Proteins are one of the four main classes of molecules--along with carbohydrates, fats, and nucleic acids--that underlie all life. A protein is a chain of amino acids, which are smaller molecules of some twenty different kinds. Each amino acid has a common root and a side group that gives it its distinctive chemical properties. When the roots are joined chemically, with what is called a peptide bond, the result is a peptide chain, consisting of repeating patterns of two carbon atoms alternating with a nitrogen atom, from which bristle the side groups of amino acids. Amino acids can join in any order, and peptide chains may contain from several to hundreds or even thousands of amino acids. A protein is made up of one or more peptide chains that have a biological function. Proteins may be enzymes, which catalyze nearly all important chemical reactions in the body, may serve on the cell surface as receptors for other molecules--as in the immune system--or may provide structure, as in hair and fingernails.
Understanding the structure and function of amino acid (also called polypeptide) chains was a key question for both biochemists and geneticists in the 1950s who were engaged in biochemical studies of ribonucleic acid (RNA). While deoxyribonucleic acid (DNA) transmits hereditary information and determines the production of different proteins in different species, RNA is copied from DNA to be a "working copy" or "messenger copy" of DNA. Because RNA is used to combine the amino acids in the proper order to make a protein, understanding its chemical structure is vital to the study of genetics.
Upon his arrival at the NIH, Anfinsen wanted to expand his study of the amino acid structure of proteins. Why, Anfinsen wondered, does a protein fold into its distinctive three-dimensional shape? Was it helped by other enzymes? Why do they take this particular form? He chose as his model bovine pancreatic ribonuclease (RNase), an enzyme that facilitates the DNA-RNA interaction in the pancreas cells of cows. Partly, Anfinsen's choice of this enzyme was practical: the Armour meat packing company of Chicago could provide Anfinsen's laboratory with a ready supply of raw material. Anfinsen and postdoctoral students Michael Sela and Fred White observed in laboratory experiments that amino acid chains in the active RNase enzyme fold spontaneously into what Anfinsen later called the enzyme's "native conformation." In an important article in the Journal of Biological Chemistry in 1954, Anfinsen showed that the sequence of amino acids in a peptide chain determines the folding pattern. The mysteriously complex process of protein folding could be explained entirely simply by the physical and chemical interactions among the amino acid side groups.
In 1954, the Rockefeller Foundation awarded Anfinsen a one-year postdoctoral fellowship at the Carlsberg Laboratory in Copenhagen, the same lab where he had worked fifteen years earlier, in 1939-40. Working with the Danish biochemist Kaj Linderstrøm-Lang, Anfinsen conducted in-depth physical analyses of the structure of RNase, and published a preliminary study of his findings on the topic in Biochimica et Biophysica Acta in 1955. To a great degree, his work was influenced by the laboratory successes of the British biochemist Frederick Sanger, who won the 1958 Nobel Prize in Chemistry for his work on the protein structure of the hormone insulin. Still, Anfinsen admitted later that his work during this period was not without its flaws. Reflecting on this article in 1989, he commented that it is "a beautiful example of how an entirely acceptable conclusion can be reached that is entirely wrong because of the paucity of knowledge at that particular time. I spent the following 15 years or so completely disproving the conclusions reached in this communication."
By 1962, Anfinsen had developed what he called his "thermodynamic hypothesis" of protein folding to explain the native conformation of amino acid structures. He theorized that the native or natural conformation occurs because this particular shape is thermodynamically the most stable in the intracellular environment. That is, it takes this shape as a result of the constraints of the peptide bonds as modified by the other chemical and physical properties of the amino acids. To test this hypothesis, Anfinsen unfolded the RNase enzyme under extreme chemical conditions and observed that the enzyme's amino acid structure refolded spontaneously back into its original form when he returned the chemical environment to natural cellular conditions. As he stated ten years later, in his 1972 Nobel acceptance speech, "The native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment."