Insulin covalent structure

The covalent structure of insulin was established by Frederick Sanger in 1953 after a 10-year effort. This was the first protein sequence determination.237 238 Sanger used partial hydrolysis of peptide chains whose amino groups had been labeled by reaction with 2,4-dinitrofluorobenzene239 to form shorter end-labeled fragments. These were analyzed for their amino acid composition and labeled and hydrolyzed again as necessary. Many peptides had to be analyzed to deduce the sequence of the 21-residue and 30-residue chains that are joined by disulfide linkages in insulin.237 238... 

The studies of Sanger and co-workers (1945-1955) on the amino acid sequence of insulin provide the best example of the use of partial acid hydrolysis for determination of the covalent structure of polypeptides. When oxidized A- or B-chains were submitted to hydrolysis in 11-12 iV HCl... 

Quaternary structure. Due to non-covalent interactions, many proteins assemble to form symmetrical complexes (oligomers). The individual components of oligomeric proteins (usually 2-12) are termed subunits or monomers. Insulin also forms quaternary structures. In the blood, it is partly present as a dimer. In addition, there are also hexamers stabilized by Zn ions (light blue) (3), which represent the form in which insulin is stored in the pancreas (see p.l60). 

This process involves the covalent locking in of structures formed by reversible self-assembly. The irreversible, post-assembly step switches off the equilibrium process involved in the self-assembly. As we will see in the following sections, self assembly with covalent postmodification is involved in a range of biochemistry (e.g. insulin synthesis) and elegant abiotic supramolecular synthesis as in the formation of catenanes and knots. 

The receptor, particularly the /3 subunit, is extremely sensitive to proteolysis [14] and can give rise to the apparent association of peptides of lower molecular mass being associated with receptor preparations. Nevertheless, there are indications, from both immunoprecipitation [15,16] and cross-linking [17] studies, that other distinct protein subunits may be associated with the insulin receptor. It is possible that these proteins represent species that are functionally or structurally capable of interacting with the insulin receptor, yet are not covalently attached to the receptor itself. Hence their association with the receptor would be expected to be easily disrupted by the manipulative processes used in purifying the solubilized receptor. The... 

Because this tyrosine kinase is a component of the receptor itself, the insulin receptor is referred to as a receptor tyrosine kinase. Second, the insulin receptor kinase is in an inactive conformation when the domain is not covalently modified. The kinase is rendered inactive by the position of an unstructured loop (called the activation loop) that lies in the center of the structure. 

The globular proteins sometimes consist of an assembly of a small number of identical or very similar sub-units, not linked together by covalent bonds. This is usually designated as the quaternary structure. Hemoglobin consists of 4 units of the type indicated in Figure 10.18b. Insulin readily forms a dimer from the two units in Figure 10.19. 

In a series of ingenious experiments on ACTH and dynorphin Schwyzer has also shown that their pharmacological potency and hydrophobic membrane interactions are critically dependent on the covalent linkage of message and address to form amphiphilic molecules. Amphiphilicity is caused in these cases by the amphiphilic primary structures, in contrast to the amphiphilicity resulting from the secondary and tertiary structure of some other peptide hormones. Such an amphiphilicity was postulated recently by Kaiser and Kezdy [32] for hormones such as insulin and -endorphin [33]. 

The primary protein structure is the sequence of amino acids in its chain. Primary structure is maintained by the covalent peptide bonds between individual amino acids. For example, one section of the insulin protein has the sequence ... 

We shall begin a discussion of the structural studies of insulin by referring to the covalent details worked out by Sanger and co-workers (Ryle et al., 1955) (Fig. 131). The molecule consists of two chains, A and B, of 21 and 30 residues each, respectively, with three disulfide bridges as indicated in Fig. 131. 

The polypeptide chains of all proteins are synthesized by the process described above. This mechanism gives rise to primary polypeptide chains, which are often further modified—for example, by cleavage into smaller peptides, by stmctural modification of selected amino acid residues, by splicing of the polypeptide chain, or by the formation of covalent bonds between polypeptide chains. Some of these secondary modifications are related to the correct folding of polypeptide chains and to the production of active enzymes or peptide hormones from inactive precursors (e.g., insulin from proinsulin). Also, the transport of proteins within the cell or the secretion of extracellular proteins is often linked to structural changes in polypeptide chains either during or after completion of synthesis. 

An entirely different principle underlies the method we have developed in our laboratory for combining insulin chains (6) In this method, shown in Figure 3, an excess of the sulfhydryl form of the A chain reacts with the S-sulfonated form of the B chain. The yield of insulin obtained by this procedure, based on the amount of B chain S sulfonate used is about 70 percent of the theoretical prediction. The implication therefore arises from the high combination yields that the necessary information for complementarity and covalent linking of the insulin chains to produce the protein is contained within the primary structure of the chains. 

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