Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology

Raschdorf, F.; Dahinden, R.; Maerki, W.; Richter, Wilhelm J.; Merryweather, James P.

doi:10.1002/bms.1200160102

Cited by 64 publications

(37 citation statements)

References 14 publications

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“…Mass spectrometry is a very attractive possibility for this: one would be asking questions about a known peptide, rather than establishing complete sequence de novo. It has already been used for disulfide analysis in a number of cases (Raschdorf et al, 1988;Hidaka et al, 1990;Zhou & Smith, 1990) and seems especially appropriate for handling complex digests of larger proteins. Combined with the method described here for differentially labeling the bridges, it may be feasible to analyze undigested peptides of moderate size.…”

Section: Scope and Limitations Of The Methodsmentioning

confidence: 99%

Disulfide structures of highly bridged peptides: A new strategy for analysis

1993

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A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide Chain be split between close or adjacent Cys residues. The water-soluble tris-(2-~arboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra-and interchain disulfides; -Cys.Cys-pair), endothelin and apamin (two disulfides; -Cys.x.Cys-pair), conotoxin GI and isomers (two disulfides; -Cys.Cys-pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys-pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.

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Section: Scope and Limitations Of The Methodsmentioning

confidence: 99%

Disulfide structures of highly bridged peptides: A new strategy for analysis

1993

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“…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%

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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“…This procedure presents the unique advantages that separation of the proteolytic mixture and sequencing of peptides are often unnecessary, so that low amounts of the original protein are required. This procedure has been successfully applied to model proteins such as insulin [8], hen egg-white lysozyme and bovine ribonuclease A [9], duck egg-white lysozyme [lo], as well as to the identification of disulfide bridges in a bacterial inhibitor of amylase [Ill, in human insulin-like growth factor [12] and very recently in a human tissue inhibitor of metalloproteinases [13].…”

mentioning

confidence: 99%

Assignment of the five disulfide bridges in an α‐amylase inhibitor from wheat kernel by fast‐atom‐bombardment mass spectrometry and Edman degradation

Poerio

Caporale

Carrano

et al. 1991

European Journal of Biochemistry

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The assignment of the five disulfide bridges in an a-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.

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Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology

Cited by 64 publications

References 14 publications

Disulfide structures of highly bridged peptides: A new strategy for analysis

Disulfide structures of highly bridged peptides: A new strategy for analysis

The Unfolding Pathway and Conformational Stability of Potato Carboxypeptidase Inhibitor

Assignment of the five disulfide bridges in an α‐amylase inhibitor from wheat kernel by fast‐atom‐bombardment mass spectrometry and Edman degradation

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