Three-dimensional structures insulin

A small number of proteins, and again insulin is an example, are synthesized as pro-proteins with an additional amino acid sequence which dictates the final three-dimensional structure. In the case of proinsulin, proteolytic attack cleaves out a stretch of 35 amino acids in the middle of the molecule to generate insulin. The peptide that is removed is known as the C chain. The other chains, A and B, remain crosslinked and thus locked in a stable tertiary stiucture by the disulphide bridges formed when the molecule originally folded as proinsulin. Bacteria have no mechanism for specifically cutting out the folding sequences from pro-hormones and the way of solving this problem is described in a later section. 

Figure 11.5 Three-dimensional structure of the engineered fast-acting insulin, Insulin lispro. Structural details courtesy of the Protein Data Bank, http //www.rcsb.org/pdb/...

The three-dimensional structure of insulin remained recalcitrant in spite of the knowledge of its primary sequence. The early crystals had been found by Scott (1936) to contain zinc which could be replaced by other divalent metals. The zinc atom is not heavy enough to be unambiguously distinguishable. Eventually it proved possible to introduce uranyl acetate and uranyl fluoride into the insulin molecule and to obtain the three-dimensional structure, first at 2.8 A resolution and then at 1.9 A (see Blundell, Dodson, Hodgkin, and Mercola, 1972). 

Blundell, Dodson, Hodgkin and Mercola reported the three-dimensional structure of insulin. 

The formation of three disulfide bonds in bovine insulin brings parts of the chain that are distant in terms of amino acid sequence into close proximity in the three-dimensional structure of the protein. 

Now we can ask what is likely to happen to the three-dimensional structure of a protein if we make a conservative replacement of one amino acid for another in the primary structnre. A conservative replacement involves, for example, substitution of one nonpolar amino acid for another, or replacement of one charged amino acid for another. Intnitively, one would expect that conservative replacements would have rather little effect on three-dimensional protein structure. If an isoleucine is replaced by a valine or leucine, the structnral modification is modest. The side chains of all of these amino acids are hydrophobic and will be content to sit in the molecnlar interior. This expectation is borne out in practice. We have noted earlier that there are many different molecnles of cytochrome c in nature, all of which serve the same basic function and all of which have similar three-dimensional structnres. We have also noted the species specificity of insulins among mammalian species. Here too we find a number of conservative changes in the primary structure of the hormone. Although there are exceptions, as a general rule conservative changes in the primary structnre of proteins are consistent with maintenance of the three-dimensional structures of proteins and the associated biological functions. 

Structure-Activity Correlations. This detailed knowledge of the three-dimensional structure of insulin led to the recognition that its biological activity resides in an area of the molecule rather than in specific amino acid residues, just as dimerization and further association of the molecule also depend on an intact spatial structure. The foregoing concept is corroborated by structural modifications of the hormone. The last three amino acids of the B chain can be removed without a loss of activity, but cleavage of the C-terminal of the A chain (Asn ) results in a total loss of activity. Amino acids can be replaced inside the chains only if such substitution does not change the overall geometry of the molecule. The structure-activity relationships of insulin derivatives are inconsistent and not always comparable. 

PTB domains (phosphotyrosine-binding domains) also bind (P)-Tyr in partner proteins, but their critical sequences and three-dimensional structures distinguish them from SH2 domains. The human genome encodes 24 proteins that contain PTB domains, including IRS-1, which we have already met in its role as a scaffold protein in insulin-signal transduction (Fig. 12-6). 

I The most important properties of a protein are deter-f mined by the sequence of amino acids in the polypeptide chain. This sequence is called the primary structure of the protein. We know the sequences for thousands of peptides and proteins, largely through the use of methods developed in Fred Sanger s laboratory and first used to determine the sequence of the peptide hormone insulin in 1953. Knowledge of the amino acid sequence is extremely useful in a number of ways (1) it permits comparisons between normal and mutant proteins (see chapter 5) (2) it permits comparisons between comparable proteins in different species and thereby has been instrumental in positioning different organisms on the evolutionary tree (see fig. 1.24) (3) finally and most important, it is a vital piece of information for determining the three-dimensional structure of the protein. 

In an effort to develop an effective bioadhesive system for buccal administration, insulin was encapsulated into polyacrylamide nanoparticles by the emulsion solvent evaporation method [98]. Though nanoparticle formation ensures even distribution of the drug, pelleting of the nanoparticles was performed to obtain three-dimensional structural conformity. In addition, it was hypothetized that the pelletized particles will remain adhered to the mucosa, leading to good absorption. While studying bioadhesion and drug release profiles, it was found that the... 

Relaxin is another peptide that can be extracted from the ovary. The three-dimensional structure of relaxin is related to that of growth-promoting peptides and is similar to that of insulin. Although the amino acid sequence differs from that of insulin, this hormone, like insulin, consists of two chains linked by disulfide bonds, cleaved from a prohormone. It is found in the ovary, placenta, uterus, and blood. Relaxin synthesis has been demonstrated in luteinized granulosa cells of the corpus luteum. 

For almost a decade there was a continuing dispute over the exact nature of insulin s chemical composition until it was finally accepted that the hormone was in fact a protein. Sanger established the amino acid sequence of insulin in 1960, and this led to the complete synthesis of the protein in 1963, and to the elucidation of its three-dimensional structure in 1972. Insulin was the first hormone for which a radioimmunoassay was developed (1978) and the first to be produced by genetic engineering (given to human volunteers in the summer of 1980). 

In native proteins of known three-dimensional structure about 7% of all prolyl peptide bonds are cis (Stewart et al., 1990 MacArthur and Thornton, 1991). Usually, the conformational state of each peptide bond is clearly defined. It is either cis or trans in every molecule, depending on the structural framework imposed by the folded protein chain. There are a few exceptions to this rule. In the native states of staphylococcal nuclease (Evans et al., 1987), insulin (Higgins et al., 1988), and calbindin (Chazin et al., 1989) cis-trans equilibria at particular Xaa-Pro bonds have been detected in solution by NMR. In staphylococcal nuclease, the cis conformer of the Lys 116-Pro 117 bond can be selectively stabilized by bind-... 

Shuffle test. An enzyme that catalyzes disulfide-sulfhydryl exchange reactions, called protein disulfide isomerase (PDl), has been isolated. PDI rapidly converts inactive scrambled ribonuclease into enzymatically active ribonuclease. In contrast, insulin is rapidly inactivated by PDI. What does this important observation imply about the relation between the amino acid sequence of insulin and its three-dimensional structure ... 

Other chemical degradation pathways include -elimination, which can lead to racemization and disulphide exchange reactions. The amino acids that may undergo P-elimination include Cys, Ser, Thr, Phe, and Lys (40) and occur especially at alkaline pH (64). The reduction and oxidation of the disulphide bonds are often accompanied by a considerable change in the protein conformation (23,33). An example is the secondary structure of insulin that is disrupted or completely lost when the disulphide bonds are broken (23). The disulphide bond disruption or interchange can also result in an altered three-dimensional structure and therefore a possible loss of activity (40) or aggregation (29). For further details, the reader is directed to the following reviews and book chapters (30,40,62,65). 

Insulin is the principal drug used to prevent ketosis and sustain life in the treatment of patients with type I (insulin-dependent) diabetes mellitus. Delivery of proteins such as insulin is a challenge because of the molecular size and the sensitivity of the molecule to the loss of its biological activity through minor alterations in the three-dimensional structure. The normal mode of delivery of insulin to patients at the present time is through intramuscular, subcutaneous, or intravenous injections. These delivery methods are not ideal because of (1) the need for training of the patient or the caretaker in the basic steps of injection, (2) the fear of needles by patients, and (3) the feeling of pain and possible fibrotic formation at the injection site. These inconveniences could lead to noncompliance. A variety of approaches for insulin delivery have been... 

The abundant IGF-I (somatomedin C), a 70-residue single-chain basic peptide with a sequence and three-dimensional structure homologous to that of proinsu-lin,206.207 jg considered a major mediator of the action of fhe pifuitary growth hormone (GH, somatotropin). Studies in cell culture suggest that GH may induce differentiation of cells, and that IGF-I may then cause a rapid proliferation of the newly differentiated cells. The homologous 67-residue IGF-II may have a similar function in fetal development. TTie cell surface receptor for IGF-I is similar to the insulin receptor, but IGF-II receptor is structurally different. It is a monomeric 250-kDa protein and although it is a substrate for a fyrosine kinase, it has no kinase activity of its... 

Species variations in primary stmctnre are also important in medicine, as illustrated by the comparison of human, beef, and pork insulin. Insulin is one of the hormones that are highly conserved between species, with very few amino acid substitutions and none in the regions that affect activity. Insulin is a polypeptide hormone of 51 amino acids that is composed of two polypeptide chains (Fig. 6.13). It is synthesized as a single polypeptide chain, but is cleaved in three places before secretion to form the C peptide and the active insulin molecule containing the A and B chains. The folding of the A and B chains into the correct three-dimensional structure is promoted by the presence of one intrachain and two interchain disulfide bonds formed by cysteine residues. The invariant residues consist of the cysteine residues engaged in disulfide bonds and the residues that form the surface of the insulin molecule that binds to the insulin receptor. The amino acid substitutions in bovine and porcine insulin (shown in blue in Fig. 6.13.) are not in amino acids that affect its activity. Consequently, bovine and pork insulin were used for many years for the treatment of diabetes mellitus. However, even with only a few different amino acids, some patients developed an immune response to these insulins. 

---