Disulfide exchange folding of insulin-like growth factor I

Hober, Sophia; Forsberg, Goeran; Palm, Gunnar; Hartmanis, Maris; Nilsson, Björn M.

doi:10.1021/bi00121a024

Cited by 105 publications

(235 citation statements)

References 42 publications

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“…6). The structure of X-PCI-h was deduced from the identification of two major thermolytic peptides, h-4 and h-10 (Table I) 34 was confirmed by the structures of thermolytic peptides a-5, a-6, and a-11 (Table I). The third disulfide, Cys 8 -Cys 12 , was found in the peptide a-1 generated by Lys-C digestion.…”

Section: Std X-pci)mentioning

confidence: 72%

“…The third disulfide, Cys 8 -Cys 12 , was found in the peptide a-1 generated by Lys-C digestion. Two minor peptides (a-7 and a-8) containing Cys 27 -Cys 34 were also found within the Lys-C digest of X-PCI-a. This is because of nonspecific cleavage, possibly by a contaminant in thermolysin.…”

Section: Std X-pci)mentioning

confidence: 92%

“…There are similar examples. The insulin-like growth factor-1 folds into two distinct disulfide isomers, one native and one scrambled, with a molar ratio of 60:40 under physiological conditions (15,(33)(34)(35)(36). The inability of PCI to refold quantitatively to the native state could be related to the fact that this protein is synthesized in vivo as a precursor protein, which has both N-terminal and C-terminal proregions (37).…”

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confidence: 99%

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The Unfolding Pathway and Conformational Stability of Potato Carboxypeptidase Inhibitor

Chang¹,

Li²,

Canals³

et al. 2000

Journal of Biological Chemistry

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The unfolding and denaturation curves of potato carboxypeptidase inhibitor (PCI) were investigated using the technique of disulfide scrambling. In the presence of denaturant and thiol initiator, the native PCI denatures by shuffling its native disulfide bonds and converts to form a mixture of scrambled PCI that consists of 9 out of a possible 14 isomers. The denaturation curve is determined by the fraction of native PCI converted to scrambled isomers under increasing concentrations of denaturant. The concentration of guanidine thiocyanate, guanidine hydrochloride, and urea required to denature 50% of the native PCI was found to be 0.7, 1.45, and 8 M, respectively. The PCI unfolding curve was constructed through the analysis of structures of scrambled isomers that were denatured under increasing concentrations of denaturant. These results reveal the existence of structurally defined unfolding intermediates and a progressive expansion of the polypeptide chain. ) as a fraction of total denatured PCI was shown to be directly proportional to the strength of the denaturing condition. Furthermore, the PCI sequence was unable to fold quantitatively into a single native structure. Under physiological conditions, the scrambled isomers of PCI that constitute about 4% of the protein were in equilibrium with native PCI.The conformational stability of proteins has been regularly studied with denaturation curves, where changes in parameters that can be correlated with the three-dimensional structure of the protein are measured as a function of denaturant concentration in the protein solution. The measured parameter is usually a physicochemical property such as UV absorbance, fluorescence, or circular dichroism of the protein of interest (1, 2). Whereas denaturation of a protein can be assessed by many different parameters, most conventional approaches are not able to determine the degree of unfolding of a protein in a given denatured state, the presence and concentration of intermediates, or the characteristics of the denatured state. There is growing evidence that within the ensemble of conformations that constitute the denatured states of a protein, there is a population of molecules that still possess residual structure (3-7), i.e. the denatured state does not imply a completely unfolded conformation. Characterizing the denatured state of proteins is a subject of increasing interest for the understanding of protein folding and stability (8 -10).Recently, we have proposed a new methodology for studying stability toward denaturants and the unfolding pathway of disulfide-containing proteins (11,12). This approach is based on the observation that when disulfide-containing proteins are treated with denaturants in the presence of a trace amount of a thiol initiator, proteins can reversibly shuffle their disulfide bonds, leading to a mixture of native and disulfide-scrambled species (fully oxidized species that have at least two non-native disulfide bridges) (13). The composition of this mixture depends on the type of denaturant a...

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Section: Std X-pci)mentioning

confidence: 72%

Section: Std X-pci)mentioning

confidence: 92%

mentioning

confidence: 99%

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The Unfolding Pathway and Conformational Stability of Potato Carboxypeptidase Inhibitor

Chang¹,

Li²,

Canals³

et al. 2000

Journal of Biological Chemistry

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“…Like chain combination, the yield of proinsulin folding is also enhanced at pH 9.5-11 because of reduced aggregation of unfolded and partially folded species (4,48). Redox-coupled folding mechanisms of a single-chain proinsulin analog (porcine insulin precursor; PIP) and IGF-I are notable for populated one-and two-disulfide intermediates identified by peptide mapping (5,49,50). A key role is played in each case by formation of cystine A20 -B19 (or IGF-I homolog 18 -61).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Insulin Chain Combination

Hua

Chen

Jia

et al. 2002

Journal of Biological Chemistry

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The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal ␣-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the Nterminal ␣-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity.1 H NMR studies of a representative analog lacking invariant side chains Ile A2 and Val A3 (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise nativelike structure. That this and related partial folds retain efficient disulfide pairing suggests that the native Nterminal ␣-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.

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“…Insulin can fold into one unique threedimensional structure, while IGF-1 is a protein that encodes two thermodynamically stable folding structures. In 1992, Hober et al (14) observed that in vitro refolding of IGF-1 resulted in two major products with different disulfide pairing. One product was native IGF-1, the other product was named swap IGF-1 because of its nonnative disulfide bonds (6-47 and 48-52).…”

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confidence: 99%

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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Disulfide exchange folding of insulin-like growth factor I

Cited by 105 publications

References 42 publications

The Unfolding Pathway and Conformational Stability of Potato Carboxypeptidase Inhibitor

The Unfolding Pathway and Conformational Stability of Potato Carboxypeptidase Inhibitor

Mechanism of Insulin Chain Combination

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

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