Insulin Hexamer Research Articles

Insulin-metal ion interactions: the binding of divalent cations to insulin hexamers and tetramers and the assembly of insulin hexamers.

An insulin hexamer containing one B10-bound Co(III) ion and one unoccupied B10 site has been synthesized. The properties of the monosubstituted hexamer show that occupancy of only one B10 site by Co3+ is sufficient to stabilize the hexameric form under the conditions of pH and concentration used in these studies. The experimentally determined, second-order rate constants for the binding of Zn2+ and Co2+ to the unoccupied B10 site are consistent with literature rate constants for the rate of association of these divalent metal ions with similar small molecule ligands. These findings indicate that the rate-limiting steps for Zn2+ and Co2+ binding involve the removal of the first aqua ligand. The rate constant for the binding of Cd2+ is significantly lower than the literature values for small molecule chelators, which suggests that some other protein-related process is rate-limiting for Cd2+ binding to the unoccupied, preformed B10 site. The kinetics of the assembly of insulin in the presence of limiting metal ion provides strong evidence indicating that the B13 site of the tetramer species can bind Zn2+, Cd2+, or Ca2+ prior to hexamer formation and that such binding assists hexamer formation. Both the tetramer and the hexamer B13 sites were found to exhibit similar affinities for Zn2+ and Cd2+ (Kd congruent to 9 microM), whereas the tetramer B13 sites bind Ca2+ much more weakly (Kd congruent to 1 mM for tetramer vs 83 microM for hexamer). The second-order rate constants estimated for the association of Zn2+ and Cd2+ to the tetrameric site indicate that the loss of the first inner-sphere aqua ligand is the rate-limiting step for binding.

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change.

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.

Adsorption of insulin on metal surfaces in relation to association behavior

The adsorption of insulin on metal surfaces from aqueous solutions was monitored by in situ ellipsometry. Clean (hydrophilic) chromium and titanium surfaces as well as chromium surfaces treated to be hydrophobic were used. The adsorbed amount was found to be higher on the hydrophilic than on the hydrophobic surfaces. The presence of the divalent metal ions zinc and calcium increased the adsorbed amount and also the fraction desorbable upon rinsing. This was related to the behavior in the bulk solution, where zinc and calcium increase the association. The increase in adsorbed amount was observed also when zinc was added to an adsorbed layer of Zn-free insulin in contact with insulin solution. Addition of citrate to an adsorbed layer of insulin from a solution containing four zinc ions per hexamer had an effect similar to that of rinsing, indicating a removal of zinc in both cases. The effect of the pH was investigated in the range from pH 7 to pH 8, showing an increase in adsorbed amounts at lower pH, especially pronounced for the samples with a high zinc content, four zinc ions per insulin hexamer. The calcium-containing samples did not show such a strong pH dependence.

1H NMR Studies of insulin: Histidine residues, metal binding, and dissociation in alkaline solution

The shifts of the H2 histidine B5 and B10 resonances of 2-Zn insulin hexamer were followed in 2H 2O by 1H NMR spectroscopy at 270 MHz from pH 9.85 to 7. The two resonances present at high pH, previously assigned to H2 histidine B5 and B10 residues, moved slightly downfield and split into four resonances at pH 8.95 and also at pH 7. By use of a paramagnetic broadening probe (Mn 2+) and the addition of Zn 2+ to metal-free insulin, it was deduced that the four resonances arose from histidines B10 and B5 in two different magnetic environments, probably either bound to Zn 2+ or not bound to Zn 2+. The pK′ values of the B5 and B10 histidines were determined in 60% 2H 2O-40% dioxan, in which insulin was soluble throughout the pH range, to be 7.1 and 6.8, respectively at 37 °C. Studies at higher pH indicated that at a concentration level suitable for 1H NMR (~1 m m) at 37 °C in 2H 2O the 2-Zn hexamer was largely dissociated to dimer at pH 10.3 and to monomer at pH 10.8. Addition of paramagnetic shift probe Ni 2+ to metal-free insulin caused changes to the spectrum similar to those produced on addition of diamagnetic Zn 2+. Addition of Co 2+ gave a different result, but there was no paramagnetic shift of the H2 histidine B10 resonance, probably because of rapid exchange at the binding site. Addition of Cd 2+ and of Cd 2+ and Ca 2+ produced changes that were similar to each other but were different from those observed on addition of Zn 2+, probably due to the binding of Cd 2+ and Ca 2+ at glutamate B13.

Effects of calcium ion on ternary complexes formed between 4-(2-pyridylazo)resorcinol and the two-zinc insulin hexamer.

As a means for probing the microenvironment of zinc in the insulin hexamer and to investigate the effects of calcium ion on the assembly and the structure of the two-zinc insulin hexamer, the thermodynamics and kinetics of the reaction between the chromophoric divalent metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) and zinc-insulin have been investigated over a wide range of conditions. For [PAR]0 much greater than [Zn2+]0 and [Zn2+]/[In] less than or equal to 0.33, the reaction leads to the sequestering and ultimate removal of all of the insulin-bound Zn2+; for [Zn2+]0 much greater than [PAR]0, two stable ternary complexes are formed where Zn2+ has ligands derived from PAR as well as from hexameric insulin. For [Zn2+]/[In] ratios below 0.33, the equilibrium distribution between the two ternary complexes is dependent on the [Zn2+]/[In] ratio. One of the complexes is assigned to the monoanion of PAR coordinated to Zn2+ that resides in a His-B10 site. The other complex is proposed to involve the coordination of (PAR)Zn to the site formed by the alpha-NH2 group of Phe-B1 and the gamma-carboxylate ion of Glu-A17 across the dimer-dimer interface on the surface of the hexamer. With either PAR or zinc-insulin in large excess, the kinetics of the PAR optical density changes are remarkably similar and biphasic. The faster step is first order in PAR and first order in insulin-bound Zn2+ (k congruent to 3 X 10(3) M-1 s-1) and involves the formation of an intermediate in which PAR is coordinated to insulin-bound zinc at the His-B10 site.(ABSTRACT TRUNCATED AT 250 WORDS)

C-13 NMR spectral studies of the thyroid hormone transport protein, transthyretin and the pancreatic insulin storage moiety, the zinc-insulin hexamer

The DEPT spectral editing technique was used to separate the CH, CH 2 and CH 3 resonances in the C-13 NMR spectra of transthyretin and the porcine zinc insulin hexamer. The advantages of this technique in terms of spectral simplification and sensitivity enhancement for 13C NMR of proteins is discussed. Spin-lattice relaxation time and nuclear Overhauser effect measurements of the backbone C-α and aliphatic sidechain carbons provided information about the dynamics of the proteins in solution and the relative mobility of some sidechain groups.

1H n.m.r. studies of insulin

1H n.m.r. studies at 270 MHz were made of the transformation of 2 Zn insulin hexamer to 4 Zn hexamer produced by the addition of anions (thiocyanate ion). Four separate H2 histidine resonances were observed for the B5 and B10 histidines in 2 Zn hexamer at pH 7 and 9 and four separate resonances also occurred in the 4 Zn hexamer. The observation of these resonances and others from phenylalanine, tyrosine and leucine residues showed that the 2 Zn to 4 Zn transformation probably occurred in solution in a similar manner to that observed in the crystal. Furthermore as occurred in the crystal, it was found that in solution the transformation was reversible (on removal of thiocyanate) and that 2 Cd insulin was unable to undergo the transformation. Des-Phe-Bl-insulin did not undergo the transformation. Addition of SCN- to Zn-free insulin (mainly dimer) produced only a small transformation, consistent with the idea that Zn2+ promotes formation of hexamer from dimer but probably does not otherwise affect the transformation.

Structure-function relationships in the free insulin monomer.

The chemical properties of the functional groups of insulin were determined at a concentration (0.5 microM) where the predominant species of insulin is the free (unassociated) monomeric unit. The glycine N-terminus and the four tyrosine phenolic groups had the same properties as in the associated forms of insulin. On the other hand the lysine epsilon-amino group and the two histidine imidazole groups had substantially altered properties. Some alteration in the properties of the phenylalanine N-terminus was also observed. The reactivity-pH profile for the imidazole groups showed a second ionization with a pKa of 10.1 in addition to an ionization with a pKa of 6.8. On the basis of the X-ray-crystallographic structure of hexameric insulin the observed changes can be accounted for by disruption of monomer-monomer or dimer-dimer interactions in the associated states of insulin. It is concluded that the conformation of the monomeric unit of insulin is essentially the same in its free and associated states in solution.

ChemInform Abstract: Design, Synthesis and Testing of Insulin Hexamer‐Stabilizing Agents.

The addition of zinc to insulin solution leads to a long-acting insulin preparation because the zinc stabilizes the less soluble hexameric form of the hormone. It is clear from the crystal structure of dizinc insulin that there is a space at the center of the hexamer, between the two zinc atoms, that could accommodate a small organic molecule. It should thus be possible to design a structure that could further stabilize the insulin hexamer by binding at this site. Computer graphic techniques have been used to design several molecules capable of forming multiple bonds to the six histidine residues surrounding the site. Synthesis and testing of one of these compounds, benzene-1,4-disulfonic acid, show a significant increase in weight-average molecular weight of insulin in solution, and control experiments with related structures suggest that this effect is due to the proposed binding mechanism.

Design, synthesis, and testing of insulin hexamer-stabilizing agents.

The addition of zinc to insulin solution leads to a long-acting insulin preparation because the zinc stabilizes the less soluble hexameric form of the hormone. It is clear from the crystal structure of dizinc insulin that there is a space at the center of the hexamer, between the two zinc atoms, that could accommodate a small organic molecule. It should thus be possible to design a structure that could further stabilize the insulin hexamer by binding at this site. Computer graphic techniques have been used to design several molecules capable of forming multiple bonds to the six histidine residues surrounding the site. Synthesis and testing of one of these compounds, benzene-1,4-disulfonic acid, show a significant increase in weight-average molecular weight of insulin in solution, and control experiments with related structures suggest that this effect is due to the proposed binding mechanism.

X-RAY STUDIES ON THE STRUCTURE OF DES-PENTAPEPTIDE INSULIN AT 2.4 RESOLUTION——THE DPI MOLECULE AND ITS STRUCTURAL RELATIONSHIP WITH INSULIN

The structure of the monomeric insulin analogue des(B26—B30) insulin is presented.; A detailed comparison with the 2Zn insulin structures shows that while there are some large changes in the structure, the basic secondary structural units maintain their integrity. The DPI structure is broadly similar to molecule Ⅰ in the 2Zn structure, and in this respect is like other crystal forms of insulin. In addition to changes on the surface of the structure there are some subtle but extensive changes in the heart of the molecule. The molecules are closely packed in the crystal with many and varied contacts, including a complex network of protein-cadmium interactions and a considerable number of water mediated contacts. The molecular surface has an unusually large number of hydrophobic groups which tend to cluster in a thick band running around the protein. The crystal structure is well ordered, indeed the clarity of some side chains and the definition of the water molecules is superior to that found in the more liquid rhombohedral crystals of 2Zn insulin hexamers.

Structural stability in the 4-zinc human insulin hexamer.

X-ray studies on human insulins prepared by semisynthetic and biosynthetic methods have recently been undertaken. Human insulin differs from porcine insulin only at the COOH terminus of the B-chain. The present study reports the crystal structure of 4-zinc human insulin, which is used clinically as a slow-acting preparation. The structure has been refined, using 1.85-A resolution data, to a residual of 0.173. The unit cell is rhombohedral, space group R3, with hexagonal cell constants a = 80.953 and c = 37.636 A, and it is nearly isomorphous with that of 4-zinc porcine insulin. As a result of a conformational change of the first eight residues of the B-chain of molecule 1 from an extended conformation observed in the 2-zinc structure to an alpha-helical one, the coordination around one of the zinc ions on the 3-fold axis has changed, an additional zinc ion in a general position is bound by the hexamer, and additional hydrogen-bonded interactions help stabilize dimer and hexamer formation. Unlike the surface of the 2-zinc insulin hexamer, which possesses a shallow depression containing a zinc ion and its coordinating water molecules, the 4-zinc human insulin hexamer contains a zinc and chloride ion at the bottom of an 8-A tunnel produced by three parallel alpha-helices. These alpha-helices shield the zinc ion from the environment, decreasing the rate of dissociation of the hexamer, and provide an explanation for the slow-acting aspect of the 4-zinc crystalline form.

Conformational flexibility in the 4-zinc human insulin hexamer as determined by X-ray crystallography

Conformational flexibility in the 4-zinc human insulin hexamer as determined by X-ray crystallography

Dogfish insulin. Primary structure, conformation and biological properties of an elasmobranchial insulin.

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid-ethanol extraction and salt precipitation. The amino acid sequences of the performic-acid-oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co-ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln leads to His, B21Glu leads to Pro, B22Arg leads to Lys, B25Phe leads to Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half-maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin. The use of interactive computer graphics model-building predicts that the dogfish insulin can attain a three-dimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model-building studies.

500-MHz1H NMR studies of insulin: Complete assignment of histidine resonances

The two histidines of the insulin monomer play a vital role in the organization of insulin into insulin hexamers. The B10 histidines bind to zinc to form two-zinc insulin hexamer, and both the B5 and B10 histidines are implicated in the formation of four-zinc insulin hexamer. These two histidines are both accessible to solvent in the dimeric form of insulin, the predominant species present at pH 2–3. In the present work we report the first 500-MHz1H NMR studies of insulin. At this frequency all four proton resonances from the two histidines of each equivalent monomer are resolved. The resonances are assigned to the C(2)- and C(4)-imidazole protons of B5 His and B10 His employing Carr-Purcell pulse sequences to detect singlets and to observe approximateT 2 relaxation times. Zinc-free bovine insulin at pH 2.9 was examined at temperatures up to 60°C in acetate buffer and in urea of varying concentrations. The environments of B5 His in molecule I and molecule II of the dimer must be the same, with the same being true for B10 His, since a total of only four sharp resonances are seen. Our assignments for the two C(2) protons are consistent with those determined from recent studies of human (B5 Ala) insulin.

The internal pH and membrane potential of the insulin-secretory granule.

The membrane potential (DeltaPsi) and the pH gradient (DeltapH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[(14)C]cyanate or the permeant base [(14)C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a DeltaPsi positive inside. Addition of MgATP(2-) under these conditions did not alter the DeltapH, but produced a marked increase in the DeltaPsi. Conversely, when a permeant anion was also included, ATP produced a marked increase in the DeltapH and a lesser increment in the DeltaPsi. NH(4) (+) salts reduced the DeltapH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the DeltaPsi. A similar reciprocity was evident when nigericin was added together with K(+) or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(+), and by the Mg(2+)-dependent ATPase inhibitor tributyltin. Uptakes of (14)C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg(2+)-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.

Examination of insulin formulations using quasi-elastic light scattering.

A selection of neutral insulin formulations from commercially available vials was examined by quasi-elastic light scattering. In preliminary investigation, significant differences in the particulate nature of insulins from different manufacturers were apparent. Some preparations contained only small diameter species believed to be insulin hexamer while others showed evidence of larger aggregates and microcrystals. Addition of small amounts of bicarbonate had no detectable effect on the small species but dissolves the microcrystals. Speculation on further and more quantitative use of this new technique for analysis of insulin solutions is reported.

1H nuclear magnetic resonance study of the histidine residues of insulin

Insulin has proved difficult to study by nuclear magnetic resonance spectroscopy because of its complex aggregation behaviour in solution and its insolubility between pH 4 and 7. Now for the first time it has been possible to assign the 1H nuclear magnetic resonances of the H-2 histidine protons of residues B5 and B10 of bovine 2 Zn insulin and Zn-free insulin, and the B5 and A8 residues of hagfish insulin. As expected, the addition of Zn to Zn-free insulin causes virtually no change in the chemical shift or the rate of H-D exchange of the H-2 proton of histidine B5, which is not involved in Zn binding in the 2 Zn insulin hexamer. The rate of H-D exchange of the H-2 proton of histidine B10 is decreased markedly on Zn binding at this residue, but the chemical shift of the resonance remains virtually constant owing to the balancing of an upfield ring current shift of the ordered histidine residues by a downfield shift due to electron withdrawal from the ring nitrogen by the Zn binding.

Cadmium-113 nuclear magnetic resonance studies of bovine insulin: two-zinc insulin hexamer specifically binds calcium.

By use of cadmium-113 nuclear magnetic resonance spectroscopy, a specific calcium ion binding site has been identified in the bovine two-zinc insulin hexamer. This site is composed of six glutamyl carboxylate groups clustered in the center of the hexamer, and is distinct from the normal zinc ion binding sites.

Role of zinc in insulin biosynthesis. Some possible zinc-insulin interactions in the pancreatic B-cell.

The behaviour of proinsulin and insulin in the presence of zinc suggests it plays an important role in insulin's production in the B-cell for the vast majority of animal species. The postulate that proinsulin forms a zinc containing hexamer soon after its synthesis and that this organization of the molecule is maintained through all the subsequent processes is supported by our observation that the proinsulin hexamer is converted readily into the insulin hexamer. In addition the zinc ions enhance proinsulin's solubility and render insulin insoluble. Zinc ions also appear to play an important role in the microcrystalline character of the precipitated insulin granule. There may be advantages in condensing the stored material in this way; it will reduce contact with the surrounding membrane where the converting, and possibly other enzymes, are thought to be located, and it will tend to exclude incompletely converted hexamers.