PHONETIC LAW
Note 13.I have in mind, e.g., the presence of postpositions in Upper Chinook, a feature that is clearly due to the influence of neighboring Sahaptin languages; or the use by
After working on the molecular structure of simple substances, Pauling had the idea as early as 1934 of explaining the properties of hemoglobin in terms of its molecular structure—even though it is a very complicated molecule of 10,000 atoms. Karl Landsteiner at the Rockefeller Institute asked Pauling how he would explain the properties of antibodies in terms of their molecular structure. Pauling discovered that he and Landsteiner thought about the serologic problem in very different ways.
I
n November 1949, an article appeared in Science which would eventually play a fundamental role in the establishment of molecular biology and molecular medicine. Linus Pauling and his collaborators published a paper with the unusual title “Sickle-cell Anemia, a Molecular Disease,” showing that the hemoglobin molecules of patients suffering from this deadly hereditary affliction had a different electrical charge than those of healthy patients. The paper had a powerful impact on the biomedical community and the public at large. Indeed, it soon became a “citation classic.”Pauling’s paper was important and novel in two different ways. On one hand, it showed for the very first time that the cause of a disease could be traced to an altered molecular structure, raising hopes that all diseases might eventually be explained in a similar fashion. On the other hand, since this disease was known to be heritable, the paper argued that genes determined precisely the structure of proteins. These two points have become so obvious today, that it might seem surprising that they have a history.
Linus Pauling, who spent more than forty years at the California Institute of Technology, exemplifies better than anyone else the emerging
“molecular vision of life” of the middle third of our century. As early as 1956, for example, Pauling endorsed the view that “man is simply a collection of molecules,” and “can be understood in terms of molecules”—
a view that gave him “great pleasure and satisfaction.” Indeed, after his pioneering studies on the nature of the chemical bond in the 1920s and
1930s, which earned him a world-wide reputation, Pauling started to investigate molecules of biological interest—which at that time essentially meant proteins. As he put it in 1937, “the secret of life itself [is] how a protein molecule is able to form, from an amorphous substrate, new protein molecules which are made after its own image.”
Pauling’s attention was drawn to sickle-cell anemia, a hereditary disease found mainly among people of African descent, in 1945 by William B.
Castle, a clinician from Harvard Medical School. Both were serving on the Medical Advisory Committee which assisted Vannevar Bush in the elaboration of his famous report, Science, the Endless Frontier. Pauling had been involved in hemoglobin studies in war-related research on blood substitutes, and had investigated the magnetic properties of hemoglobin since 1935. He was thus already familiar with hemoglobin when Castle told him that only venous—deoxygenated—blood of sickle-cell anemia patients showed, upon microscopic inspection, sickle-shaped red blood cells. This indicated that the hemoglobin molecule was probably involved in the sickling process, causing the cells to acquire their distorted shape.
Pauling then thought that for these patients, “perhaps the Hb [hemoglobin]
molecule changes shape.”He had been searching avidly for nearly ten years for a medical problem to solve in order to demonstrate the power of his physico-chemical approach to biology and medicine. Like many other scientists, he was also eager to convert wartime support—from the Office of Scientific Research and Development (OSRD), for example—into peace-time money, along the lines of Bush’s Endless Frontier, which called for a more obvious relevance of scientific research to American public needs. Thus, the sickle-cell anemia project represented for Pauling a timely convergence of political, financial and intellectual interests.
Linus Pauling lecturing on sickle-cell anemia, Tokyo, February 1955
Pauling assigned the sickle-cell anemia project to Harvey A. Itano, a young M.D. hired in 1946, as a thesis topic for his Ph.D. Drawing on the knowledge and resources of several medical practitioners, Itano tried, without success, several different physical and chemical methods to distinguish normal from sickle-cell anemia hemoglobin. He then turned to electrophoresis, a then-new technique designed to separate molecules according to their electrical charge, which had already been used to analyze blood proteins. Caltech was one of the few institutes in the world to own an electrophoresis apparatus, an instrument not yet commercially available at that time. This proved to be a good choice, and Itano was able, in 1948, to find a slight electrophoretic difference between normal and sickle-cell anemia hemoglobins.
Not only was Pauling’s group able to demonstrate that patients with sickle-cell anemia had a different hemoglobin than healthy persons, but he also showed that blood taken from persons suffering from “sicklemia,” a milder form of the disease, contained a mixture of normal and pathological hemoglobin—in about equal amounts. They thus concluded that
“sicklemia” reflected a heterozygous condition and sickle-cell anemia, a homozygous condition. They reached this conclusion apparently independently of James Neel at Ann Arbor, who, on genetic grounds, arrived at the same result, which he published a few months earlier.
By the time Pauling et al.’s paper appeared, it was well known that human hemoglobins (adult and fetal) differed electrophoretically, and several diseases had been correlated with altered electrophoretic patterns of blood proteins. So what was new about the Science paper? Beadle and Tatum had elaborated the “one gene-one enzyme” hypothesis in the 1940s, but it was not yet clear what it was that genes control, beyond the absence or presence of a particular enzyme. Pauling’s sickle-cell anemia work demonstrated that genes could alter qualitatively the structure of proteins—
in this case, with dramatic consequences for human health. It also proposed a causal link—not a mere correlation—”between the existence of
‘defective’ hemoglobin molecules and the pathological consequences of sickle-cell disease.”
But the sickle-cell anemia success did much more. Under Pauling’s energetic advertisement in numerous speeches and papers, the discovery became emblematic of how basic science could solve medical problems. In 1956, for example, he asserted, “I believe that chemistry can be applied effectively to medical problems, and that through this application we may look forward to significant progress in the field of medicine, as it is transformed from its present empirical form into the science of molecular medicine.” Immediately after the 1949 paper, Pauling tried to establish a medical research institute at Caltech devoted to “molecular medicine.”
Public and private funding agencies remained skeptical of Pauling’s approach, however, and he was unable to attract the necessary funds.
Based on their knowledge of the molecular nature of sickle-cell anemia, Pauling and Itano proposed several treatments to prevent sickling. After
Pauling was a more
two years of clinical trials performed by George Burch, a physician from New Orleans, the results turned out to be disappointing and were never published. Unfortunately, this would not be the last of such failures. Even today, our extremely detailed understanding of the molecular etiology of sickle-cell anemia has led to new diagnostic possibilities, but little in the way of significant improvements in therapy.
In the 1950s, Itano and others moved on to generalize their approach to other blood pathologies. But for Pauling, the main question was to pinpoint the origin of the electrophoretic difference—presumably a difference in the amino acid composition of the normal and pathological hemoglobins. With the chemist Walter A. Schroeder, he performed chromatographic analyses of normal and sickle-cell anemia hemoglobin and was surprised to find, in 1950, that there was no difference in amino acid content, which could explain the electrophoresis result—a conclusion soon confirmed by others. Pauling thus thought that the electrophoretic difference resulted “from a difference in folding of the polypeptide chain.”
With the immunologist Dan Campbell, he found a serological difference between the two forms of hemoglobin. Furthermore, when denatured, hemoglobin no longer showed the electrophoretic difference. The most likely conclusion from these pieces of evidence was that the same polypeptide was folded, under genetic control, in two different ways which affected the electrophoretic mobility. This conclusion fitted perfectly in the
“molding model” of protein synthesis. In the following years, Pauling often used the example of sickle-cell anemia hemoglobin to support his views on protein synthesis. In 1954, for example, in his Harvey lecture, he said
“the gene responsible for the sickle-cell abnormality is one that determines the nature of the folding of polypeptide chains, rather than their composition.”
This conception of protein synthesis thus gained unsuspected support from the results of sickle-cell anemia research. However, by the end of the 1950s, this conception would be completely abandoned, and replaced by the model we have adopted today, whereby genes determine the amino-acid sequences of proteins, and do not serve to direct their three dimensional folding. In this reversal, sickle-cell anemia research was again involved. Thus the case of sickle-cell anemia conveniently highlights the terms of this debate, which settled in the mid-1950s and only reopened recently, to some extent, with the discovery of chaperones.
Around 1950, the debate focused around the following questions: what deter mines the three dimensional structure of a protein? Does it
“automatically” follow from its amino acid composition and sequence, or is some other component, genetic or non-genetic, involved in giving it its final configuration?
The idea that some substance other than the protein itself, such as the antigen for antibodies, the substrate for enzymes, or even the gene itself, was directing protein folding was favored by many researchers until the mid-1950s. Pauling, in a lecture held in 1948, summarized this theory of protein synthesis:
The mechanism of obtaining [immunological specificity] is one of moulding a plastic material, the coiling chain, into a die or mould, the surface of the antigen molecule. I believe that the same process of moulding of plastic materials into a configuration complementary to that of another molecule which serves as a template, is responsible for all biological specificity. I believe that the genes serve as the templates on which are moulded the enzymes which are responsible for the chemical characters of the organism.
Some people, however, had remained skeptical of Pauling and Schroeder’s results. Francis Crick, for example, recalled that Schroeder’s
“method was in fact too crude to detect such a single change in amino acid composition. I clearly realized this at the time. … I was convinced (perhaps rashly) that there would be a change in amino acid composition.”
Such “moulding models” of protein synthesis were not advocated by
“outsider” scientists, nor were they only theoretical speculations. Similar views were held around the same time by influential figures like the microbiologists Jacques Monod (Nobel prize in 1965) and Sol Spiegelman, the biochemists John Northrop (Nobel prize 1946), John Synge (Nobel prize 1952), and Felix Haurowitz, and the geneticist George Beadle (Nobel prize in 1958). The strongest empirical support for these ideas came from the study of antibody formation and enzymatic induction in bacteria.
Indeed, in 1957 big news came from Cambridge, England. Vernon Ingram, who had taken up Crick’s skepticism, was able to point to a single amino acid difference between normal and sickle-cell hemoglobin that explained the electrophoretic difference. His success was the result of a new method he had devised, combining paper chromatography with electrophoresis for the separation of peptides—”fingerprinting,” as he called it. The importance of this result went far beyond the etiology of a particular disease. Indeed, for the first time it was demonstrated, as Ingram wrote in 1957, that “an alteration in a Mendelian gene causes an alteration in the amino acid sequence of the corresponding polypeptide chain.” He had brought the understanding of the role of genes one step further than Pauling. Not since the proposed double helix structure for DNA in 1953 had the research interests of geneticists, biochemists, and structuralists merged so closely in a single project.
Pauling immediately and radically changed his views about the mechanism of protein synthesis:
It is likely that the principal function of the gene involved in the
manufacture of a protein is to determine the sequence of amino acids in the polypeptide chain of the protein molecules. … it is probable that the polypeptide chain folds into its stable configuration automatically, that the stable configuration is determined by the amino-acid sequence.
This idea was forcefully elaborated by Crick as the “Central Dogma” in his famous 1957 lecture “On Protein Synthesis.” It allowed many
researchers to concentrate on how DNA sequences determine protein sequences, the “coding problem,” or, to use Crick’s new terminology, how DNA “information” is passed into protein.
Consensus spread rapidly. Indeed, when concluding the Cold Spring Harbor Symposia on Quantitative Biology of 1960, Jacques Monod and François Jacob (Nobel prize 1965) wrote:
A few years ago, the question was often debated whether any further (non-genetic) structural information needed to be furnished, or might conceivably be used in some cases, at the stage of tertiary folding in protein synthesis.
[This] issue was not discussed during the conference, evidently because it is considered as settled.
Sickle-cell anemia was not the only research line involved in this denouement. Most importantly, the work of Christian Anfinsen during the late 1950s and 1960s showed that denatured ribonuclease could regain its secondary and tertiary structure spontaneously, earning him the Nobel Prize for chemistry in 1972. Monod’s model for enzymatic induction, where the substrate determined the enzyme’s folding, was facing increasing conflicting experimental evidence, and started to be abandoned in the late 1950s, to be eventually replaced by the operon model. Similarly, the
“molding model” of antibody formation, which Pauling had done much to popularize, was challenged by Frank Macfarlane Burnet (Nobel prize 1960) with his clonal selection theory of 1957, in which each cell produces antibodies with only one specificity. Joshua Lederberg (Nobel prize 1958) rapidly drew the conclusions for protein specificity, namely that antibodies with different specificities must have different amino acid sequences.
Finally, the wider recognition during the 1950s that genes were nucleic acids, and not proteins, made it increasingly difficult to devise a mechanism by which they would act as three-dimensional templates for protein folding.
Thus, very different research programs, in biochemistry, immunology, genetics, physical chemistry, and hematology all converged in the second half of the 1950s to redefine one of the foundations of molecular biology:
the relationships between sequences, structures, and functions. This shows how much what we today call “molecular biology” was, and still is, a highly interdisciplinary field, lacking methodological unity and resisting any subsumption under a coherent disciplinary label. Finally, this redefinition led to the primacy of sequences in explaining biological and pathological processes as well. The “Central Dogma,” the genetic code, and finally the present excitement for sequencing and genomics, show how much this idea has become central in our understanding of life.
Just how it was that sequences of DNA, the hereditary material, determine the amino acid sequences of proteins—the “coding problem”—
became a major focus of molecular biologists and biochemists in the following years. They deciphered the genetic code by 1966, and it was
finally clear, as Crick put it, how DNA “information” was passed into protein. The “sequencing culture” has grown by leaps and bounds ever since, as shown by its most recent and visible example, the Human Genome Project. The sickle-cell anemia project represented a turning point in Pauling’s career. From the mid-1950s, after he had received the Nobel Prize in chemistry (1954), he became increasingly involved in political activities, leaving him less time in the laboratory. He shifted his remaining research toward medical problems such as the molecular basis of mental deficiencies and his controversial vitamin C crusade. His medical research resonated with his peace activism, as in his claim that nuclear bomb testing was the source of an increased mutation rate, causing innumerable “molecular diseases.”
The legacy of sickle-cell anemia research in the middle of our century can hardly be underestimated. It rapidly became a favorite example, in news editorials and textbooks, of how a molecular approach could explain biological and pathological processes. However, the story has often been told without regard to the fact that Pauling did not succeed simply by applying physical chemistry to a medical problem, but rather by relying on skills from the clinic as well as the laboratory, from biologists, biochemists, and physicians. Pauling’s grand vision of molecularizing biology and medicine has been realized to an extent he could never have foreseen, even if our therapeutic power does not yet match our ever more profound understanding of the molecular basis of health and disease.
134