Atomic Positions in Rhombohedral 2-Zinc Insulin Crystals

Blundell, T.L.; Jf, Cutfield; Sm, Cutfield; Ej, Dodson; Gg, Dodson; Dc, Hodgkin; Da, Mercola; Vijayan, M.

doi:10.1038/231506a0

Cited by 310 publications

(245 citation statements)

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“…The polypeptide hormone insulin has a mainly helical native structure, with its two polypeptide chains linked by two interchain and one intra-chain disulfide bonds (18). In vitro, insulin is readily converted to an inactive fibrillar form by incubation at high insulin concentrations, low pH and high temperatures (19,20).…”

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confidence: 99%

The protofilament structure of insulin amyloid fibrils

Jiménez

Nettleton

Bouchard

et al. 2002

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

779

822

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Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-␤ structure. However, there is a lack of information relating the 4.8 Å ␤-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-␤ ribbon is composed of relatively flat ␤-sheets rather than being the highly twisted, ␤-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in ␤-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.U nder conditions that destabilize the native state, proteins can self-aggregate into insoluble, fibrillar assemblies (1-3). In the form of amyloid fibrils or fibril precursors, the proteins not only lack their original biological function but also may be harmful to organisms, causing pathologies such as Alzheimer's and prion diseases. Although amyloid precursor proteins do not share any sequence or structural homology, amyloid fibrils are typically unbranched, protease-resistant filaments approximately 100 Å in diameter and composed of Ϸ20-35 Å wide protofilaments, which are sometimes arranged around an electron lucent core (4, 5). Recently, several nonpathological proteins and short peptides have been shown to self-assemble into amyloid-like fibrils (6-9), leading to the suggestion that amyloid formation is a generic property of polypeptide chains (3, 10).The overall morphology of amyloid aggregates depends on the conditions in which fibrillogenesis takes place, and different fibril morphologies are often observed in the same preparation (7,(11)(12)(13)(14). Variable structures also are seen in ex vivo fibrils extracted from amyloidotic tissue (4, 15). The morphological variation seems to be caused by fibrils with a variable number and arrangement of protofilaments. X-ray fiber diffraction studies reveal a characteristic cross-␤ structure with ␤-strands of the precursor protein arranged perpendicular to, and ribbon-like ␤-sheets parallel to, the fibril axis (2, 16, 17). The ␤-strand repeat has also been directly visualized by cryo-EM (8). However, there is a lack of three-dimensional (3D) structural information on how the 4.8 Å ␤-strand repeat relates to the overall fibril assembly.The polypeptide hormone insulin has a mainly helical native structure, with its two polypeptide chains linked by two interchain and one intra-chain disulfide bonds (18). In vitro, insulin is readily converted to an inactiv...

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mentioning

confidence: 99%

The protofilament structure of insulin amyloid fibrils

Jiménez

Nettleton

Bouchard

et al. 2002

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

779

822

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“…However, many biochemical and structural properties of insulin and proinsulin have evolved in response to differential requirements of biosynthesis, processing, transport, and storage in the ␤-cells of Langerhans islets (2,3). Importantly, insulin and insulin analogues are readily adapted for expression in yeast as single-chain proinsulin-like precursors lacking the connecting peptide in the following configuration: amino acid residues 1-29 of the B-chain connected to the A-chain by a short removable mini-C-peptide and fused to the yeast prepro-␣ factor through a single dibasic cleavage site (secretory expression of insulin in yeast and in vitro enzymatic conversion of the precursor to insulin has recently been reviewed (4,5)).…”

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confidence: 99%

Engineering-enhanced Protein Secretory Expression in Yeast with Application to Insulin

Kjeldsen

Ludvigsen

Diers

et al. 2002

Journal of Biological Chemistry

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Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp B28 ) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin.Administration of the two-chain 51-amino acid peptide hormone insulin is life sustaining for many of the 150 million diabetic patients in the world. Classical pancreatic extraction results in 10 -15 mg of insulin per pancreas and the number of porcine or bovine pancreases to produce current requirements of insulin (in excess of 7 metric tons) is simply not available. Consequently, a substantial fraction of insulin is today produced by eukaryotic secretory expression in the yeast Saccharomyces cerevisiae. Recently, second generation fast-acting genetically engineered insulin analogues have been engineered for improved diabetes therapy. This is exemplified by the clinically relevant fast-acting insulin, which features an amino acid substitution in position 28 of the B-chain from proline to aspartic acid and is used for treatment of diabetes mellitus (1).However, many biochemical and structural properties of insulin and proinsulin have evolved in response to differential requirements of biosynthesis, processing, transport, and storage in the ␤-cells of Langerhans islets (2, 3). Importantly, insulin and insulin analogues are readily adapted for expression in yeast as single-chain proinsulin-like precursors lacking the connecting peptide in the following configuration: amino acid residues 1-29 of the B-chain connected to the A-chain by a short removable mini-C-peptide and fused to the yeast prepro-␣ factor through a single dibasic cleavage site (secretory expression of insulin in yeast and in vitro enzymatic conversion of the precursor to insulin has recently been reviewed (4, 5)). Removal of the prepro-␣ factor by endoproteolytic cleavage by the Kex2 endoprotease in a late Golgi compartment generates a singlechain insulin precursor with self-association properties and structure essentially similar to that of human two-chain insulin (6, 7).Proteins exit the lumen of the endoplasmic reticulum (ER) 1 after folding, and frequently this is a rate-limiting step in secretion (8 -10). Herein we describe an improvement in eukaryotic secretion efficiency with application to insulin, using a strategy of structure-based rational engineering under the assumption that a more stably folded precursor molecule is ...

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“…In the years since the determination of the amino acid sequence of bovine insulin [I] and the subsequent elucidation of the three-dimensional structure of the protein hormone [2], many insulins from different species have been studied. These investigations have attempted to corrclate the physical and biological properties of the hormone to specific amino acid residues in the sequence [3,4], as well as to understand thc evolution of the hormone [5,6].…”

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confidence: 99%

Dogfish insulin

Bajaj

Blundell

Pitts

et al. 1983

European Journal of Biochemistry

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Insulin from an elasmobranch, the spiny dogfish (Squalus acantlzias) 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 A,, asparagine. The B,, 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 (A,Gln+ His, B, Glu +Pro, B,,Arg+Lys, B,,Phe+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 threedimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model-building studies.In the years since the determination of the amino acid sequence of bovine insulin [I] and the subsequent elucidation of the three-dimensional structure of the protein hormone [2], many insulins from different species have been studied. These investigations have attempted to corrclate the physical and biological properties of the hormone to specific amino acid residues in the sequence [3,4], as well as to understand thc evolution of the hormone [5,6].Immunocytochemical and biological studies suggest the presence of an insulin-like activity in prokaryotes such as Escherichia coli K12 [7], in various unicellular eukaryotes [S], in the protostomian [9] and the deuterostomian [lo] lines of invertebrate evolution [5]. However, the complete chemical characterization of these insulin immunoreactivities has not yet been possible. Though representative insulin sequences from teleosts reptiles, aves and mammals, are available, 21 lack of information exists at the level of lowermost gnathastomian vertebrates, namely the cartilaginous fish [5].Insulin from the hagfish (Myuine glutinosa) a jawless fish, is the phylogenically most original insulin to be scqucnced [5,1 I]. X-ray analysis [I21 suggests that this insulin has a spatial structure very similar to porcine insulin, even though the cyclostomian line seems to have diverged from the main line of vertebrate evolution nearly 500 million years ago [13]. Hagfish insulin has a very low biological potency when assayed in mammalian systems. Further it lacks the histidine at B,, which is responsible for the formation of zinc-insulin hexamers in most mammalian insulins. In contrast, several teleost insulins that have been studied can for...

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Atomic Positions in Rhombohedral 2-Zinc Insulin Crystals

Cited by 310 publications

References 6 publications

The protofilament structure of insulin amyloid fibrils

The protofilament structure of insulin amyloid fibrils

Engineering-enhanced Protein Secretory Expression in Yeast with Application to Insulin

Dogfish insulin

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