Structure of the pig insulin dimer in the cubic crystal

Badger, John; Harris, Michael; Reynolds, C. D.; Evans, Alan C.; Dodson, E.J.; Dodson, Guy; North, A. C. T.

doi:10.1107/s0108768190009570

Cited by 68 publications

(53 citation statements)

References 20 publications

(22 reference statements)

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“…Insulin (51 amino acids, 5.8 kDa) crystallizes from zinc-free alkaline solutions as a symmetric dimer arranged in orthogonal cross-connected rows in the cubic space group I213 (a = 78.9 A) (1,2). The crystal lattice, which contains 65% solvent volume, is stable from pH 7-10 in 0.1 M monovalent cation salt solutions (3) and from pH 5-11 in 1 M Na2SO4.…”

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confidence: 99%

Stereospecific dihaloalkane binding in a pH-sensitive cavity in cubic insulin crystals.

Gursky

Fontano

Bhyravbhatla

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Crystallographic analysis at 2-A resolution of the selective binding of dihalogenated methane, ethane, and ethylene compounds in the cavity on the cubic insulin dimer axis provides a model for anesthetic-protein interactions. At pH 6-11, 1,2-dichloroethane binds isomorphically in the righthanded cis-conformation, displacing four water molecules from the invariant cavity. Lowering the pH to 5.7 in 1 M Na2SO4 without dihaloalkanes induces a cooperative structural transition in which the dyad cavities between B13 glutamate pairs are constricted, and SO2-ions are bound by rearranged triads of Bi NH' groups. In the presence of dichloroethane at pH 5-5.5, the equilibrium is shifted to a mixture of the ligand-bound and ligand-excluding cavity structures, with halfoccupancy of the sulfate sites, exemplifying how a volatile anesthetic can act as an allosteric effector. Measurements at pH 9 of the occupancies of structurally similar dihaloalkanes demonstrate a high degree of binding selectivity. Induced polarization of the ligand and bound water by the charge distribution in the binding cavity apparently provides the selective electrostatic interactions that discriminate between dihaloalkanes of comparable size and polarity.Insulin (51 amino acids, 5.8 kDa) crystallizes from zinc-free alkaline solutions as a symmetric dimer arranged in orthogonal cross-connected rows in the cubic space group I213 (a = 78.9 A) (1, 2). The crystal lattice, which contains 65% solvent volume, is stable from pH 7-10 in 0.1 M monovalent cation salt solutions (3) and from pH 5-11 in 1 M Na2SO4. The pH-dependent local conformational changes and coupled binding of monovalent cations, which have been crystallographically characterized in pH range 7-11 (4), demonstrate the usefulness of this system for studying the effects of altered electrostatic interactions on the protein and solvent structure. Following our observation (4) that cubic insulin crystals in alkaline 0.1-1 M salt solutions bind 1,2-dichloroethane (ClH2C-CH2CI) with unit occupancy at a site on the dimer axis ( Fig. 1), we have characterized this binding at pH 5-11 and compared the binding of similar dihaloalkanes at pH 9. § Such binding exemplifies the type of hydrophobic ligand-protein interactions that may underlie the action of inhalational general anesthetics (6, 7). MATERIALS AND METHODSCrystal Preparation and X-Ray Data Collection. Cubic Zn-free crystals of bovine insulin, grown from phosphate buffer at pH 9 (1, 4), were equilibrated by dialysis against sodium salt solutions that varied in pH from 5 to 11 and subsequently equilibrated with liquid dihaloalkanes. Dihaloalkane binding was discovered by exposing the crystals to 1,2-dichloroethane as solvent for Formvar, which was applied in the capillary to cast an encasing film that prevents crystal slippage (8). Binding at the dyad site, as detected by x-ray diffraction, was complete after 30-sec direct exposure to liquid dichloroethane or after overnight soaking in a saturated aqueous solution. The x-ray data were collect...

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confidence: 99%

Stereospecific dihaloalkane binding in a pH-sensitive cavity in cubic insulin crystals.

Gursky

Fontano

Bhyravbhatla

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Human insulin was shown to contain 51 amino acids, arranged in two chains (an acidic A-chain of 21 residues and a basic B-chain of 30 residues). Insulin is a globular protein containing two chains; its structure as a monomer in solution resembles a crystallographic T-state protomer [2,3].…”

Section: Introductionmentioning

confidence: 99%

Neural Network Modeling in Dithiothreitol Reduction and Ion Treatment of Recombinant Human Insulin Obtained from the Circular Dichroism (CD) Spectral Information

Sardari¹,

Soltani²

2009

TOBIOIJ

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Abstract:In this study, induced changes in the secondary structure of the human insulin were carried out by addition of various reagents causing modification in the disulfide bond such as dithiothreitol (DTT) three dimensional structure of insulin. CD spectra were taken accordingly and the spectra recorded. There are methods to predict and estimate spectral changes of a peptide molecule, however there is no method to process CD spectral data and correlate them with that of inducing factor. Artificial intelligence backpropagation algorithm, as a strong model building tool was used here for prediction and data mining. Therefore, artificial neural network (ANN) methodology was used to build a model to study the effect of selected biochemical factors in the downstream process of a recombinant peptide.

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“…After the pressure release the crystals slowly (about 4 minutes) disappeared. It is worth to notice that dimeric and hexameric insulin crystallizes at ambient pressure into cubic structures of similar dimensions [7]. …”

Section: Resultsmentioning

confidence: 99%