The use of insulin as an injected therapeutic agent for the treatment of diabetes has been one of the outstanding successes of modern medicine. The therapy has, however, had its associated problems, not least because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood. This is especially true at meal times when absorption from subcutaneous tissue is too slow to mimic the normal rapid increments of insulin in the blood. In the neutral solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers and this self-association, which under normal physiological circumstances functions to facilitate proinsulin transport, conversion and intracellular storage, may limit the rate of absorption. We now report that it is possible, by single amino-acid substitutions, to make insulins which are essentially monomeric at pharmaceutical concentrations (0.6 mM) and which have largely preserved their biological activity. These monomeric insulins are absorbed two to three times faster after subcutaneous injection than the present rapid-acting insulins. They are therefore capable of giving diabetic patients a more physiological plasma insulin profile at the time of meal consumption.

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A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.

Three X-ray crystallographic studies have been carried out on pig insulin in the presence of three ligands, thiocyanate, methylparaben (methyl p-hydroxybenzoate), and phenol. In each case, rhombohedral crystals were obtained, which diffracted to 1.8, 1.9, and 2.3 A, respectively. Each crystal structure was very similar to that of 4-zinc pig insulin, which was used as a starting model for PROLSQ refinement (Collaborative Computational Project, Number 4, 1994). The R factors for the refined structures of thiocyanate insulin, methylparaben insulin, and phenol insulin were 19.6, 18.4, and 19.1, respectively. Each crystal structure consists of T3R3f insulin hexamers with two zinc ions per hexamer. In the R3f trimer of the thiocyanate insulin hexamer, one thiocyanate ion is coordinated to the zinc on the hexamer 3-fold axis, but there is no evidence of zinc ion binding in the off-axis zinc ion sites seen in the 4-zinc pig insulin structure. In the methylparaben insulin and phenol insulin hexamers, the phenolic ligands are bound at the dimer-dimer interfaces in the R3f trimers in a manner similar to that of phenol in R6 phenol insulin. The binding of methylparaben appears to make the hexamer more compact by drawing the A and the B chains closer together in the binding site. In all three structures presented herein, the conformations of the first three residues of the B chain in the R3f trimer are extended rather than alpha-helical, as is seen in R6 phenol insulin. The energetics of ligand binding in the insulin hexamer are discussed.