In 1943, a young biochemist named Fredrick Sanger, who had just obtained his Ph.D. at the University of Cambridge, began postdoctoral research to “investigate the structure of insulin.” More specifically, he was assigned by his supervisor to devise a method to determine which amino acid residue lay at the N-terminal end of a protein. Sanger did a great deal more than that, becoming one of the two individuals ever to win two Nobel Prizes in the same category (Chemistry).
Insulin was an obvious choice because it was known to be one of the smaller protein molecules, and it was available in a relatively pure form, as it was already used in the treatment of diabetes. Sanger soon solved his initial problem; he found the amino acids which lay at the N-terminal tails of the two peptide chains in insulin, using a colored compound that selectively reacted to those sites. Then, by completely digesting the protein in acid, he could pick out the colored terminal amino acids from all the rest by a new technique called chromatography. There turned out to be two poly peptide chains in the molecule. Sanger soon realized that one might determine the internal sequence of each chain, by using mild acid treatment and enzyme digestion to make many overlapping peptides (Figure 1), which allowed him to deduce the entire sequence of both chains. This required nearly a decade of laborious, careful work, much of which was carried out by Sanger himself.
Fig1. The use of overlapping peptides to sequence proteins: the case for insulin.
When the entire sequence was completed, it gave an unequivocal result: there was no evidence for any repetitive sequences and bovine insulin was defined as a unique sequence of amino acid residues. Subsequent studies showed that all insulin molecules from a given species of animal had the same sequence, although there were small interspecies differences. Sanger’s work established once and for all the “molecularity” of proteins; it completed the work of Fischer and Hofmeister from half a century earlier. In addition, these findings pointed directly to the necessity of some fundamental cellular mechanism to provide the information to dictate protein sequences. Thus, this is one of the crucial studies in the evolution of molecular biology. In 1958, Frederick Sanger was awarded the Nobel Prize in Chemistry “for his work on the structure of proteins, especially that of insulin.”
Thus, proteins are polymers of α-amino acid monomers. The general structure of an α-amino acid is shown in Figure 2. These are amino acids in which the amine group is attached to the α-carbon or Cα, the one next to the carboxyl group. All amino acids contain this core, but they differ in the side chain R that is also attached to the Cα. Proline is technically an imino acid as a result of its cyclic side-chain structure. Glycine is an unusual amino acid because its side chain R is a single hydrogen atom and thus its Cα is a center of symmetry. The Cα of all other amino acids is an asymmetric center and therefore they have stereoisomers, designated D and L (see Figure 2). Only the L-isomer is found in native proteins, although some D-isomer amino acids do exist in living cells. Why nature chose only the L-isomer to make proteins remains a mystery.
Fig2. Chemical structure of α-amino acids. R denotes a chemical group that is unique to each amino acid. The asymmetric C atom satisfied its four valencies by the attachment of four different groups. Molecules that contain such an atom, known as a chiral center and designated here by a star, are optically active as they rotate the plane of polarized light in different directions. C-O-R-N law: Place the hydrogen behind the chiral center and count around from the –COOH group to the –R group to the –NH2 group. If these groups are arranged counterclockwise, the molecule is the L-stereoisomer; if clock wise, it is the D-stereoisomer. Proteins consist exclusively of L-amino acids, but D-amino acids are also found in living organisms. For example, D-alanine is a component of bacterial cell walls.
The structures of the 20 classical amino acids that DNA codes for and specifies in the proteins of all living organisms are shown in Figure 3.3. Recently it has been shown that selenocysteine and pyrrolysine are also DNA-coded amino acids that are included in proteins, albeit rarely. These two unusual amino acids and their incorporation into protein chains are discussed in Chapter 15. The variety of side chains in the different amino acids allows a great variety of interactions with the solvent environment, other protein molecules, or other groups within the same protein molecule. Some side chains are aliphatic or aromatic; these will tend to be hydrophobic and so are typically packed into the interior of protein molecules. Others, such as those with acidic or basic groups, or their carboxamides such as glutamine, will be hydrophilic, preferring to associate with the water surrounding a protein. Other amino acids contain specialized groups, such as hydroxyl or sulfhydryl. Altogether, the variety in the side chains of their amino acids provides protein molecules with a formidable tool kit for molecular interactions.
Fig3. Classical amino acids found in proteins. The amino acids are grouped according to the chemical properties of their side chains. Each amino acid is presented by its full name, its three-letter abbreviation, and its one-letter code.
