Solution Structure of an Engineered Insulin Monomer at Neutral pH

Olsen, Henrik Baare; Ludvigsen, Svend; Kaarsholm, Niels C.

doi:10.1021/bi960292+

Cited by 129 publications

(221 citation statements)

References 51 publications

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“…As a control, both isomers are stable in similar buffer without free thiol catalyst (data not shown). This disulfide rearrangement experiment further confirmed that these two isomers of [1][2][3][4][5][6][7][8][9]-PIP are thermodynamically controlled folding products with different disulfide linkages. If we compare the disulfide reshuffling process of isomers 1 and 2 at different time points, we can see that the conversion from isomer 1 into isomer 2 is faster than the reverse direction (data not shown).…”

Section: [1-9]pip Was Expressed In Yeast Assupporting

confidence: 52%

“…The expression plasmids encoding Expression, Purification, and Identification of [1][2][3][4][5][6][7][8][9]PIP, [1][2][3][4]PIP, [1][2][3][4][5][6][7][8][9][10]Mini-IGF-1, and [1][2][3][4][5]…”

Section: Methodsmentioning

confidence: 99%

“…, and pVT102-U/RMFL- [1][2][3][4][5]mini-IGF-1 were transformed into S. cereVisiae cells of strain XV700-6B (leu2 ura3 pep4). The transformed yeast cells were cultured in a 16-L fermenter and the secreted target protein was purified from the medium in four steps (16,19).…”

Section: Methodsmentioning

confidence: 99%

“…The purified isomers 1 and 2 of [1-9]-PIP, as well as [1][2][3][4][5][6][7][8][9][10]mini-IGF-1, were dissolved in 0.1 M Tris-HCl and 1 mM EDTA buffer (pH 8.7) with 8 M urea. The final protein concentration was 1 mg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…The sample, [1][2][3][4][5][6][7][8][9]PIP (isomer 2), [1][2][3][4][5][6][7][8][9][10]mini-IGF-1, native form of mini-IGF-1, and PI, were dissolved in the different redox buffer (0.1 M Tris-HCl and 1 mM EDTA, pH 8.7) at a final concentration of 0.1 mg/ mL. In the redox buffer, the ratio of GSH and GSSG was 5 mM/5 mM.…”

Section: Methodsmentioning

confidence: 99%

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Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

et al. 2004

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Although insulin and insulin-like growth factor-1 (IGF-1) belong to one family, insulin folds into one thermodynamically stable structure, while IGF-1-folds into two thermodynamically stable structures (native and swap forms). We have demonstrated previously that the bifurcating folding behavior of IGF-1 is mainly controlled by its B-domain. To further elucidate which parts of the sequences determine their different folding behavior, by exchanging the N-terminal sequences of mini-IGF-1 and recombinant porcine insulin precursor (PIP), we prepared four peptide models: [1-9]PIP, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1 by means of protein engineering, and their disulfide rearrangement, V8 digestion, circular dichroic spectra, disulfide stability, and in vitro refolding were investigated. Among them only [1-9]-PIP, like mini-IGF-1/IGF-1, was expressed in yeast as two isomers: isomer 1 (corresponding to swap IGF-1) and isomer 2 (corresponding to native IGF-1), which are supported by the experimental results of disulfide rearrangements, peptide mapping of V8 endoprotenase digests, circular dichroic analysis, in vitro refolding, and disulfide stability analysis. The other peptide models, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1, fold into one stable structure as PIP does, which indicates that sequence 1-4 of mini-IGF-1 is important for the folding behavior of mini-IGF-1/IGF-1 but not sufficient to lead to a bifurcating folding. The results demonstrated that the folding information, by which mini-IGF-1/IGF-1-folds into two thermodynamically structures, is encoded/written in its sequence 1-9, while sequences 1-10 of B chain in insulin/PIP play an important role in the guide of its unique disulfide pairing during the folding process.Insulin is a structurally and functionally well-characterized small globular protein with A-and B-chains linked by three disulfides. Its three-dimensional structure has been well defined by X-ray crystallography (1) and NMR (2-4) since the 1970s. Insulin-like growth factor-1 (IGF-1) 1 is a 70-residue single-chain globular protein composed of B-, C-, A-, and D-domains from the N-terminus to the C-terminus (5). Its B-and A-domains are homologous to the B-and A-chains of insulin, respectively; its 12-residue C-domain is analogous to the C-peptide of proinsulin, but they share no homology; its C-terminal 8-residue D-domain has no counterpart in insulin. The three-dimensional structure of IGF-1 has also been well-defined by X-ray crystallography (6, 7) and NMR (8, 9). Insulin and IGF-1 belong to the same superfamily. They have a common structural motif (10, 11) and share homologous sequence, similar three-dimensional structure (1,6,7), and a common ancestor (12,13). Both mainly consist of three R-helical segments (A2-A8, A13-A19, and B9-B19 in insulin; 8-18, 42-49, and 54-61 in IGF-1) in the A-and B-chains/domains; the three R-helical segments are stabilized by three identical disulfides (A6-A11, A7-B7, and A20-B19 in insulin; 47-52, 6-48, and 18-61 in IGF-1); and when IGF-1...

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Section: [1-9]pip Was Expressed In Yeast Assupporting

confidence: 52%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

et al. 2004

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Growth factor receptors: Structure, mechanism, and drug discovery

McInnes

Sykes

1997

Biopolymers

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The focus of this review is the relationship between the three‐dimensional structure of ligands of the various members of the growth factor receptor tyrosine kinase superfamily and their interaction with the cognate receptor. Particular attention is given to the transforming growth factor—α, epidermal growth factor (EGF); nerve growth factor, neurotrophin; and insulin‐like growth factor—1 (IGF‐1), insulin systems since these have been extensively studied in recent years. The three receptor types, which bind these ligands, are the epidermal growth factor receptor family (erb B receptors), the neurotrophin or Trk receptor family, and IGF‐1/insulin receptors, respectively, and represent three distinct members of the tyrosine kinase superfamily. For each of these, formation of the ligand‐receptor complex initiates the signal transduction cascade through autophosphorylation by the intracellular tyrosine kinase domain. The extracellular portion of the receptor that contains the ligand binding domain in these systems varies significantly in organization in each case. For the EGF receptor system, ligand binding induces homo‐ and heterodimerization of the receptor leading to activation of the intracellular kinase. For the Trk receptor system, homodimerization of receptors has been shown to occur, although a second receptor, p75, is also required for high affinity binding of neurotrophins and for enhanced sensitivity of tyrosine kinase activation at low ligand concentrations. The IGF‐1 and insulin receptors exist as covalent cross‐linked dimers where each monomer is composed of two subunits. The aim of this review is also to discuss the mechanism of ligand‐receptor interaction for each of these cases; however, since no structural information is yet available for the ligand‐receptor complex, the discussion will largely be centered on the molecular requirements of ligand binding. As these receptors are activated through the ligand binding site on the extracellular domain, this represents a possible target for pharmacological intervention by inhibition or stimulation of this portion of the receptor. Thus from a drug design perspective, the focus of this review is to discuss progress in the development of agonists or antagonists of the ligand for these receptors. © 1998 John Wiley & Sons, Inc. Biopoly 43: 339–366, 1997

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Growth factor receptors: Structure, mechanism, and drug discovery

McInnes¹,

Sykes²

1997

Biopolymers

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The focus of this review is the relationship between the three-dimensional structure of ligands of the various members of the growth factor receptor tyrosine kinase superfamily and their interaction with the cognate receptor. Particular attention is given to the transforming growth factor-alpha, epidermal growth factor (EGF); nerve growth factor, neurotrophin; and insulin-like growth factor-1 (IGF-1), insulin systems since these have been extensively studied in recent years. The three receptor types, which bind these ligands, are the epidermal growth factor receptor family (erb B receptors), the neurotrophin or Trk receptor family, and IGF-1/insulin receptors, respectively, and represent three distinct members of the tyrosine kinase superfamily. For each of these, formation of the ligand-receptor complex initiates the signal transduction cascade through autophosphorylation by the intracellular tyrosine kinase domain. The extracellular portion of the receptor that contains the ligand binding domain in these systems varies significantly in organization in each case. For the EGF receptor system, ligand binding induces homo- and heterodimerization of the receptor leading to activation of the intracellular kinase. For the Trk receptor system, homodimerization of receptors has been shown to occur, although a second receptor, p75, is also required for high affinity binding of neurotrophins and for enhanced sensitivity of tyrosine kinase activation at low ligand concentrations. The IGF-1 and insulin receptors exist as covalent cross-linked dimers where each monomer is composed of two subunits. The aim of this review is also to discuss the mechanism of ligand-receptor interaction for each of these cases; however, since no structural information is yet available for the ligand-receptor complex, the discussion will largely be centered on the molecular requirements of ligand binding. As these receptors are activated through the ligand binding site on the extracellular domain, this represents a possible target for pharmacological intervention by inhibition or stimulation of this portion of the receptor. Thus from a drug design perspective, the focus of this review is to discuss progress in the development of agonists or antagonists of the ligand for these receptors.

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Solution Structure of an Engineered Insulin Monomer at Neutral pH

Cited by 129 publications

References 51 publications

Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

Growth factor receptors: Structure, mechanism, and drug discovery

Growth factor receptors: Structure, mechanism, and drug discovery

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