History of molecular biology
Encyclopedia
The history of molecular biology begins in the 1930s with the convergence of various, previously distinct biological disciplines: biochemistry
, genetics
, microbiology
, and virology
. With the hope of understanding life at its most fundamental level, numerous physicists and chemists also took an interest in what would become molecular biology
.
In its modern sense, molecular biology attempts to explain the phenomena of life
starting from the macromolecular properties that generate them. Two categories of macromolecules in particular are the focus of the molecular biologist: 1) nucleic acid
s, among which the most famous is deoxyribonucleic acid (or DNA
), the constituent of gene
s, and 2) protein
s, which are the active agents of living organisms. One definition of the scope of molecular biology therefore is to characterize the structure, function and relationships between these two types of macromolecules. This relatively limited definition will suffice to allow us to establish a date for the so-called "molecular revolution", or at least to establish a chronology of its most fundamental developments.
of the Rockefeller Foundation
in 1938—was an idea of physical and chemical explanations of life, rather than a coherent discipline. Following the advent of the Mendelian-chromosome theory of heredity
in the 1910s and the maturation of atomic theory
and quantum mechanics
in the 1920s, such explanations seemed within reach. Weaver and others encouraged (and funded) research at the intersection of biology, chemistry and physics, while prominent physicists such as Niels Bohr
and Erwin Schrödinger
turned their attention to biological speculation. However, in the 1930s and 1940s it was by no means clear which—if any—cross-disciplinary research would bear fruit; work in colloid chemistry, biophysics
and radiation biology, crystallography
, and other emerging fields all seemed promising.
In 1940, George Beadle and Edward Tatum demonstrated the existence of a precise relationship between genes and proteins. In the course of their experiments connecting genetics with biochemistry, they switched from the genetics mainstay Drosophila
to a more appropriate model organism
, the fungus Neurospora
; the construction and exploitation of new model organisms would become a recurring theme in the development of molecular biology. In 1944, Oswald Avery
, working at the Rockefeller Institute of New York
, demonstrated that genes are made up of DNA(see Avery-MacLeod-McCarty experiment
). In 1952, Alfred Hershey
and Martha Chase
confirmed that the genetic material of the bacteriophage
, the virus which infects bacteria, is made up of DNA (see Hershey-Chase experiment
). In 1953, James Watson
and Francis Crick
discovered the double helical structure of the DNA molecule. In 1961, Francois Jacob
and Jacques Monod
hypothesized the existence of an intermediary between DNA and its protein products, which they called messenger RNA
. Between 1961 and 1965, the relationship between the information contained in DNA and the structure of proteins was determined: there is a code, the genetic code
, which creates a correspondence between the succession of nucleotide
s in the DNA sequence and a series of amino acids in proteins. At the beginning of the 1960s, Monod and Jacob also demonstrated how certain specific proteins, called regulative proteins
, latch onto DNA at the edges of the genes and control the transcription
of these genes into messenger RNA; they direct the "expression
" of the genes.
The chief discoveries of molecular biology took place in a period of only about twenty-five years. Another fifteen years were required before new and more sophisticated technologies, united today under the name of genetic engineering
, would permit the isolation and characterization of genes, in particular those of highly complex organisms.
in the 19th century, developed by the German chemist Justus von Liebig
and following the birth of biochemistry at the beginning of the 20th, thanks to another German chemist Eduard Buchner
. Between the molecules studied by chemists and the tiny structures visible under the optical microscope, such as the cellular nucleus or the chromosomes, there was an obscure zone, "the world of the ignored dimensions," as it was called by the chemical-physicist Wolfgang Ostwald
. This world is populated by colloids, chemical compounds whose structure and properties were not well defined.
The successes of molecular biology derived from the exploration of that unknown world by means of the new technologies developed by chemists and physicists: X-ray diffraction, electron microscopy, ultracentrifugization
, and electrophoresis
. These studies revealed the structure and function of the macromolecules.
A milestone in that process was the work of Dr. Linus Pauling in 1949, which for the first time linked the specific genetic mutation
in patients with sickle cell disease to a demonstrated change in an individual protein, the hemoglobin
in the erythrocytes of heterozygous or homozygous individuals.
were described: the process of digestion
and the absorption of the nutritive elements derived from alimentation, such as the sugars. Every one of these processes is catalyzed
by a particular enzyme
. Enzymes are proteins, like the antibodies present in blood or the proteins responsible for muscular contraction. As a consequence, the study of proteins, of their structure and synthesis, became one of the principal objectives of biochemists.
The second discipline of biology which developed at the beginning of the 20th century is genetics. After the rediscovery of the laws of Mendel
through the studies of Hugo de Vries
, Carl Correns
and Erich von Tschermak
in 1900, this science began to take shape thanks to the adoption by Thomas Hunt Morgan
, in 1910, of a model organism for genetic studies, the famous fruit fly (Drosophila melanogaster
). Shortly after, Morgan showed that the genes are localized on chromosomes. Following this discovery, he continued working with Drosophila and, along with numerous other research groups, confirmed the importance of the gene in the life and development of organisms. Nevertheless, the chemical nature of genes and their mechanisms of action remained a mystery. Molecular biologists committed themselves to the determination of the structure, and the description of the complex relations between, genes and proteins.
The development of molecular biology was not just the fruit of some sort of intrinsic "necessity" in the history of ideas, but was a characteristically historical phenomenon, with all of its unknowns, imponderables and contingencies: the remarkable developments in physics at the beginning of the 20th century highlighted the relative lateness in development in biology, which became the "new frontier" in the search for knowledge about the empirical world. Moreover, the developments of the theory of information
and cybernetics
in the 1940s, in response to military exigencies, brought to the new biology a significant number of fertile ideas and, especially, metaphors.
The choice of bacteria and of its virus, the bacteriophage, as models for the study of the fundamental mechanisms of life was almost natural - they are the smallest living organisms known to exist - and at the same time the fruit of individual choices. This model owes its success, above all, to the fame and the sense of organization of Max Delbrück
, a German physicist, who was able to create a dynamic research group, based in the United States, whose exclusive scope was the study of the bacteriophage: the School of the Phage.
The geographic panorama of the developments of the new biology was conditioned above all by preceding work. The US, where genetics had developed the most rapidly, and the UK, where there was a coexistence of both genetics and biochemical research of highly advanced levels, were in the avant-garde. Germany, the cradle of the revolutions in physics, with the best minds and the most advanced laboratories of genetics in the world, should have had a primary role in the development of molecular biology. But history decided differently: the arrival of the Nazis
in 1933 - and, to a less extreme degree, the rigidification of totalitarian measures in fascist
Italy - caused the emigration of a large number of Jewish and non-Jewish scientists. The majority of them fled to the US or the UK, providing an extra impulse to the scientific dynamism of those nations. These movements ultimately made molecular biology a truly international science from the very beginnings.
and the other deoxyribose
. It was this subsequent discovery that led to the identification and naming of DNA as a substance distinct from RNA.
Friedrich Miescher
(1844–1895) discovered a substance he called "nuclein" in 1869. Somewhat later, he isolated a pure sample of the material now known as DNA from the sperm of salmon, and in 1889 his pupil, Richard Altmann
, named it "nucleic acid". This substance was found to exist only in the chromosomes.
In 1919 Phoebus Levene
at the Rockefeller Institute identified the components (the four bases, the sugar and the phosphate chain) and he showed that the components of DNA were linked in the order phosphate-sugar-base. He called each of these units a nucleotide
and suggested the DNA molecule consisted of a string of nucleotide units linked together through the phosphate groups, which are the 'backbone' of the molecule. However Levene thought the chain was short and that the bases repeated in the same fixed order. Torbjorn Caspersson and Einar Hammersten showed that DNA was a polymer.
proposed that inherited traits would be inherited via a "giant hereditary molecule" which would be made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template". Max Delbrück
, Nikolai V. Timofeeff-Ressovsky, and Karl G. Zimmer published results in 1935 suggesting that chromosomes are very large molecules the structure of which can be changed by treatment with X-ray
s, and that by so changing their structure it was possible to change the heritable characteristics governed by those chromosomes. In 1937 William Astbury
produced the first X-ray diffraction patterns from DNA. He was not able to propose the correct structure but the patterns showed that DNA had a regular structure and therefore it might be possible to deduce what this structure was.
In 1943, Oswald Theodore Avery and a team of scientists discovered that traits proper to the "smooth" form of the Pneumococcus could be transferred to the "rough" form of the same bacteria merely by making the killed "smooth" (S) form available to the live "rough" (R) form. Quite unexpectedly, the living R Pneumococcus bacteria were transformed into a new strain of the S form, and the transferred S characteristics turned out to be heritable. Avery called the medium of transfer of traits the transforming principle; he identified DNA as the transforming principle, and not protein
as previously thought. He essentially redid Frederick Griffith
's experiment. In 1953, Alfred Hershey
and Martha Chase
did an experiment (Hershey-Chase experiment
) that showed, in T2 phage, that DNA is the genetic material (Hershey shared the Nobel prize with Luria).
and was led by Maurice Wilkins
and was later joined by Rosalind Franklin
. Another group consisting of Francis Crick
and James D. Watson
was at Cambridge
. A third group was at Caltech and was led by Linus Pauling
. Crick and Watson built physical models using metal rods and balls, in which they incorporated the known chemical structures of the nucleotides, as well as the known position of the linkages joining one nucleotide to the next along the polymer. At King's College Maurice Wilkins and Rosalind Franklin examined X-ray diffraction
patterns of DNA fibers. Of the three groups, only the London group was able to produce good quality diffraction patterns and thus produce sufficient quantitative data about the structure.
) shapes. Pauling had deduced this structure from X-ray patterns and from attempts to physically model the structures. (Pauling was also later to suggest an incorrect three chain helical DNA structure based on Astbury's data.) Even in the initial diffraction data from DNA by Maurice Wilkins, it was evident that the structure involved helices. But this insight was only a beginning. There remained the questions of how many strands came together, whether this number was the same for every helix, whether the bases pointed toward the helical axis or away, and ultimately what were the explicit angles and coordinates of all the bonds and atoms. Such questions motivated the modeling efforts of Watson and Crick.
visited Cambridge and inspired Crick with a description of experiments Chargaff had published in 1947. Chargaff had observed that the proportions of the four nucleotides vary between one DNA sample and the next, but that for particular pairs of nucleotides — adenine and thymine, guanine and cytosine — the two nucleotides are always present in equal proportions.
Using X-ray diffraction, as well as other data from Rosalind Franklin
and her information that the bases were paired, James D. Watson
and Francis Crick
arrived at the first accurate model of DNA's molecular structure in 1953, which was accepted through inspection by Rosalind Franklin. The discovery was announced on February 28, 1953; the first Watson/Crick paper appeared in Nature
on April 25, 1953. Sir Lawrence Bragg, the director of the Cavendish Laboratory
, where Watson and Crick worked, gave a talk at Guys Hospital Medical School in London on Thursday, May 14, 1953 which resulted in an article by Ritchie Calder in The News Chronicle
of London, on Friday, May 15, 1953, entitled "Why You Are You. Nearer Secret of Life." The news reached readers of The New York Times
the next day; Victor K. McElheny, in researching his biography, "Watson and DNA: Making a Scientific Revolution", found a clipping of a six-paragraph New York Times article written from London and dated May 16, 1953 with the headline "Form of `Life Unit' in Cell Is Scanned." The article ran in an early edition and was then pulled to make space for news deemed more important.(The New York Times subsequently ran a longer article on June 12, 1953). The Cambridge University undergraduate newspaper
also ran its own short article on the discovery on Saturday, May 30, 1953. Bragg's original announcement at a Solvay conference
on proteins in Belgium
on 8 April 1953 went unreported by the press. In 1962 Watson, Crick, and Maurice Wilkins
jointly received the Nobel Prize
for Physiology or Medicine for their determination of the structure of DNA.
. Work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons, and Har Gobind Khorana and others deciphered the genetic code
not long afterward (1966). These findings represent the birth of molecular biology
.
The tRNAPHE structure is notable in the field of nucleic acid structure in general, as it represented the first solution of a long-chain nucleic acid structure of any kind - RNA or DNA - preceding Dickerson's solution of a B-form dodecamer by nearly a decade. Also, tRNAPHE demonstrated many of the tertiary interactions observed in RNA architecture which would not be categorized and more thoroughly understood for years to come, providing a foundation for all future RNA structural research.
Subsequent to Tom Cech's publication implicating the Tetrahymena group I intron as an autocatalytic ribozyme, and Sidney Altman's report of catalysis by ribonuclease P RNA, several other catalytic RNAs were identified in the late 1980s, including the hammerhead ribozyme. In 1994, McKay et al. published the structure of a 'hammerhead RNA-DNA ribozyme-inhibitor complex' at 2.6 Ångström resolution, in which the autocatalytic activity of the ribozyme was disrupted via binding to a DNA substrate. The conformation of the ribozyme published in this paper was eventually shown to be one of several possible states, and although this particular sample was catalytically inactive, subsequent structures have revealed its active-state architecture. This structure was followed by Doudna's publication of the structure of the P4-P6 domains of the Tetrahymena group I intron, a fragment of the ribozyme originally made famous by Cech. The second clause in the title of this publication - Principles of RNA Packing - concisely evinces the value of these two structures: for the first time, comparisons could be made between well described tRNA structures and those of globular RNAs outside the transfer family. This allowed the framework of categorization to be built for RNA tertiary structure. It was now possible to propose the conservation of motifs, folds, and various local stabilizing interactions. For an early review of these structures and their implications, see RNA FOLDS: Insights from recent crystal structures, by Doudna and Ferre-D'Amare.
In addition to the advances being made in global structure determination via crystallography, the early 1990s also saw the implementation of NMR as a powerful technique in RNA structural biology. Coincident with the large-scale ribozyme structures being solved crystallographically, a number of structures of small RNAs and RNAs complexed with drugs and peptides were solved using NMR. In addition, NMR was now being used to investigate and supplement crystal structures, as exemplified by the determination of an isolated tetraloop-receptor motif structure published in 1997. Investigations such as this enabled a more precise characterization of the base pairing and base stacking interactions which stabilized the global folds of large RNA molecules. The importance of understanding RNA tertiary structural motifs was prophetically well described by Michel and Costa in their publication identifying the tetraloop motif: "..it should not come as a surprise if self-folding RNA molecules were to make intensive use of only a relatively small set of tertiary motifs. Identifying these motifs would greatly aid modeling enterprises, which will remain essential as long as the crystallization of large RNAs remains a difficult task".
, Venkatraman Ramakrishnan and Thomas Steitz for their structural work on the ribosome, demonstrating the prominent role RNA structural biology has taken in modern molecular biology.
and others. Members of this class (called the "albuminoids", Eiweisskörper, or matières albuminoides) were recognized by their ability to coagulate or flocculate
under various treatments such as heat or acid; well-known examples at the start of the nineteenth century included albumen from egg white
s, blood
serum albumin
, fibrin
, and wheat
gluten
. The similarity between the cooking of egg whites and the curdling of milk was recognized even in ancient times; for example, the name albumen for the egg-white protein was coined by Pliny the Elder
from the Latin albus ovi (egg white).
With the advice of Jöns Jakob Berzelius
, the Dutch chemist Gerhardus Johannes Mulder carried out elemental analyses
of common animal and plant proteins. To everyone's surprise, all proteins had nearly the same empirical formula
, roughly C400H620N100O120 with individual sulfur and phosphorus atoms. Mulder published his findings in two papers (1837,1838) and hypothesized that there was one basic substance (Grundstoff) of proteins, and that it was synthesized by plants and absorbed from them by animals in digestion. Berzelius was an early proponent of this theory and proposed the name "protein" for this substance in a letter dated 10 July 1838
Mulder went on to identify the products of protein degradation such as the amino acid
, leucine
, for which he found a (nearly correct) molecular weight of 131 Da
.
, hundreds of times larger than other molecules being studied. Hence, the chemical structure of proteins (their primary structure
) was an active area of research until 1949, when Fred Sanger sequenced insulin
. The (correct) theory that proteins were linear polymers of amino acid
s linked by peptide bond
s was proposed independently and simultaneously by Franz Hofmeister
and Emil Fischer
at the same conference in 1902. However, some scientists were sceptical that such long macromolecule
s could be stable in solution. Consequently, numerous alternative theories of the protein primary structure
were proposed, e.g., the colloidal hypothesis that proteins were assemblies of small molecules, the cyclol
hypothesis of Dorothy Wrinch
, the diketopiperazine hypothesis of Emil Abderhalden
and the pyrrol/piperidine hypothesis of Troensgard (1942). Most of these theories had difficulties in accounting for the fact that the digestion of proteins yielded peptide
s and amino acid
s.
Proteins were finally shown to be macromolecules of well-defined composition (and not colloidal mixtures) by Theodor Svedberg
using analytical ultracentrifugation. The possibility that some proteins are non-covalent associations of such macromolecules was shown by Gilbert Smithson Adair
(by measuring the osmotic pressure
of hemoglobin
) and, later, by Frederic M. Richards
in his studies of ribonuclease S. The mass spectrometry
of proteins has long been a useful technique for identifying posttranslational modification
s and, more recently, for probing protein structure.
Most proteins are difficult to purify
in more than milligram quantities, even using the most modern methods. Hence, early studies focused on proteins that could be purified in large quantities, e.g., those of blood
, egg white
, various toxin
s, and digestive/metabolic enzymes obtained from slaughterhouse
s. Many techniques of protein purification were developed during World War II
in a project led by Edwin Joseph Cohn
to purify blood proteins to help keep soldiers alive. In the late 1950s, the Armour Hot Dog Co.
purified 1 kg (= one million milligrams) of pure bovine pancreatic ribonuclease A
and made it freely available to scientists around the world. This generous act made RNase A the main protein for basic research for the next few decades, resulting in several Nobel Prizes.
of a protein was composed of two distinct processes: the precipitation
of a protein from solution was preceded by another process called denaturation
, in which the protein became much less soluble, lost its enzymatic activity and became more chemically reactive. In the mid-1920s, Tim Anson
and Alfred Mirsky
proposed that denaturation was a reversible process, a correct hypothesis that was initially lampooned by some scientists as "unboiling the egg". Anson also suggested that denaturation was a two-state ("all-or-none") process, in which one fundamental molecular transition resulted in the drastic changes in solubility, enzymatic activity and chemical reactivity; he further noted that the free energy changes upon denaturation were much smaller than those typically involved in chemical reactions. In 1929, Hsien Wu
hypothesized that denaturation was protein unfolding, a purely conformational change that resulted in the exposure of amino acid side chains to the solvent. According to this (correct) hypothesis, exposure of aliphatic and reactive side chains to solvent rendered the protein less soluble and more reactive, whereas the loss of a specific conformation caused the loss of enzymatic activity. Although considered plausible, Wu's hypothesis was not immediately accepted, since so little was known of protein structure and enzymology and other factors could account for the changes in solubility, enzymatic activity and chemical reactivity. In the early 1960s, Chris Anfinsen
showed that the folding of ribonuclease A
was fully reversible with no external cofactors needed, verifying the "thermodynamic hypothesis" of protein folding that the folded state represents the global minimum of free energy
for the protein.
The hypothesis of protein folding was followed by research into the physical interactions that stabilize folded protein structures. The crucial role of hydrophobic interactions was hypothesized by Dorothy Wrinch
and Irving Langmuir
, as a mechanism that might stabilize her cyclol
structures. Although supported by J. D. Bernal
and others, this (correct) hypothesis was rejected along with the cyclol hypothesis, which was disproven in the 1930s by Linus Pauling
(among others). Instead, Pauling championed the idea that protein structure was stabilized mainly by hydrogen bond
s, an idea advanced initially by William Astbury
(1933). Remarkably, Pauling's incorrect theory about H-bonds resulted in his correct models for the secondary structure
elements of proteins, the alpha helix
and the beta sheet
. The hydrophobic interaction was restored to its correct prominence by a famous article in 1959 by Walter Kauzmann
on denaturation
, based partly on work by Kaj Linderstrøm-Lang. The ionic nature of proteins was demonstrated by Bjerrum, Weber and Arne Tiselius
, but Linderstrom-Lang showed that the charges were generally accessible to solvent and not bound to each other (1949).
The secondary
and low-resolution tertiary structure
of globular proteins was investigated initially by hydrodynamic methods, such as analytical ultracentrifugation and flow birefringence
. Spectroscopic methods to probe protein structure (such as circular dichroism
, fluorescence, near-ultraviolet and infrared absorbance) were developed in the 1950s. The first atomic-resolution structures of proteins were solved by X-ray crystallography
in the 1960s and by NMR
in the 1980s. , the Protein Data Bank
has nearly 40,000 atomic-resolution structures of proteins. In more recent times, cryo-electron microscopy
of large macromolecular assemblies and computational protein structure prediction
of small protein domains are two methods approaching atomic resolution.
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
, genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
, microbiology
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...
, and virology
Virology
Virology is the study of viruses and virus-like agents: their structure, classification and evolution, their ways to infect and exploit cells for virus reproduction, the diseases they cause, the techniques to isolate and culture them, and their use in research and therapy...
. With the hope of understanding life at its most fundamental level, numerous physicists and chemists also took an interest in what would become molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
.
In its modern sense, molecular biology attempts to explain the phenomena of life
Life
Life is a characteristic that distinguishes objects that have signaling and self-sustaining processes from those that do not, either because such functions have ceased , or else because they lack such functions and are classified as inanimate...
starting from the macromolecular properties that generate them. Two categories of macromolecules in particular are the focus of the molecular biologist: 1) nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s, among which the most famous is deoxyribonucleic acid (or DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
), the constituent of gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s, and 2) protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s, which are the active agents of living organisms. One definition of the scope of molecular biology therefore is to characterize the structure, function and relationships between these two types of macromolecules. This relatively limited definition will suffice to allow us to establish a date for the so-called "molecular revolution", or at least to establish a chronology of its most fundamental developments.
General overview
In its earliest manifestations, molecular biology—the name was coined by Warren WeaverWarren Weaver
Warren Weaver was an American scientist, mathematician, and science administrator...
of the Rockefeller Foundation
Rockefeller Foundation
The Rockefeller Foundation is a prominent philanthropic organization and private foundation based at 420 Fifth Avenue, New York City. The preeminent institution established by the six-generation Rockefeller family, it was founded by John D. Rockefeller , along with his son John D. Rockefeller, Jr...
in 1938—was an idea of physical and chemical explanations of life, rather than a coherent discipline. Following the advent of the Mendelian-chromosome theory of heredity
Mendelian inheritance
Mendelian inheritance is a scientific description of how hereditary characteristics are passed from parent organisms to their offspring; it underlies much of genetics...
in the 1910s and the maturation of atomic theory
Atomic theory
In chemistry and physics, atomic theory is a theory of the nature of matter, which states that matter is composed of discrete units called atoms, as opposed to the obsolete notion that matter could be divided into any arbitrarily small quantity...
and quantum mechanics
Quantum mechanics
Quantum mechanics, also known as quantum physics or quantum theory, is a branch of physics providing a mathematical description of much of the dual particle-like and wave-like behavior and interactions of energy and matter. It departs from classical mechanics primarily at the atomic and subatomic...
in the 1920s, such explanations seemed within reach. Weaver and others encouraged (and funded) research at the intersection of biology, chemistry and physics, while prominent physicists such as Niels Bohr
Niels Bohr
Niels Henrik David Bohr was a Danish physicist who made foundational contributions to understanding atomic structure and quantum mechanics, for which he received the Nobel Prize in Physics in 1922. Bohr mentored and collaborated with many of the top physicists of the century at his institute in...
and Erwin Schrödinger
Erwin Schrödinger
Erwin Rudolf Josef Alexander Schrödinger was an Austrian physicist and theoretical biologist who was one of the fathers of quantum mechanics, and is famed for a number of important contributions to physics, especially the Schrödinger equation, for which he received the Nobel Prize in Physics in 1933...
turned their attention to biological speculation. However, in the 1930s and 1940s it was by no means clear which—if any—cross-disciplinary research would bear fruit; work in colloid chemistry, biophysics
Biophysics
Biophysics is an interdisciplinary science that uses the methods of physical science to study biological systems. Studies included under the branches of biophysics span all levels of biological organization, from the molecular scale to whole organisms and ecosystems...
and radiation biology, crystallography
Crystallography
Crystallography is the experimental science of the arrangement of atoms in solids. The word "crystallography" derives from the Greek words crystallon = cold drop / frozen drop, with its meaning extending to all solids with some degree of transparency, and grapho = write.Before the development of...
, and other emerging fields all seemed promising.
In 1940, George Beadle and Edward Tatum demonstrated the existence of a precise relationship between genes and proteins. In the course of their experiments connecting genetics with biochemistry, they switched from the genetics mainstay Drosophila
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
to a more appropriate model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
, the fungus Neurospora
Neurospora crassa
Neurospora crassa is a type of red bread mold of the phylum Ascomycota. The genus name, meaning "nerve spore" refers to the characteristic striations on the spores. The first published account of this fungus was from an infestation of French bakeries in 1843. N...
; the construction and exploitation of new model organisms would become a recurring theme in the development of molecular biology. In 1944, Oswald Avery
Oswald Avery
Oswald Theodore Avery ForMemRS was a Canadian-born American physician and medical researcher. The major part of his career was spent at the Rockefeller University Hospital in New York City...
, working at the Rockefeller Institute of New York
Rockefeller University
The Rockefeller University is a private university offering postgraduate and postdoctoral education. It has a strong concentration in the biological sciences. It is also known for producing numerous Nobel laureates...
, demonstrated that genes are made up of DNA(see Avery-MacLeod-McCarty experiment
Avery-MacLeod-McCarty experiment
The Avery–MacLeod–McCarty experiment was an experimental demonstration, reported in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, that DNA is the substance that causes bacterial transformation...
). In 1952, Alfred Hershey
Alfred Hershey
Alfred Day Hershey was an American Nobel Prize-winning bacteriologist and geneticist.He was born in Owosso, Michigan and received his B.S. in chemistry at Michigan State University in 1930 and his Ph.D. in bacteriology in 1934, taking a position shortly thereafter at the Department of Bacteriology...
and Martha Chase
Martha Chase
Martha Cowles Chase , also known as Martha C. Epstein, was an American geneticist famously known for being a member of the 1952 team which experimentally showed that DNA rather than protein is the genetic material of life. She was greatly respected as a geneticist. Chase was born in 1927 in...
confirmed that the genetic material of the bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
, the virus which infects bacteria, is made up of DNA (see Hershey-Chase experiment
Hershey-Chase experiment
The Hershey–Chase experiments were a series of experiments conducted in 1952 by Alfred Hershey and Martha Chase, which helped to confirm that DNA was the genetic material. While DNA had been known to biologists since 1869, a few scientists still assumed at the time that proteins carried the...
). In 1953, James Watson
James D. Watson
James Dewey Watson is an American molecular biologist, geneticist, and zoologist, best known as one of the co-discoverers of the structure of DNA in 1953 with Francis Crick...
and Francis Crick
Francis Crick
Francis Harry Compton Crick OM FRS was an English molecular biologist, biophysicist, and neuroscientist, and most noted for being one of two co-discoverers of the structure of the DNA molecule in 1953, together with James D. Watson...
discovered the double helical structure of the DNA molecule. In 1961, Francois Jacob
François Jacob
François Jacob is a French biologist who, together with Jacques Monod, originated the idea that control of enzyme levels in all cells occurs through feedback on transcription. He shared the 1965 Nobel Prize in Medicine with Jacques Monod and André Lwoff.-Childhood and education:François Jacob is...
and Jacques Monod
Jacques Monod
Jacques Lucien Monod was a French biologist who was awarded a Nobel Prize in Physiology or Medicine in 1965, sharing it with François Jacob and Andre Lwoff "for their discoveries concerning genetic control of enzyme and virus synthesis"...
hypothesized the existence of an intermediary between DNA and its protein products, which they called messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
. Between 1961 and 1965, the relationship between the information contained in DNA and the structure of proteins was determined: there is a code, the genetic code
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
, which creates a correspondence between the succession of nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s in the DNA sequence and a series of amino acids in proteins. At the beginning of the 1960s, Monod and Jacob also demonstrated how certain specific proteins, called regulative proteins
Regulation of gene expression
Gene modulation redirects here. For information on therapeutic regulation of gene expression, see therapeutic gene modulation.Regulation of gene expression includes the processes that cells and viruses use to regulate the way that the information in genes is turned into gene products...
, latch onto DNA at the edges of the genes and control the transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of these genes into messenger RNA; they direct the "expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
" of the genes.
The chief discoveries of molecular biology took place in a period of only about twenty-five years. Another fifteen years were required before new and more sophisticated technologies, united today under the name of genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
, would permit the isolation and characterization of genes, in particular those of highly complex organisms.
The exploration of the molecular dominion
If we evaluate the molecular revolution within the context of biological history, it is easy to note that it is the culmination of a long process which began with the first observations through a microscope. The aim of these early researchers was to understand the functioning of living organisms by describing their organization at the microscopic level. From the end of the 18th century, the characterization of the chemical molecules which make up living beings gained increasingly greater attention, along with the birth of physiological chemistryPhysiological chemistry
Physiological chemistry is the chemistry of the organs and tissues of the body and of the various physiological processes incident to life. Physiological chemistry is essentially the precursor to modern biochemistry. In the nineteenth century, physiological chemistry dealt primarily with...
in the 19th century, developed by the German chemist Justus von Liebig
Justus von Liebig
Justus von Liebig was a German chemist who made major contributions to agricultural and biological chemistry, and worked on the organization of organic chemistry. As a professor, he devised the modern laboratory-oriented teaching method, and for such innovations, he is regarded as one of the...
and following the birth of biochemistry at the beginning of the 20th, thanks to another German chemist Eduard Buchner
Eduard Buchner
Eduard Buchner was a German chemist and zymologist, awarded with the 1907 Nobel Prize in Chemistry thanks to his work on fermentation.-Early years:...
. Between the molecules studied by chemists and the tiny structures visible under the optical microscope, such as the cellular nucleus or the chromosomes, there was an obscure zone, "the world of the ignored dimensions," as it was called by the chemical-physicist Wolfgang Ostwald
Wolfgang Ostwald
Carl Wilhelm Wolfgang Ostwald was a German chemist and biologist researching colloids.Ostwald was born in Riga, the son of the 1909 winner of the Nobel Prize in Chemistry, Wilhelm Ostwald, and died in Dresden.-Works:...
. This world is populated by colloids, chemical compounds whose structure and properties were not well defined.
The successes of molecular biology derived from the exploration of that unknown world by means of the new technologies developed by chemists and physicists: X-ray diffraction, electron microscopy, ultracentrifugization
Ultracentrifuge
The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 2,000,000 g . There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge...
, and electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
. These studies revealed the structure and function of the macromolecules.
A milestone in that process was the work of Dr. Linus Pauling in 1949, which for the first time linked the specific genetic mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
in patients with sickle cell disease to a demonstrated change in an individual protein, the hemoglobin
Hemoglobin
Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...
in the erythrocytes of heterozygous or homozygous individuals.
The encounter between biochemistry and genetics
The development of molecular biology is also the encounter of two disciplines which made considerable progress in the course of the first thirty years of the twentieth century: biochemistry and genetics. The first studies the structure and function of the molecules which make up living things. Between 1900 and 1940, the central processes of metabolismMetabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
were described: the process of digestion
Digestion
Digestion is the mechanical and chemical breakdown of food into smaller components that are more easily absorbed into a blood stream, for instance. Digestion is a form of catabolism: a breakdown of large food molecules to smaller ones....
and the absorption of the nutritive elements derived from alimentation, such as the sugars. Every one of these processes is catalyzed
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
by a particular enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
. Enzymes are proteins, like the antibodies present in blood or the proteins responsible for muscular contraction. As a consequence, the study of proteins, of their structure and synthesis, became one of the principal objectives of biochemists.
The second discipline of biology which developed at the beginning of the 20th century is genetics. After the rediscovery of the laws of Mendel
Gregor Mendel
Gregor Johann Mendel was an Austrian scientist and Augustinian friar who gained posthumous fame as the founder of the new science of genetics. Mendel demonstrated that the inheritance of certain traits in pea plants follows particular patterns, now referred to as the laws of Mendelian inheritance...
through the studies of Hugo de Vries
Hugo de Vries
Hugo Marie de Vries ForMemRS was a Dutch botanist and one of the first geneticists. He is known chiefly for suggesting the concept of genes, rediscovering the laws of heredity in the 1890s while unaware of Gregor Mendel's work, for introducing the term "mutation", and for developing a mutation...
, Carl Correns
Carl Correns
Carl Erich Correns was a German botanist and geneticist, who is notable primarily for his independent discovery of the principles of heredity, and for his rediscovery of Gregor Mendel's earlier paper on that subject, which he achieved simultaneously but independently of the botanists Erich...
and Erich von Tschermak
Erich von Tschermak
Erich von Tschermak-Seysenegg was an Austrian agronomist who developed several new disease-resistant crops, including wheat-rye and oat hybrids. He was a son of the Moravia-born mineralogist Gustav Tschermak von Seysenegg...
in 1900, this science began to take shape thanks to the adoption by Thomas Hunt Morgan
Thomas Hunt Morgan
Thomas Hunt Morgan was an American evolutionary biologist, geneticist and embryologist and science author who won the Nobel Prize in Physiology or Medicine in 1933 for discoveries relating the role the chromosome plays in heredity.Morgan received his PhD from Johns Hopkins University in zoology...
, in 1910, of a model organism for genetic studies, the famous fruit fly (Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
). Shortly after, Morgan showed that the genes are localized on chromosomes. Following this discovery, he continued working with Drosophila and, along with numerous other research groups, confirmed the importance of the gene in the life and development of organisms. Nevertheless, the chemical nature of genes and their mechanisms of action remained a mystery. Molecular biologists committed themselves to the determination of the structure, and the description of the complex relations between, genes and proteins.
The development of molecular biology was not just the fruit of some sort of intrinsic "necessity" in the history of ideas, but was a characteristically historical phenomenon, with all of its unknowns, imponderables and contingencies: the remarkable developments in physics at the beginning of the 20th century highlighted the relative lateness in development in biology, which became the "new frontier" in the search for knowledge about the empirical world. Moreover, the developments of the theory of information
Information theory
Information theory is a branch of applied mathematics and electrical engineering involving the quantification of information. Information theory was developed by Claude E. Shannon to find fundamental limits on signal processing operations such as compressing data and on reliably storing and...
and cybernetics
Cybernetics
Cybernetics is the interdisciplinary study of the structure of regulatory systems. Cybernetics is closely related to information theory, control theory and systems theory, at least in its first-order form...
in the 1940s, in response to military exigencies, brought to the new biology a significant number of fertile ideas and, especially, metaphors.
The choice of bacteria and of its virus, the bacteriophage, as models for the study of the fundamental mechanisms of life was almost natural - they are the smallest living organisms known to exist - and at the same time the fruit of individual choices. This model owes its success, above all, to the fame and the sense of organization of Max Delbrück
Max Delbrück
Max Ludwig Henning Delbrück was a German-American biophysicist and Nobel laureate.-Biography:Delbrück was born in Berlin, German Empire...
, a German physicist, who was able to create a dynamic research group, based in the United States, whose exclusive scope was the study of the bacteriophage: the School of the Phage.
The geographic panorama of the developments of the new biology was conditioned above all by preceding work. The US, where genetics had developed the most rapidly, and the UK, where there was a coexistence of both genetics and biochemical research of highly advanced levels, were in the avant-garde. Germany, the cradle of the revolutions in physics, with the best minds and the most advanced laboratories of genetics in the world, should have had a primary role in the development of molecular biology. But history decided differently: the arrival of the Nazis
Nazism
Nazism, the common short form name of National Socialism was the ideology and practice of the Nazi Party and of Nazi Germany...
in 1933 - and, to a less extreme degree, the rigidification of totalitarian measures in fascist
Fascism
Fascism is a radical authoritarian nationalist political ideology. Fascists seek to rejuvenate their nation based on commitment to the national community as an organic entity, in which individuals are bound together in national identity by suprapersonal connections of ancestry, culture, and blood...
Italy - caused the emigration of a large number of Jewish and non-Jewish scientists. The majority of them fled to the US or the UK, providing an extra impulse to the scientific dynamism of those nations. These movements ultimately made molecular biology a truly international science from the very beginnings.
History of DNA biochemistry
The study of DNA is a central part of molecular biology.First isolation of DNA
Working in the 19th century, biochemists initially isolated DNA and RNA (mixed together) from cell nuclei. They were relatively quick to appreciate the polymeric nature of their "nucleic acid" isolates, but realized only later that nucleotides were of two types—one containing riboseRibose
Ribose is an organic compound with the formula C5H10O5; specifically, a monosaccharide with linear form H––4–H, which has all the hydroxyl groups on the same side in the Fischer projection....
and the other deoxyribose
Deoxyribose
Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...
. It was this subsequent discovery that led to the identification and naming of DNA as a substance distinct from RNA.
Friedrich Miescher
Friedrich Miescher
Johannes Friedrich Miescher was a Swiss physician and biologist. He was the first researcher to isolate and identify nucleic acid.-Biography:...
(1844–1895) discovered a substance he called "nuclein" in 1869. Somewhat later, he isolated a pure sample of the material now known as DNA from the sperm of salmon, and in 1889 his pupil, Richard Altmann
Richard Altmann
Richard Altmann was a German pathologist and histologist from Deutsch Eylau in the Province of Prussia. He studied medicine in Greifswald, Königsberg, Marburg, and Giessen, and earned his doctorate at the University of Giessen in 1877...
, named it "nucleic acid". This substance was found to exist only in the chromosomes.
In 1919 Phoebus Levene
Phoebus Levene
Phoebus Aaron Theodore Levene, M.D. was a Russian-American biochemist who studied the structure and function of nucleic acids...
at the Rockefeller Institute identified the components (the four bases, the sugar and the phosphate chain) and he showed that the components of DNA were linked in the order phosphate-sugar-base. He called each of these units a nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
and suggested the DNA molecule consisted of a string of nucleotide units linked together through the phosphate groups, which are the 'backbone' of the molecule. However Levene thought the chain was short and that the bases repeated in the same fixed order. Torbjorn Caspersson and Einar Hammersten showed that DNA was a polymer.
Chromosomes and inherited traits
In 1927 Nikolai KoltsovNikolai Koltsov
Nikolai Konstantinovich Koltsov was a Russian biologist. He was one of the creators of modern genetics. Nikolai Koltsov was a teacher of Nikolay Timofeeff-Ressovsky.-Scientific career:...
proposed that inherited traits would be inherited via a "giant hereditary molecule" which would be made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template". Max Delbrück
Max Delbrück
Max Ludwig Henning Delbrück was a German-American biophysicist and Nobel laureate.-Biography:Delbrück was born in Berlin, German Empire...
, Nikolai V. Timofeeff-Ressovsky, and Karl G. Zimmer published results in 1935 suggesting that chromosomes are very large molecules the structure of which can be changed by treatment with X-ray
X-ray
X-radiation is a form of electromagnetic radiation. X-rays have a wavelength in the range of 0.01 to 10 nanometers, corresponding to frequencies in the range 30 petahertz to 30 exahertz and energies in the range 120 eV to 120 keV. They are shorter in wavelength than UV rays and longer than gamma...
s, and that by so changing their structure it was possible to change the heritable characteristics governed by those chromosomes. In 1937 William Astbury
William Astbury
William Thomas Astbury FRS was an English physicist and molecular biologist who made pioneering X-ray diffraction studies of biological molecules. His work on keratin provided the foundation for Linus Pauling's discovery of the alpha helix...
produced the first X-ray diffraction patterns from DNA. He was not able to propose the correct structure but the patterns showed that DNA had a regular structure and therefore it might be possible to deduce what this structure was.
In 1943, Oswald Theodore Avery and a team of scientists discovered that traits proper to the "smooth" form of the Pneumococcus could be transferred to the "rough" form of the same bacteria merely by making the killed "smooth" (S) form available to the live "rough" (R) form. Quite unexpectedly, the living R Pneumococcus bacteria were transformed into a new strain of the S form, and the transferred S characteristics turned out to be heritable. Avery called the medium of transfer of traits the transforming principle; he identified DNA as the transforming principle, and not protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
as previously thought. He essentially redid Frederick Griffith
Frederick Griffith
Frederick Griffith was a British bacteriologist whose focus was the epidemiology and pathology of bacterial pneumonia. In January 1928 he reported what is now known as Griffith's Experiment, the first widely accepted demonstrations of bacterial transformation, whereby a bacterium distinctly...
's experiment. In 1953, Alfred Hershey
Alfred Hershey
Alfred Day Hershey was an American Nobel Prize-winning bacteriologist and geneticist.He was born in Owosso, Michigan and received his B.S. in chemistry at Michigan State University in 1930 and his Ph.D. in bacteriology in 1934, taking a position shortly thereafter at the Department of Bacteriology...
and Martha Chase
Martha Chase
Martha Cowles Chase , also known as Martha C. Epstein, was an American geneticist famously known for being a member of the 1952 team which experimentally showed that DNA rather than protein is the genetic material of life. She was greatly respected as a geneticist. Chase was born in 1927 in...
did an experiment (Hershey-Chase experiment
Hershey-Chase experiment
The Hershey–Chase experiments were a series of experiments conducted in 1952 by Alfred Hershey and Martha Chase, which helped to confirm that DNA was the genetic material. While DNA had been known to biologists since 1869, a few scientists still assumed at the time that proteins carried the...
) that showed, in T2 phage, that DNA is the genetic material (Hershey shared the Nobel prize with Luria).
Discovery of the structure of DNA
In the 1950s, three groups made it their goal to determine the structure of DNA. The first group to start was at King's College LondonKing's College London
King's College London is a public research university located in London, United Kingdom and a constituent college of the federal University of London. King's has a claim to being the third oldest university in England, having been founded by King George IV and the Duke of Wellington in 1829, and...
and was led by Maurice Wilkins
Maurice Wilkins
Maurice Hugh Frederick Wilkins CBE FRS was a New Zealand-born English physicist and molecular biologist, and Nobel Laureate whose research contributed to the scientific understanding of phosphorescence, isotope separation, optical microscopy and X-ray diffraction, and to the development of radar...
and was later joined by Rosalind Franklin
Rosalind Franklin
Rosalind Elsie Franklin was a British biophysicist and X-ray crystallographer who made critical contributions to the understanding of the fine molecular structures of DNA, RNA, viruses, coal and graphite...
. Another group consisting of Francis Crick
Francis Crick
Francis Harry Compton Crick OM FRS was an English molecular biologist, biophysicist, and neuroscientist, and most noted for being one of two co-discoverers of the structure of the DNA molecule in 1953, together with James D. Watson...
and James D. Watson
James D. Watson
James Dewey Watson is an American molecular biologist, geneticist, and zoologist, best known as one of the co-discoverers of the structure of DNA in 1953 with Francis Crick...
was at Cambridge
University of Cambridge
The University of Cambridge is a public research university located in Cambridge, United Kingdom. It is the second-oldest university in both the United Kingdom and the English-speaking world , and the seventh-oldest globally...
. A third group was at Caltech and was led by Linus Pauling
Linus Pauling
Linus Carl Pauling was an American chemist, biochemist, peace activist, author, and educator. He was one of the most influential chemists in history and ranks among the most important scientists of the 20th century...
. Crick and Watson built physical models using metal rods and balls, in which they incorporated the known chemical structures of the nucleotides, as well as the known position of the linkages joining one nucleotide to the next along the polymer. At King's College Maurice Wilkins and Rosalind Franklin examined X-ray diffraction
Crystallography
Crystallography is the experimental science of the arrangement of atoms in solids. The word "crystallography" derives from the Greek words crystallon = cold drop / frozen drop, with its meaning extending to all solids with some degree of transparency, and grapho = write.Before the development of...
patterns of DNA fibers. Of the three groups, only the London group was able to produce good quality diffraction patterns and thus produce sufficient quantitative data about the structure.
Helix structure
In 1948 Pauling discovered that many proteins included helical (see alpha helixAlpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
) shapes. Pauling had deduced this structure from X-ray patterns and from attempts to physically model the structures. (Pauling was also later to suggest an incorrect three chain helical DNA structure based on Astbury's data.) Even in the initial diffraction data from DNA by Maurice Wilkins, it was evident that the structure involved helices. But this insight was only a beginning. There remained the questions of how many strands came together, whether this number was the same for every helix, whether the bases pointed toward the helical axis or away, and ultimately what were the explicit angles and coordinates of all the bonds and atoms. Such questions motivated the modeling efforts of Watson and Crick.
Complementary nucleotides
In their modeling, Watson and Crick restricted themselves to what they saw as chemically and biologically reasonable. Still, the breadth of possibilities was very wide. A breakthrough occurred in 1952, when Erwin ChargaffErwin Chargaff
Erwin Chargaff was an American biochemist who emigrated to the United States during the Nazi era. Through careful experimentation, Chargaff discovered two rules that helped lead to the discovery of the double helix structure of DNA...
visited Cambridge and inspired Crick with a description of experiments Chargaff had published in 1947. Chargaff had observed that the proportions of the four nucleotides vary between one DNA sample and the next, but that for particular pairs of nucleotides — adenine and thymine, guanine and cytosine — the two nucleotides are always present in equal proportions.
Rosalind Franklin
Rosalind Elsie Franklin was a British biophysicist and X-ray crystallographer who made critical contributions to the understanding of the fine molecular structures of DNA, RNA, viruses, coal and graphite...
and her information that the bases were paired, James D. Watson
James D. Watson
James Dewey Watson is an American molecular biologist, geneticist, and zoologist, best known as one of the co-discoverers of the structure of DNA in 1953 with Francis Crick...
and Francis Crick
Francis Crick
Francis Harry Compton Crick OM FRS was an English molecular biologist, biophysicist, and neuroscientist, and most noted for being one of two co-discoverers of the structure of the DNA molecule in 1953, together with James D. Watson...
arrived at the first accurate model of DNA's molecular structure in 1953, which was accepted through inspection by Rosalind Franklin. The discovery was announced on February 28, 1953; the first Watson/Crick paper appeared in Nature
Nature
Nature, in the broadest sense, is equivalent to the natural world, physical world, or material world. "Nature" refers to the phenomena of the physical world, and also to life in general...
on April 25, 1953. Sir Lawrence Bragg, the director of the Cavendish Laboratory
Cavendish Laboratory
The Cavendish Laboratory is the Department of Physics at the University of Cambridge, and is part of the university's School of Physical Sciences. It was opened in 1874 as a teaching laboratory....
, where Watson and Crick worked, gave a talk at Guys Hospital Medical School in London on Thursday, May 14, 1953 which resulted in an article by Ritchie Calder in The News Chronicle
News Chronicle
The News Chronicle was a British daily newspaper. It ceased publication on 17 October 1960, being absorbed into the Daily Mail. Its offices were in Bouverie Street, off Fleet Street, London, EC4Y 8DP, England.-Daily Chronicle:...
of London, on Friday, May 15, 1953, entitled "Why You Are You. Nearer Secret of Life." The news reached readers of The New York Times
The New York Times
The New York Times is an American daily newspaper founded and continuously published in New York City since 1851. The New York Times has won 106 Pulitzer Prizes, the most of any news organization...
the next day; Victor K. McElheny, in researching his biography, "Watson and DNA: Making a Scientific Revolution", found a clipping of a six-paragraph New York Times article written from London and dated May 16, 1953 with the headline "Form of `Life Unit' in Cell Is Scanned." The article ran in an early edition and was then pulled to make space for news deemed more important.(The New York Times subsequently ran a longer article on June 12, 1953). The Cambridge University undergraduate newspaper
Varsity (Cambridge)
Varsity is the oldest of Cambridge University's main student newspapers. It has been published continuously since 1947, and is one of only three fully independent student newspapers in the UK. It appears every Friday around Cambridge...
also ran its own short article on the discovery on Saturday, May 30, 1953. Bragg's original announcement at a Solvay conference
Solvay Conference
The International Solvay Institutes for Physics and Chemistry, located in Brussels, were founded by the Belgian industrialist Ernest Solvay in 1912, following the historic invitation-only 1911 Conseil Solvay, the turning point in world physics...
on proteins in Belgium
Belgium
Belgium , officially the Kingdom of Belgium, is a federal state in Western Europe. It is a founding member of the European Union and hosts the EU's headquarters, and those of several other major international organisations such as NATO.Belgium is also a member of, or affiliated to, many...
on 8 April 1953 went unreported by the press. In 1962 Watson, Crick, and Maurice Wilkins
Maurice Wilkins
Maurice Hugh Frederick Wilkins CBE FRS was a New Zealand-born English physicist and molecular biologist, and Nobel Laureate whose research contributed to the scientific understanding of phosphorescence, isotope separation, optical microscopy and X-ray diffraction, and to the development of radar...
jointly received the Nobel Prize
Nobel Prize
The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...
for Physiology or Medicine for their determination of the structure of DNA.
"Central Dogma"
Watson and Crick's model attracted great interest immediately upon its presentation. Arriving at their conclusion on February 21, 1953, Watson and Crick made their first announcement on February 28. In an influential presentation in 1957, Crick laid out the "Central Dogma", which foretold the relationship between DNA, RNA, and proteins, and articulated the "sequence hypothesis." A critical confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 in the form of the Meselson-Stahl experimentMeselson-Stahl experiment
The Meselson–Stahl experiment was an experiment by Matthew Meselson and Franklin Stahl in 1958 which supported the hypothesis that DNA replication was semiconservative. Semiconservative replication means that when the double stranded DNA helix was replicated, each of the two double stranded DNA...
. Work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons, and Har Gobind Khorana and others deciphered the genetic code
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
not long afterward (1966). These findings represent the birth of molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
.
Pre-history: the helical structure of RNA
The earliest work in RNA structural biology coincided, more or less, with the work being done on DNA in the early 1950s. In their seminal 1953 paper, Watson and Crick suggested that van der Waals crowding by the 2`OH group of ribose would preclude RNA from adopting a double helical structure identical to the model they proposed - what we now know as B-form DNA. This provoked questions about the three dimensional structure of RNA: could this molecule form some type of helical structure, and if so, how? As with DNA, early structural work on RNA centered around isolation of native RNA polymers for fiber diffraction analysis. In part because of heterogeneity of the samples tested, early fiber diffraction patterns were usually ambiguous and not readily interpretable. In 1955, Grunberg-Manago et al. published a paper describing the enzyme polynucleotide phosphorylase, which cleaved a phosphate group from nucleotide diphosphates to catalyze their polymerization. This discovery allowed researchers to synthesize homogenous nucleotide polymers, which they then combined to produce double stranded molecules. These samples yielded the most readily interpretable fiber diffraction patterns yet obtained, suggesting an ordered, helical structure for cognate, double stranded RNA that differed from that observed in DNA. These results paved the way for a series of investigations into the various properties and propensities of RNA. Through the late 1950s and early 1960s, numerous papers were published on various topics in RNA structure, including RNA-DNA hybridization, triple stranded RNA, and even small-scale crystallography of RNA di-nucleotides - G-C, and A-U - in primitive helix-like arrangements. For a more in-depth review of the early work in RNA structural biology, see the article The Era of RNA Awakening: Structural biology of RNA in the early years by Alexander Rich.The beginning: crystal structure of tRNAPHE
In the mid-1960s, the role of tRNA in protein synthesis was being intensively studied. At this point, ribosomes had been implicated in protein synthesis, and it had been shown that an mRNA strand was necessary for the formation of these structures. In a 1964 publication, Warner and Rich showed that ribosomes active in protein synthesis contained tRNA molecules bound at the A and P sites, and discussed the notion that these molecules aided in the peptidyl transferase reaction. However, despite considerable biochemical characterization, the structural basis of tRNA function remained a mystery. In 1965, Holley et al. purified and sequenced the first tRNA molecule, initially proposing that it adopted a cloverleaf structure, based largely on the ability of certain regions of the molecule to form stem loop structures. The isolation of tRNA proved to be the first major windfall in RNA structural biology. Following Holley's publication, numerous investigators began work on isolation tRNA for crystallographic study, developing improved methods for isolating the molecule as they worked. By 1968 several groups had produced tRNA crystals, but these proved to be of limited quality and did not yield data at the resolutions necessary to determine structure. In 1971, Kim et al. achieved another breakthrough, producing crystals of yeast tRNAPHE that diffracted to 2-3 Ångström resolutions by using spermine, a naturally occurring polyamine, which bound to and stabilized the tRNA. Despite having suitable crystals, however, the structure of tRNAPHE was not immediately solved at high resolution; rather it took pioneering work in the use of heavy metal derivatives and a good deal more time to produce a high-quality density map of the entire molecule. In 1973, Kim et al. produced a 4 Ångström map of the tRNA molecule in which they could unambiguously trace the entire backbone. This solution would be followed by many more, as various investigators worked to refine the structure and thereby more thoroughly elucidate the details of base pairing and stacking interactions, and validate the published architecture of the molecule.The tRNAPHE structure is notable in the field of nucleic acid structure in general, as it represented the first solution of a long-chain nucleic acid structure of any kind - RNA or DNA - preceding Dickerson's solution of a B-form dodecamer by nearly a decade. Also, tRNAPHE demonstrated many of the tertiary interactions observed in RNA architecture which would not be categorized and more thoroughly understood for years to come, providing a foundation for all future RNA structural research.
The renaissance: the hammerhead ribozyme and the group I intron: P4-6
For a considerable time following the first tRNA structures, the field of RNA structure did not dramatically advance. The ability to study an RNA strcuture depended upon the potential to isolate the RNA target. This proved limiting to the field for many years, in part owing to the fact that other know targets - i.e. the ribosome - were significantly more difficult to isolate and crystallize. Further, because other interesting RNA targets had simply not been identified, or were not sufficiently understood to be deemed interesting, there was simply a lack of things to study structurally. As such, for some twenty years following the original publication of the tRNAPHE structure, the structures of only a handful of other RNA targets were solved, with almost all of these belonging to the transfer RNA family. This unfortunate lack of scope would eventually be overcome largely because of two major advancements in nucleic acid research: the identification of ribozymes, and the ability to produce them via in vitro transcription.Subsequent to Tom Cech's publication implicating the Tetrahymena group I intron as an autocatalytic ribozyme, and Sidney Altman's report of catalysis by ribonuclease P RNA, several other catalytic RNAs were identified in the late 1980s, including the hammerhead ribozyme. In 1994, McKay et al. published the structure of a 'hammerhead RNA-DNA ribozyme-inhibitor complex' at 2.6 Ångström resolution, in which the autocatalytic activity of the ribozyme was disrupted via binding to a DNA substrate. The conformation of the ribozyme published in this paper was eventually shown to be one of several possible states, and although this particular sample was catalytically inactive, subsequent structures have revealed its active-state architecture. This structure was followed by Doudna's publication of the structure of the P4-P6 domains of the Tetrahymena group I intron, a fragment of the ribozyme originally made famous by Cech. The second clause in the title of this publication - Principles of RNA Packing - concisely evinces the value of these two structures: for the first time, comparisons could be made between well described tRNA structures and those of globular RNAs outside the transfer family. This allowed the framework of categorization to be built for RNA tertiary structure. It was now possible to propose the conservation of motifs, folds, and various local stabilizing interactions. For an early review of these structures and their implications, see RNA FOLDS: Insights from recent crystal structures, by Doudna and Ferre-D'Amare.
In addition to the advances being made in global structure determination via crystallography, the early 1990s also saw the implementation of NMR as a powerful technique in RNA structural biology. Coincident with the large-scale ribozyme structures being solved crystallographically, a number of structures of small RNAs and RNAs complexed with drugs and peptides were solved using NMR. In addition, NMR was now being used to investigate and supplement crystal structures, as exemplified by the determination of an isolated tetraloop-receptor motif structure published in 1997. Investigations such as this enabled a more precise characterization of the base pairing and base stacking interactions which stabilized the global folds of large RNA molecules. The importance of understanding RNA tertiary structural motifs was prophetically well described by Michel and Costa in their publication identifying the tetraloop motif: "..it should not come as a surprise if self-folding RNA molecules were to make intensive use of only a relatively small set of tertiary motifs. Identifying these motifs would greatly aid modeling enterprises, which will remain essential as long as the crystallization of large RNAs remains a difficult task".
The modern era: the age of RNA structural biology
The resurgence of RNA structural biology in the mid-1990s has caused a veritable explosion in the field of nucleic acid structural research. Since the publication of the hammerhead and P4-6 structures, numerous major contributions to the field have been made. Some of the most noteworthy examples include the structures of the Group I and Group II introns, and the Ribosome. It should be noted that the first three structures were produced using in vitro transcription, and that NMR has played a role in investigating partial components of all four structures - testaments to the indispensability of both techniques for RNA research. Most recently, the 2009 Nobel Prize in Chemistry was awarded to Ada YonathAda Yonath
Ada E. Yonath is an Israeli crystallographer best known for her pioneering work on the structure of the ribosome. She is the current director of the Helen and Milton A. Kimmelman Center for Biomolecular Structure and Assembly of the Weizmann Institute of Science. In 2009, she received the Nobel...
, Venkatraman Ramakrishnan and Thomas Steitz for their structural work on the ribosome, demonstrating the prominent role RNA structural biology has taken in modern molecular biology.
First isolation and classification
Proteins were recognized as a distinct class of biological molecules in the eighteenth century by Antoine FourcroyAntoine François, comte de Fourcroy
Antoine François, comte de Fourcroy was a French chemist and a contemporary of Antoine Lavoisier. Fourcroy collaborated with Lavoisier, Guyton de Morveau, and Claude Berthollet on the Méthode de nomenclature chimique, a work that helped standardize chemical nomenclature.-Life and work:Fourcroy...
and others. Members of this class (called the "albuminoids", Eiweisskörper, or matières albuminoides) were recognized by their ability to coagulate or flocculate
Flocculation
Flocculation, in the field of chemistry, is a process wherein colloids come out of suspension in the form of floc or flakes by the addition of a clarifying agent. The action differs from precipitation in that, prior to flocculation, colloids are merely suspended in a liquid and not actually...
under various treatments such as heat or acid; well-known examples at the start of the nineteenth century included albumen from egg white
Egg white
Egg white is the common name for the clear liquid contained within an egg. In chickens it is formed from the layers of secretions of the anterior section of the hen's oviduct during the passage of the egg. It forms around either fertilized or unfertilized egg yolks...
s, blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
serum albumin
Serum albumin
Serum albumin, often referred to simply as albumin is a protein that in humans is encoded by the ALB gene.Serum albumin is the most abundant plasma protein in mammals. Albumin is essential for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular...
, fibrin
Fibrin
Fibrin is a fibrous, non-globular protein involved in the clotting of blood. It is a fibrillar protein that is polymerised to form a "mesh" that forms a hemostatic plug or clot over a wound site....
, and wheat
Wheat
Wheat is a cereal grain, originally from the Levant region of the Near East, but now cultivated worldwide. In 2007 world production of wheat was 607 million tons, making it the third most-produced cereal after maize and rice...
gluten
Gluten
Gluten is a protein composite found in foods processed from wheat and related grain species, including barley and rye...
. The similarity between the cooking of egg whites and the curdling of milk was recognized even in ancient times; for example, the name albumen for the egg-white protein was coined by Pliny the Elder
Pliny the Elder
Gaius Plinius Secundus , better known as Pliny the Elder, was a Roman author, naturalist, and natural philosopher, as well as naval and army commander of the early Roman Empire, and personal friend of the emperor Vespasian...
from the Latin albus ovi (egg white).
With the advice of Jöns Jakob Berzelius
Jöns Jakob Berzelius
Jöns Jacob Berzelius was a Swedish chemist. He worked out the modern technique of chemical formula notation, and is together with John Dalton, Antoine Lavoisier, and Robert Boyle considered a father of modern chemistry...
, the Dutch chemist Gerhardus Johannes Mulder carried out elemental analyses
Elemental analysis
Percent Composition is a process where a sample of some material is analyzed for its elemental and sometimes isotopic composition. Elemental analysis can be qualitative , and it can be quantitative...
of common animal and plant proteins. To everyone's surprise, all proteins had nearly the same empirical formula
Empirical formula
In chemistry, the empirical formula of a chemical compound is the simplest positive integer ratio of atoms of each element present in a compound. An empirical formula makes no reference to isomerism, structure, or absolute number of atoms. The empirical formula is used as standard for most ionic...
, roughly C400H620N100O120 with individual sulfur and phosphorus atoms. Mulder published his findings in two papers (1837,1838) and hypothesized that there was one basic substance (Grundstoff) of proteins, and that it was synthesized by plants and absorbed from them by animals in digestion. Berzelius was an early proponent of this theory and proposed the name "protein" for this substance in a letter dated 10 July 1838
The name protein that I propose for the organic oxide of fibrinFibrinFibrin is a fibrous, non-globular protein involved in the clotting of blood. It is a fibrillar protein that is polymerised to form a "mesh" that forms a hemostatic plug or clot over a wound site....
and albuminAlbuminAlbumin refers generally to any protein that is water soluble, which is moderately soluble in concentrated salt solutions, and experiences heat denaturation. They are commonly found in blood plasma, and are unique to other blood proteins in that they are not glycosylated...
, I wanted to derive from [the GreekGreek languageGreek is an independent branch of the Indo-European family of languages. Native to the southern Balkans, it has the longest documented history of any Indo-European language, spanning 34 centuries of written records. Its writing system has been the Greek alphabet for the majority of its history;...
word] πρωτειος, because it appears to be the primitive or principal substance of animal nutrition.
Mulder went on to identify the products of protein degradation such as the amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
, leucine
Leucine
Leucine is a branched-chain α-amino acid with the chemical formula HO2CCHCH2CH2. Leucine is classified as a hydrophobic amino acid due to its aliphatic isobutyl side chain. It is encoded by six codons and is a major component of the subunits in ferritin, astacin and other 'buffer' proteins...
, for which he found a (nearly correct) molecular weight of 131 Da
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
.
Purifications and measurements of mass
The minimum molecular weight suggested by Mulder's analyses was roughly 9 kDaAtomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
, hundreds of times larger than other molecules being studied. Hence, the chemical structure of proteins (their primary structure
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...
) was an active area of research until 1949, when Fred Sanger sequenced insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
. The (correct) theory that proteins were linear polymers of amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s linked by peptide bond
Peptide bond
This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...
s was proposed independently and simultaneously by Franz Hofmeister
Franz Hofmeister
Franz Hofmeister was an early protein scientist, and is famous for his studies of salts that influence the solubility and conformational stability of proteins...
and Emil Fischer
Emil Fischer
Emil Fischer may refer to:* Emil Fischer , German dramatic basso* Franz Joseph Emil Fischer , German chemist, worked with oil and coal* Hermann Emil Fischer , German Nobel laureate in chemistry...
at the same conference in 1902. However, some scientists were sceptical that such long macromolecule
Macromolecule
A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s could be stable in solution. Consequently, numerous alternative theories of the protein primary structure
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...
were proposed, e.g., the colloidal hypothesis that proteins were assemblies of small molecules, the cyclol
Cyclol
The cyclol hypothesis is the first structural model of a folded, globular protein. It was developed by Dorothy Wrinch in the late 1930s, and was based on three assumptions. Firstly, the hypothesis assumes that two peptide groups can be crosslinked by a cyclol reaction ; these crosslinks are...
hypothesis of Dorothy Wrinch
Dorothy Maud Wrinch
Dorothy Maud Wrinch was a mathematician and biochemical theorist best known for her attempt to deduce protein structure using mathematical principles....
, the diketopiperazine hypothesis of Emil Abderhalden
Emil Abderhalden
Emil Abderhalden was a Swiss biochemist and physiologist. His main findings, though disputed already in the 1920s, were not finally rejected until the late 1990s. Whether his misleading findings were based on fraud or simply the result of a lack of scientific rigor remains unclear...
and the pyrrol/piperidine hypothesis of Troensgard (1942). Most of these theories had difficulties in accounting for the fact that the digestion of proteins yielded peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
s and amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s.
Proteins were finally shown to be macromolecules of well-defined composition (and not colloidal mixtures) by Theodor Svedberg
Theodor Svedberg
Theodor H. E. Svedberg was a Swedish chemist and Nobel laureate, active at Uppsala University. His work with colloids supported the theories of Brownian motion put forward by Einstein and the Polish geophysicist Marian Smoluchowski...
using analytical ultracentrifugation. The possibility that some proteins are non-covalent associations of such macromolecules was shown by Gilbert Smithson Adair
Gilbert Smithson Adair
Gilbert Smithson Adair FRS was an early protein scientist who used osmotic pressure measurements to establish that hemoglobin was a tetramer under physiological conditions...
(by measuring the osmotic pressure
Osmotic pressure
Osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane....
of hemoglobin
Hemoglobin
Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...
) and, later, by Frederic M. Richards
Frederic M. Richards
Frederic Middlebrook Richards , or commonly referred to as Fred Richards, was Sterling Professor Emeritus of Molecular Biophysics and Biochemistry at Yale University.-Biography:...
in his studies of ribonuclease S. The mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
of proteins has long been a useful technique for identifying posttranslational modification
Posttranslational modification
Posttranslational modification is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins....
s and, more recently, for probing protein structure.
Most proteins are difficult to purify
Protein purification
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or...
in more than milligram quantities, even using the most modern methods. Hence, early studies focused on proteins that could be purified in large quantities, e.g., those of blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
, egg white
Egg white
Egg white is the common name for the clear liquid contained within an egg. In chickens it is formed from the layers of secretions of the anterior section of the hen's oviduct during the passage of the egg. It forms around either fertilized or unfertilized egg yolks...
, various toxin
Toxin
A toxin is a poisonous substance produced within living cells or organisms; man-made substances created by artificial processes are thus excluded...
s, and digestive/metabolic enzymes obtained from slaughterhouse
Slaughterhouse
A slaughterhouse or abattoir is a facility where animals are killed for consumption as food products.Approximately 45-50% of the animal can be turned into edible products...
s. Many techniques of protein purification were developed during World War II
World War II
World War II, or the Second World War , was a global conflict lasting from 1939 to 1945, involving most of the world's nations—including all of the great powers—eventually forming two opposing military alliances: the Allies and the Axis...
in a project led by Edwin Joseph Cohn
Edwin Joseph Cohn
Edwin Joseph Cohn was an early protein scientist. A graduate of Phillips Academy, Andover [1911], and the University of Chicago [1914, PhD 1917], he made important advances in the physical chemistry of proteins, and was responsible for the blood fractionation project that saved thousands of lives...
to purify blood proteins to help keep soldiers alive. In the late 1950s, the Armour Hot Dog Co.
Armour and Company
Armour & Company was an American slaughterhouse and meatpacking company founded in Chicago, Illinois, in 1867 by the Armour brothers, led by Philip Danforth Armour. By 1880, the company was Chicago's most important business and helped make the city and its Union Stock Yards the center of the...
purified 1 kg (= one million milligrams) of pure bovine pancreatic ribonuclease A
Ribonuclease A
Ribonuclease A is a pancreatic ribonuclease that cleaves single-stranded RNA. Bovine pancreatic RNase A is one of the classic model systems of protein science.-History:...
and made it freely available to scientists around the world. This generous act made RNase A the main protein for basic research for the next few decades, resulting in several Nobel Prizes.
Protein folding and first structural models
The study of protein folding began in 1910 with a famous paper by Henrietta Chick and C. J. Martin, in which they showed that the flocculationFlocculation
Flocculation, in the field of chemistry, is a process wherein colloids come out of suspension in the form of floc or flakes by the addition of a clarifying agent. The action differs from precipitation in that, prior to flocculation, colloids are merely suspended in a liquid and not actually...
of a protein was composed of two distinct processes: the precipitation
Precipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...
of a protein from solution was preceded by another process called denaturation
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
, in which the protein became much less soluble, lost its enzymatic activity and became more chemically reactive. In the mid-1920s, Tim Anson
Mortimer Louis Anson
Mortimer Louis Anson was an early protein scientist.He is famous for having proposed that protein folding was areversible, two-state reaction, and for being the foundingeditor of the journal Advances in Protein Chemistry....
and Alfred Mirsky
Alfred Mirsky
Alfred Ezra Mirsky was an American pioneer in molecular biology.Mirsky graduated from Harvard College in 1922, after which he studied for two years at the Columbia University College of Physicians and Surgeons until 1924 when he moved to the University of Cambridge on a US National Research...
proposed that denaturation was a reversible process, a correct hypothesis that was initially lampooned by some scientists as "unboiling the egg". Anson also suggested that denaturation was a two-state ("all-or-none") process, in which one fundamental molecular transition resulted in the drastic changes in solubility, enzymatic activity and chemical reactivity; he further noted that the free energy changes upon denaturation were much smaller than those typically involved in chemical reactions. In 1929, Hsien Wu
Hsien Wu
Hsien Wu was an early protein scientist who was the first to propose that protein denaturation was a purely conformational change, i.e., corresponded to protein unfolding and not to some chemical alteration of the protein...
hypothesized that denaturation was protein unfolding, a purely conformational change that resulted in the exposure of amino acid side chains to the solvent. According to this (correct) hypothesis, exposure of aliphatic and reactive side chains to solvent rendered the protein less soluble and more reactive, whereas the loss of a specific conformation caused the loss of enzymatic activity. Although considered plausible, Wu's hypothesis was not immediately accepted, since so little was known of protein structure and enzymology and other factors could account for the changes in solubility, enzymatic activity and chemical reactivity. In the early 1960s, Chris Anfinsen
Christian B. Anfinsen
Christian Boehmer Anfinsen, Jr. was an American biochemist. He shared the 1972 Nobel Prize in Chemistry with Stanford Moore and William Howard Stein for work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation...
showed that the folding of ribonuclease A
Ribonuclease A
Ribonuclease A is a pancreatic ribonuclease that cleaves single-stranded RNA. Bovine pancreatic RNase A is one of the classic model systems of protein science.-History:...
was fully reversible with no external cofactors needed, verifying the "thermodynamic hypothesis" of protein folding that the folded state represents the global minimum of free energy
Thermodynamic free energy
The thermodynamic free energy is the amount of work that a thermodynamic system can perform. The concept is useful in the thermodynamics of chemical or thermal processes in engineering and science. The free energy is the internal energy of a system less the amount of energy that cannot be used to...
for the protein.
The hypothesis of protein folding was followed by research into the physical interactions that stabilize folded protein structures. The crucial role of hydrophobic interactions was hypothesized by Dorothy Wrinch
Dorothy Maud Wrinch
Dorothy Maud Wrinch was a mathematician and biochemical theorist best known for her attempt to deduce protein structure using mathematical principles....
and Irving Langmuir
Irving Langmuir
Irving Langmuir was an American chemist and physicist. His most noted publication was the famous 1919 article "The Arrangement of Electrons in Atoms and Molecules" in which, building on Gilbert N. Lewis's cubical atom theory and Walther Kossel's chemical bonding theory, he outlined his...
, as a mechanism that might stabilize her cyclol
Cyclol
The cyclol hypothesis is the first structural model of a folded, globular protein. It was developed by Dorothy Wrinch in the late 1930s, and was based on three assumptions. Firstly, the hypothesis assumes that two peptide groups can be crosslinked by a cyclol reaction ; these crosslinks are...
structures. Although supported by J. D. Bernal
J. D. Bernal
John Desmond Bernal FRS was one of Britain’s best known and most controversial scientists, called "Sage" by his friends, and known for pioneering X-ray crystallography in molecular biology.-Origin and education:His family was Irish, of mixed Italian and Spanish/Portuguese Sephardic Jewish origin...
and others, this (correct) hypothesis was rejected along with the cyclol hypothesis, which was disproven in the 1930s by Linus Pauling
Linus Pauling
Linus Carl Pauling was an American chemist, biochemist, peace activist, author, and educator. He was one of the most influential chemists in history and ranks among the most important scientists of the 20th century...
(among others). Instead, Pauling championed the idea that protein structure was stabilized mainly by hydrogen bond
Hydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
s, an idea advanced initially by William Astbury
William Astbury
William Thomas Astbury FRS was an English physicist and molecular biologist who made pioneering X-ray diffraction studies of biological molecules. His work on keratin provided the foundation for Linus Pauling's discovery of the alpha helix...
(1933). Remarkably, Pauling's incorrect theory about H-bonds resulted in his correct models for the secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
elements of proteins, the alpha helix
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
and the beta sheet
Beta sheet
The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...
. The hydrophobic interaction was restored to its correct prominence by a famous article in 1959 by Walter Kauzmann
Walter Kauzmann
Walter J. Kauzmann was an American chemist and professor emeritus of Princeton University. He was noted for his work in both physical chemistry and biochemistry. His most important contribution was recognizing that the hydrophobic effect plays a key role in determining the three-dimensional...
on denaturation
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
, based partly on work by Kaj Linderstrøm-Lang. The ionic nature of proteins was demonstrated by Bjerrum, Weber and Arne Tiselius
Arne Tiselius
Arne Wilhelm Kaurin Tiselius was a Swedish biochemist who won the Nobel Prize in Chemistry in 1948.- Biography:Tiselius was born in Stockholm...
, but Linderstrom-Lang showed that the charges were generally accessible to solvent and not bound to each other (1949).
The secondary
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
and low-resolution tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
of globular proteins was investigated initially by hydrodynamic methods, such as analytical ultracentrifugation and flow birefringence
Flow birefringence
In biochemistry, flow birefringence is a hydrodynamic technique for measuring the rotational diffusion constants . The birefringence of a solution sandwiched between two concentric cylinders is measured as a function of the difference in rotational speed between the inner and outer cylinders...
. Spectroscopic methods to probe protein structure (such as circular dichroism
Circular dichroism
Circular dichroism refers to the differential absorption of left and right circularly polarized light. This phenomenon was discovered by Jean-Baptiste Biot, Augustin Fresnel, and Aimé Cotton in the first half of the 19th century. It is exhibited in the absorption bands of optically active chiral...
, fluorescence, near-ultraviolet and infrared absorbance) were developed in the 1950s. The first atomic-resolution structures of proteins were solved by X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
in the 1960s and by NMR
Protein nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins. The field was pioneered by Richard R. Ernst and Kurt Wüthrich, among others...
in the 1980s. , the Protein Data Bank
Protein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....
has nearly 40,000 atomic-resolution structures of proteins. In more recent times, cryo-electron microscopy
Cryo-electron microscopy
Cryo-electron microscopy , or electron cryomicroscopy, is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures...
of large macromolecular assemblies and computational protein structure prediction
Protein structure prediction
Protein structure prediction is the prediction of the three-dimensional structure of a protein from its amino acid sequence — that is, the prediction of its secondary, tertiary, and quaternary structure from its primary structure. Structure prediction is fundamentally different from the inverse...
of small protein domains are two methods approaching atomic resolution.