The wandering pathway to determining N to C synthesis of proteins: Some recollections concerning protein structure and biosynthesis
2006; Wiley; Volume: 34; Issue: 4 Linguagem: Inglês
10.1002/bmb.2006.494034042642
ISSN1539-3429
Autores Tópico(s)RNA modifications and cancer
ResumoWhen I reflect on how advances in biochemical knowledge occurred, it seems that they were often a result of “being in the right place at the right time” and “interacting with the right people.” In my own case, I can trace the cumulative effects of many such events on the sequential development of my interests and experimental efforts studying proteins, their physical structure, and their mode of biological synthesis from amino acids. The following is an outline of my recollections concerning what could be described as a random career process. My exposure to biochemical knowledge began in the 1940s at UCLA in a general biology course. We learned from our textbook that DNA is a “large repeating tetra-nucleotide polymer, whose biological function is to maintain the osmotic pressure of the cell nucleus.” In a subsequent advanced biochemistry course given by Professor Max Dunn, we learned that proteins are “colloids of random composition made of amino acids.” We studied in great experimental detail how this had been “proven” using the latest experimental technique of deuterium dilution analysis. The laboratory part of that course was excellent, having as teaching assistant a remarkable graduate student: Bruce Merrifield, who later won a Nobel Prize for devising solid state peptide synthesis. In the laboratory experiments, we learned to analyze proteins, carbohydrates, and lipids, using the primitive tools of the time. However, we were taught that proteins were merely random colloids. That perplexed me as being incompatible with the specificity of function of proteins such as enzymes. The discrepancy kindled my interest in learning more about the structure and function of proteins. On graduation in 1948 with a degree in chemistry, I chose to continue graduate education in a program dedicated to the study of proteins. I applied and was accepted as a graduate student in the Laboratory of Biophysical Chemistry at Harvard Medical School. There, intensive efforts were underway under the leadership of Edwin Cohn to fractionate and physically characterize the proteins in human blood serum. The laboratory was a dynamic place, combining the latest ideas and experimental techniques related to the physical structure and functions of proteins. The atmosphere was interactive, and the graduate students had free access to faculty for discussions and experimental work in any or all of their laboratories. Professors John L. (Larry) Oncley, John Edsall, Walter Hughes, and Barbara Low were dedicated to understanding the chemistry, structure, and function of protein molecules using the technology of the times. Their laboratories were always open to graduate students, and I was able to do extensive experimental work with all of them. My main thesis work [1] in the laboratory of Larry Oncley made use of dielectric constant measurements of solutions of human serum albumin, over a wide range of electrical frequencies, to determine the physical shape of the molecule in solution. The conclusion was that the molecule is shaped like a rigid prolate ellipsoid, similar in shape to a football. At that time, this modest conclusion was considered a significant advance in knowledge! There were few graduate students at the time, largely because of the limited availability of funding. I particularly remember discussions with fellow students Tom Thompson, Frederick Richards, and Renee Zlochover. Midway through our graduate studies (1951), Renee and I were married and somehow have managed joint careers, three children, and family responsibilities for 55 years afterward. During my last years of thesis work, I had the good fortune to hear seminars by two future Nobel Prize winners from Cambridge University that shaped the direction of my interests. One was a talk by Fred Sanger, who described his groundbreaking determination of the unique amino acid sequence of insulin. The other was a talk by Max Perutz, outlining his recent studies on the physical structure of hemoglobin using the technique of x-ray crystallography. Although he had not been able to determine the actual structure because of the “phase problem,” he made an approximation that the hemoglobin molecule might be shaped like a stack of four pancake-like objects, each with an iron atom in the center. He indicated that progress to direct determination of the actual physical structure might be possible if a sufficiently heavy atom could be incorporated into the hemoglobin crystals but said that iron was not heavy enough and possibly no atom in the periodic table might be heavy enough to solve the phase problem. After the seminar, I spoke to Max Perutz about the experience that I had during my thesis work synthesizing small organic molecules containing several mercury atoms and proposed that these might be useful in addressing the crystallographic phase problem for hemoglobin. He was very interested and suggested that I might work with him as a postdoctoral fellow, a suggestion that sounded very attractive. I immediately began a side project, along with my thesis work, reading the early papers (circa 1890) on the synthesis of organic compounds containing mercury atoms and synthesizing a number of compounds having between two and six mercury atoms for possible use in protein crystallography. When my thesis was almost finished, I applied for and received a fellowship to spend the following academic year with Professor John G. Kirkwood in the Chemistry Department at Yale, conducting experimental and theoretical studies of electrostatic forces acting between protein molecules. The experimental method employed in the laboratory was solution light-scattering analysis as a function of protein and salt concentration, which led to interesting results. I remember particularly informative discussions during the year at Yale with S. J. Singer and Serge Timasheff, concerning the behavior of protein molecules in solution. Meanwhile, it had taken over a full year to apply for, and obtain, a fellowship to work with Max Perutz in England. For the next two years (1954–1956), I was able to work in the Medical Research Council (M.R.C.)1 unit in the Cavendish Laboratory at the Cambridge University Department of Physics. Upon arrival, I soon learned that the M.R.C. unit was viewed within the physics department as a personal enthusiasm (hobby) of Sir Laurence Bragg, the Cavendish Professor of Physics, and was not considered to be “real physics” by many other members of the department. The building in which the Cavendish Laboratory was located was subject to an interesting rule, dating back to when Rutherford was Professor. He had decided in the early 1900s that scientists were doing too many experiments and not thinking enough. To remedy this misbehavior, the building was locked up at 5 p.m. each day and not reopened until 7 a.m. the following day. However, since protein crystallography was very important to Bragg, we were given special keys to enter the building at night to change films in the x-ray cameras. The shared office space for the two senior members of the M.R.C. unit, Max Perutz and John Kendrew, was a room ∼7 feet square in which they sat back to back at small desks. The laboratory space was a room about 25 feet square in which 10–12 graduate students, postdoctoral fellows, technicians, and visitors shared laboratory bench space. Office space was a similar room in which there were about a dozen desks crowded together in one open space, often noisy with conversations between occupants or between various occupants and visitors. I was fortunate to be assigned a corner desk with the immediately adjacent desk on my left occupied by a quite noisy, but always interesting, fellow named Francis Crick. People seemed to be continually dropping by to talk to Francis. The conversations were usually interesting, and occasionally, I participated. Topics covered a very broad range of physics, mathematics, and biology, analyzing theory, experimental techniques, and alternative interpretations. Emphasis continually shifted between experimental details, molecular mechanisms, and the “big picture” of biological function and evolution. I quickly learned the protein crystallization techniques used by Max Perutz for horse hemoglobin and those used by John Kendrew for sperm whale myoglobin. After becoming competent with the experimental details of x-ray data collection and analysis from single protein crystals, I began the process of searching for heavy atom derivatives to use in solving the phase problem. The x-ray diffraction pattern on a film from a crystal of the normal protein was compared visually with the pattern from a crystal containing a possible heavy atom derivative. After many unsuccessful heavy atom trials, I finally obtained numerous differences in the intensities of individual diffraction spots from a crystal, suggesting a possible success. This observation had to be confirmed by computation of a “Patterson Map” at the University Computing Center. Fortunately, at the time (1954), Cambridge University had (for one year only) the most powerful digital computer in the world, EDSAC 1. Three technicians had built it by hand, using dozens of war surplus radio chassis and hundreds of vacuum tubes, almost filling an enormous room with racks of chassis and bundled wires running everywhere. Computer “memory” consisted of an iron pipe 15 feet long filled with mercury, with a loudspeaker at one end of the pipe and a microphone at the other end. The dynamic memory consisted of the sound waves traveling down the pipe, with a cycle time of a millisecond and a total memory capacity of a kilobyte. Computer input was a punched paper tape, and output was an electric typewriter. The contraption usually ran only a few hours before one of the many hundreds of vacuum tubes burned out. Calculation then stopped until one of the three technicians who had built the computer came by to repair it. After 6 p.m., this required waiting until the next morning, or Monday morning if on weekends. Computers have improved in many ways during the subsequent half-century! Because our crystallographic computations were among the most complex done at the time, we were only allowed to use the facility after 8 p.m., when other users had finished their work. We could work during the night until we finished our calculations or a vacuum tube burned out, whichever came first. After several abortive efforts with crystals that didn't work out or with computer failures, I finally got a success with myoglobin crystals containing mercuric iodide. This was the first useful heavy atom derivative for myoglobin and caused much excitement in the laboratory since the mercury atoms had enough electrons to determine some of the phases in the crystal [2]. Vernon Ingram, who at the time was a staff member in the M.R.C., had previously reacted hemoglobin with a silver atom at an accessible cysteine side chain, but silver atoms did not have enough electrons to allow useful phase determination for hemoglobin. However, by using a number of mercurials and other high atomic number atoms in various complexes and compounds, in an assortment of binding conditions, it soon proved possible to find useful derivatives for hemoglobin also [3]. Gerhard Bodo, an Austrian chemist, who contributed several of the subsequent heavy atom derivatives, soon joined us. These were very exciting times since it was becoming clear that we were on the experimental path to determining the detailed structures of myoglobin and hemoglobin, the first proteins whose structures would be solved to atomic resolution. Knowledge had significantly progressed in the few years since I had been taught that proteins were colloids with no definite structure or composition! Even while carrying out these exciting crystallographic studies on myoglobin and hemoglobin, my interest was increasingly captured by the many thoughtful discussions involving Francis Crick and whoever else happened to be nearby. Participants randomly included Aaron Klug, Jim Watson, Hugh Huxley, Leslie Orgel, David Blow, and many others. Such ruminations often occurred within the M.R.C. unit. Some also happened at lunch in the Eagle, a nearby Pub. Among the many topics covered, a prominent theme concerned the possible nature of the “genetic code” and its relationship to protein structure. Would the code be linear or branched in structure, “punctuated” or not, and expressed in two-letter or three-letter nucleotide units? What was the relationship between DNA and RNA in synthesizing proteins? How did mutant proteins differ from normal proteins? These and many similar questions were endlessly discussed and stirred my interest and imagination. In an imaginative effort to get experimental data on the nature of protein mutation, Francis organized a search for amino acid mutations in a soluble protein, first selecting chicken egg lysozyme as a small, easily assayed protein. Starch gel electrophoresis was used to search for a charge difference in a possible mutant lysozyme, which would then be analyzed for a changed amino acid. Eggs from many farms, within Cambridgeshire or beyond, were collected and analyzed for a charge difference from normal lysozyme. However, no differences were found from hundreds of trials. Attention then shifted to human tear lysozyme, and members of the M.R.C. unit, sometimes reluctantly, donated their tears. These were obtained by holding a cut onion close to the face while small beakers placed under our eyes caught our tears. No charge differences between the lysozyme samples from many “volunteers” could be observed, and the project was eventually dropped, much to our relief. Fortunately, shortly afterward, Max Perutz received a blood sample from London, obtained from an African patient suffering with sickle cell anemia. The intent was to use x-ray methods to search for possible structural organization of the hemoglobin molecules at low oxygen pressure. However, no experimental changes were observed by x-ray analysis. The sample would probably have been discarded except for the pervasive influence of Francis Crick. He suggested that an amino acid change in the sickle cell hemoglobin molecule might be observable by using the technique that Vernon Ingram had recently developed. This involved physically separating, in two dimensions on paper, the soluble peptides produced by digestion of hemoglobin with trypsin (named fingerprinting by him). Vernon did indeed obtain a clear positive result one morning, just at the obligatory morning coffee break. The entire M.R.C. group excitedly gathered around the “fingerprint” to discuss the possible meaning of the data indicating that a peptide from the mutant hemoglobin had grossly shifted position. Within a short time, it was confirmed that an amino acid change in the wandering peptide had occurred, experimentally substantiating the idea that mutations could consist of changes in the amino acid composition of proteins [4]. My two-year fellowship period was ending while these exciting advances in knowledge of protein structure and genetics were occurring. I had to search for my next position in an almost nonexistent job market. Fortunately, two very different positions were offered at almost the last day of the fellowship period. The first offer came from Sir Lawrence Bragg, who had recently retired from his professorship in Cambridge to become head of the Royal Institution in London but continued to follow events at the M.R.C. unit closely. He was organizing a research group to study protein structure by means of x-ray crystallography and suggested that I join it. My visit with him to Michael Faraday's laboratory in the basement of the Royal Institution was one of the most inspirational moments in my life. Viewing Faraday's carefully written notebooks and highly diverse handmade laboratory equipment, including the electromagnets wound from copper wire hand-insulated with torn strips of silk dresses, made a lasting impression and seemed to set an ultimate standard for experimental ingenuity and versatility. While I was considering Bragg's offer, an alternative offer of assistant professorship of chemistry came from Linus Pauling at Cal Tech. It later became known that Peter Pauling, Linus's son, who was a graduate student in our laboratory at the M.R.C., had informed his father of the unpublished progress in the x-ray-derived structures of myoglobin and hemoglobin. The offer contained a heavy teaching load and no promise of research funding, but I quickly accepted it nevertheless. Shortly after my arrival at Cal Tech in 1956, I had an appointment with Linus Pauling to discuss my departmental duties in teaching and to outline my research opportunities. During the initial phase of the conversation, I outlined the progress that had occurred in the structures of myoglobin and hemoglobin at the M.R.C. He met my description of Ingram's exciting new findings on the amino acid change in the sickle cell hemoglobin peptide with skepticism, attributing the results to possible differential digestion of the two proteins by trypsin. Upon discussing my research plans, I was informed that it was expected that I join a research group to be directed by Pauling and Robert Corey to use a new heavy atom method that Pauling had devised to determine protein structure by x-ray analysis. The method would be based on the differential x-ray diffraction effects of two very large isomorphous anions, one containing 12 niobium atoms and the other containing 12 tantalum atoms, to determine x-ray phases in protein crystals. Rashly and naively, I told him that I thought the method would probably not work and why, based on my previous experience with protein crystals and many heavy atom compounds. I even more rashly indicated that the project was not appealing to me for that and other reasons. Only later did I realize that I did not want to take part in another group effort repeating the role I had at the M.R.C. unit with another protein or two. In my simple mind, the methodology for solving protein structure by x-ray crystallography had already been demonstrated at the M.R.C. unit in Cambridge. The road to further progress seemed in place, and it appeared to be inevitable that the physical structures of many protein molecules would soon follow. The conceptual problem that then seemed most exciting to me was protein synthesis, an understanding of the process of protein molecule assembly from amino acids. This extremely idealistic viewpoint was clearly the delayed effect of having had a desk next to Francis Crick for two years. A search of the scientific literature soon indicated that at least two major groups were working on the problem of protein biosynthesis from amino acids: Paul Zamecnik's group at Harvard and George Palade's group at the Rockefeller Institute. At the time, it was known that 14C-labeled amino acids were rapidly incorporated into peptide-like material within high molecular weight intracellular structures containing RNA and protein. The favored experimental systems involved rat pancreas or liver, both systems being less than ideal because of the multitude of different proteins being synthesized and the presence of proteolytic enzymes in cellular extracts. After a few frustrating attempts to do experimental work with liver and pancreas, I decided that both experimental systems seemed hopeless for my purposes. Having a heavy teaching load and very little funding, I was extremely fortunate to be allowed to use the laboratory facilities of Jerome Vinograd in the Biology Department for exploratory experiments. His laboratory instruments included centrifuges and an analytical ultracentrifuge, which proved essential. Vinograd was a dynamic experimentalist, and we had many vigorous discussions of the theory and experimental techniques relating to nucleic acids or protein behavior in the cell. He and his laboratory brought back memories of the intellectually stimulating atmosphere at the M.R.C. unit in Cambridge. Among the other individuals using Vinograd's laboratory and ultracentrifuge was Matthew Meselson, a graduate student, studying replication of DNA in bacteria. After following the published literature, doing a few messy experiments with rat liver and pancreas, and being quite discouraged, I was extraordinarily fortunate to learn of local experimental activity that might be of use to study protein synthesis. In the next building, Henry Borsook was investigating hematopoiesis (the process of forming new red blood cells) in anemic rabbits and trying to isolate the stimulatory factor, erythropoietin, from the anemic rabbit serum. A brief visit to Borsook's laboratory quickly confirmed the existence of an ideal experimental system, available free of cost to me. He was producing several anemic rabbits a week by phenylhydrazine injection, saving the serum to study and discarding the immature red cells, reticulocytes, which were actively synthesizing hemoglobin. He was pleased to donate the immature red cells to me if I would pick them up once a week in an ice bucket. He also wanted to know why I wanted them. When I explained the idea of studying the assembly of hemoglobin molecules by pulse labeling the immature red cells with radioactive amino acids and studying radioactivity levels associated with the particles containing messenger RNA and with hemoglobin, he seemed perplexed. To my great surprise, he didn't accept the idea that proteins were encoded from DNA or RNA but believed that they were copied from pre-existing protein molecules by a molding process. This was especially surprising to me since he had previously been a co-author, with Linus Pauling, in a classic paper on the thermodynamics of peptide bond formation. The “microsomal particles” found in immature red cells turned out to be free of membrane material, quite homogeneous in size, and easy to isolate by centrifugation. In addition, the primary, if not only, protein the reticulocytes synthesized was hemoglobin, a soluble protein composed of two separable polypeptides. The experimental system seemed ideal for studying protein assembly from amino acids. I soon began exploratory experiments, by both chemical and enzymatic techniques, to separate the amino-terminal or carboxyl-terminal amino acids in hemoglobin from the rest of the amino acids in the protein. The intent was to eventually pulse-label them differentially, using carbon-14-labeled amino acids. As the experimental efforts gradually progressed, I occasionally reported the results at national meetings. At one of these meetings in Boston, I suggested the term “ribosome” for the synthetically active, membrane-free particles found within the much larger membrane-containing microsomal particles. Richard Roberts thought the term useful and was the first to use it in a publication [5]. Experimental work was very slowly progressing when an attractive job offer came from the Biology Department at MIT. The position was free of heavy teaching requirements and would be in association with Alexander Rich and Cyrus Levinthal in a newly created, intellectually dynamic, biophysical section within the Biology Department. After brief consideration, I accepted the offer, and experiments were soon resumed (1958) in the new environment, which fortunately had many unexpected, unique, and crucial advantages. Perhaps foremost among these was the presence in the Biology Department of both the knowledge and the equipment for physically separating peptides formed by enzymatic digestion of hemoglobin. Vernon Ingram had recently moved to the Biology Department at MIT from the M.R.C. unit at Cambridge and set up equipment for “fingerprinting” various hemoglobins. In addition, Michael Naughton, who had worked with Fred Sanger in the Biochemistry Department at Cambridge University and had been involved in originally developing the two-dimensional method of separating peptides on paper in Sanger's laboratory, had also come to the Biology Department at MIT. Naughton proved to be an amazing and indispensable source of experimental expertise concerning the technology of forming and processing peptides from proteins. In this environment, emphasis quickly shifted from end-group analysis to peptide fingerprint analysis. I soon made progress in separating 14C pulse-labeled peptides formed by trypsin digestion of the hemoglobin or ribosomes from immature rabbit red cell (reticulocytes). Differential labeling of the peptides was apparent, but the results were not quantitatively reproducible and therefore not interpretable. Fortunately again, just at that time, the amino acid leucine labeled with tritium, 3H, of very high specific activity became available. This enabled double label experiments, mixing reticulocytes incubated for a short time (pulse-labeled) with tritium-labeled leucine and reticulocytes incubated for a long time (uniform-labeled) with carbon-labeled leucine. The resulting doubly labeled hemoglobin could be digested with trypsin, and peptides could be separated and quantitatively analyzed for isotope content. This technique eventually led to reproducible data and to the conclusion that the hemoglobin polypeptides were assembled from single amino acids, beginning at the free amino end and progressing stepwise to the free carboxyl end of the polypeptide. At the time (1960), the actual amino acid sequence of hemoglobin polypeptide chains was not known for any species, nor did we know the amino acid composition of the hemoglobin peptides we had separated. However, the reproducible quantitative data fit very well to an intellectually reasonable model, and in a theoretical sense, predicted the actual physical order of the tryptic peptides in the hemoglobin molecule. After more experimental work, I sent a manuscript describing the experimental findings and conclusions to John Edsall, who soon sent it for publication in PNAS [6]. At almost the same time that the page proofs for the manuscript were returned from the publisher for corrections, I received a most interesting and characteristic letter from Francis Crick. It is so illuminating, of both his thinking style and the state of knowledge at the time, that I have reproduced it here (Fig. 1). I immediately sent a copy of the page proofs to him, noting that the experimental results were not in agreement with his model. A characteristic handwritten Crick response was immediately returned and is also reproduced here (Fig. 2). Shortly afterward (1961), I moved again, taking a position as professor in the newly formed Biophysics Department at The Johns Hopkins School of Medicine. Michael Naughton soon joined me, and with his help, we repeated the fingerprinting experiments and determined the amino acid compositions of the rabbit hemoglobin tryptic peptides. By this time, the amino acid sequence of human hemoglobin had been determined, and we could match the human peptide compositions with our newly measured rabbit peptide compositions, which were almost identical. The spatial order of the tryptic peptides from human hemoglobin was then compared with the pulse-labeled order of the rabbit peptides. Fortunately, the pulse label data corresponded perfectly to the physical sequence, experimentally validating the conclusion that protein biosynthesis begins at the amino end of the polypeptide and progresses sequentially to the carboxyl end [7]. In agreement with the general notion that nothing in biology is as simple as it first appears, almost a decade passed before David Wilson and I were able to show [8] that assembly of the polypeptide chains of hemoglobin actually begins with a transient methionine. The transient methionine is very rapidly cleaved from the nascent peptide chains and is not present in the finished molecules, which were examined by the double labeling technique. Letter from Francis Crick to Howard Dintzis dated Feb. 3, 1961. Handwritten letter from Francis Crick to Howard Dintzis dated March 29, 1961. It says: Dear Howard, Very many thanks for sending the enclosed. I quite agree it makes nonsense of our idea—we shall have to think again. This will certainly be a classic paper. It will be interesting to see how it turns out when the amino acid sequence is known. In haste. Yours ever, Francis
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