would pass this idea on to Harvey Itano, who was completing his doctorate in chemistry at Caltech. Itano conducted a series of initial experiments on the hemoglobin molecule, all of which failed. After months of tedious investigation, however, Itano, Dr. S. J. Singer and Dr. Ibert C. Wells – both of them newly-minted Ph.D.’s – were able to use the techniques of electrophoresis to identify a significant distinction. The paper “Sickle Cell Anemia, a Molecular Disease” was then published in the fall of 1949 and the concept of molecular disease was instantly established.

Listen: Pauling describes the Itano, Singer and Wells electrophoresis experiments

Although Pauling wasn’t the first to think about diseases in terms of molecular aberrations, no one prior to the Pauling-Itano group had concretely demonstrated their existence. After their initial success, Singer and Itano continued to expand on the original research, eventually discovering a less-severe case of sickle cell anemia called sicklemia. The duo also described the manner in which sickle cell anemia is inherited. As such, not only did Pauling and his colleagues identify the exact source of the disease, they also provided a link to genetics and confirmed Pauling’s view that analysis on a molecular level can provide valuable information. Later, Itano would discover more abnormal hemoglobin molecules, and the molecular analysis of diseases would continue.

Since Pauling’s coining of the term “molecular disease,” many other diseases have been similarly categorized: Hemophilia, Thalassemia, Alzheimer’s Disease and Muscular Dystrophy to name a few. (Though it could also be argued that every heritable disease can be classified as a molecular disease because these diseases require a modified genetic component that can be passed from parent to child.)

Thalassemia, for example, is also a disease of the hemoglobin molecule. However, while sickle cell anemia is caused by the production of abnormal hemoglobin, Thalassemia, conversely, involves the abnormal production of hemoglobin. More specifically, in cases of Thalassemia, the rate of production of a specific globin chain is decreased, which then causes the formation of abnormal hemoglobin molecules.

Pauling’s conceptualization of sickle cell anemia as a disease of the hemoglobin molecule jump-started years of research pertaining to abnormal hemoglobins and opened many new doors in the study of inherited diseases. Although he wasn’t directly involved in the discovery of the abnormal hemoglobin molecules, Pauling’s development of the concept of molecular disease was achievement enough to significantly raise his stature in the medical community (at least for a while) and further cement his status as a scientist of world-historical importance.

For more information on molecular disease and other related topics, please visit the website “It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia.”

Pauling’s Theory of Sickle Cell Anemia

It's in the BloodWe owe to Pauling and his collaborators the realization that sickle cell anaemia is an example of an inherited ‘molecular disease’ and that it is due to an alteration in the structure of a large protein molecule, an alteration leading to a protein which is by all criteria still a haemoglobin.
– Vernon M. Ingram, 1957.

Of the four Documentary History websites that the OSU Libraries Special Collections has produced, “It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia” is, in certain respects, the most unique.

For one, “the blood site” — its usual in-house appellation — is the only of our Documentary Histories not to have been written by Pauling biographer Tom Hager.  On the contrary, the idea for the blood site arose out of a history of science master’s thesis that Melinda Gormley — then a graduate student and now a professor at OSU — developed from research done in the Ava Helen and Linus Pauling Papers. As Dr. Gormley documented in this article (PDF, see pp. 8-9) it took the better part of two years to repurpose the text of her dissertation into a format suitable for the web.

Gormley’s thesis topic was relatively broad — “The Varieties of Linus Pauling’s Work on Hemoglobin and Sickle Cell Anemia,” (PDF, 1.8 MB) — and, as a result, the swath of content covered in the website is similarly wide.  The website begins its narrative in 1930, ends it in 1994, and along the way discusses Pauling’s contributions to areas ranging from immunology to Scientific War Work to evolutionary theory to orthomolecular psychiatry.  All of these topics will be addressed in future posts on this blog.

The heart of the blood site, however, is Pauling’s research on sickle cell anemia. Sickle cell anemia is a terrible disease that predominantly effects inhabitants of sub-Saharan Africa or those who can trace their lineage to that region.  The disease is a painful one, characterized by drastically-malformed red blood cells, and manifesting itself in a host of health maladies and, often, shortened lifespans.

Many folks who are semi-acquainted with the Pauling legacy know that he was, in some way, important to the modern understanding of sickle cell anemia.  But how? Well, Linus Pauling was the first individual to correctly theorize that sickle cell anemia is a disease that locates its source to the molecular level — in the process Pauling likewise became the first individual to postulate the concept of a molecular disease.

What then, exactly, was Pauling’s theory of sickle cell anemia?  That is the question that we aim to explore in this post.

Linus Pauling probably wasn’t a true freak-of-nature genius in the manner of an Einstein or a Mozart.  On the contrary, the likely secret of his profound success as a scientist was at least threefold in nature: 1) he possessed a relentless work ethic; 2) he was a very clear and concise thinker who conceptualized his ideas well and understood the efficiencies inherent to leading teams of researchers as opposed to going it alone; 3) and most importantly, he was deeply interested in, and capable of concretely understanding, radically-disparate areas of scientific study.  All three of these traits reveal themselves in the sickle cell anemia story.

Pauling first encountered the problem of sickle cell anemia rather by accident.  At a dinner in 1945, Pauling sat in the audience of an informal presentation by physician Dr. William Castle, wherein it was noted that the shape of red blood cells in sickle cell patients varied depending on whether the blood was venous or arterial —  normal in arterial blood, sickled in venous blood.  Clearly this suggested that the oxygen content in sickle cell blood played a major role in its molecular architecture. By his own recollection, “within two seconds,” Pauling concluded that the oxygen piece of the equation suggested that hemoglobin must be involved in the sickling mechanism — a conclusion that he could reach because of his keen understanding of the structural chemistry of hemoglobin.

In 1960, Pauling provided this description of his initial thoughts on how malformed hemoglobin could lead to sickled red blood cells.

…immediately I thought, “could it be possible that this disease, which seems to be a disease of the red cell because the red cells in the patients are twisted out of shape, could really be a disease of the hemoglobin molecule?” Nobody had ever suggested that there could be molecular diseases before, but this idea popped into my head. I thought, “could it be that these patients can manufacture a special kind of hemoglobin such that the molecules are sticky and clamp on to one another to form long rods, which then line up side by side to form a long needle-like crystal, which as it grows inside of the red cell becomes longer than the diameter of the cell and thus twists the red cell out of shape?”

From here, Pauling delegated many of the details necessary to verifying his thinking on the sickle cell problem to a team of Caltech graduate students led by Harvey Itano.  (This was common practice for Pauling, and helps explain how he was able to generate over 1,100 published papers in ninety-three years of living)  Using a variety of methods including electrophoresis, the Itano team, in the words of a 1950 Caltech press release

found a difference – slight but still unmistakable – between normal hemoglobin and that of a sickle-cell anemia patient.  Sickle-cell hemoglobin proved to have a greater positive electrical charge, under the proper chemical conditions, than did the hemoglobin from a normal person.  Such a difference in electrical properties can only mean a difference in molecular architecture, in the way in which the hemoglobin molecules are constructed.

In other words, Pauling was right: sickle cell anemia was a molecular disease and malformed hemoglobin was the cause.

In 1956, an English chemist named Vernon Ingram, using a new technique called fingerprinting, (Pauling provides a rather technical description of the method here) proved conclusively that sickle cell anemia was an inherited disease as well.  Moreover, sickle cell anemia was found to be caused by an astonishingly small change at the molecular level.  Physicist John Hopfield described it this way

On the surface of the ten-thousand atom molecule, there is a slight change. A small group of a few atoms on the edge of the molecule is replaced by another small group of atoms. That’s all that happens – an exchange of a few atoms. Yet it’s enough to make people very ill. The effect of the change is to create a sticky point between an abnormal molecule and its neighbor, causing molecules to pile up on each other.

Just as Linus Pauling predicted, after dinner, in 1945.