Production of disulfide‐linked hirudin dimer by in vitro folding

Chang, Jui‐Yoa; Grossenbacher, H.; Meyhack, Bernd; Maerki, W.

doi:10.1016/0014-5793(93)81607-2

Cited by 9 publications

(7 citation statements)

References 18 publications

(9 reference statements)

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“…When the reshuffling is catalyzed by the build-in Cys, the reaction is essentially intramolecular. Similar phenomenon of build-in Cys-promoted disulfide shuffling has been observed in the production of the hirudin dimer (38) and the folding of pro BPTI (39,40).…”

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confidence: 69%

Isomers of Epidermal Growth Factor with Ser ⇒ Cys Mutation at the N-Terminal Sequence: Isomerization, Stability, Unfolding, Refolding, and Structure

Lu¹,

Jiang²,

Chang³

2005

Biochemistry

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The structure of human epidermal growth factor (EGF, 53 amino acids) comprises three distinct loops (A, B, and C) connected correspondingly by the three native disulfide bonds, Cys(6)-Cys(20), Cys(14)-Cys(31), and Cys(33)-Cys(42). The connection of Cys(6) and Cys(20) forming the N-terminal A loop is essential for the biological activity of EGF [Barnham et al. (1998) Protein Sci. 7, 1738-1749] and has also been shown to represent a major kinetic trap in the oxidative folding of EGF [Chang et al. (2001) J. Biol. Chem. 276, 4845-4852]. To further understand the chemical nature of this kinetic trap, we have prepared three EGF mutants each with a single Ser --> Cys mutation at Ser residues (Ser(2), Ser(4), and Ser(9)) flanking Cys(6). This allows competition between Cys(6) and mutated Cys(2), Cys(4), and Cys(9) to link with Cys(20) and to form EGF isomers containing different sizes of the A loop. The results show that, in the cases of EGF(S2C) and EGF(S4C), native Cys(6)-Cys(20) is favored over Cys(2)-Cys(20) and Cys(4)-Cys(20) by 4.5- and 9-fold, respectively, in the state of equilibrium. However, in the case of EGF(S9C), a non-native Cys(9)-Cys(20) is thermodynamically more stable than the native Cys(6)-Cys(20) by a free-energy difference (DeltaG degrees ) of 1.12 kcal/mol. Implications of these data in the formation of kinetic trap of EGF folding are discussed. Stabilized isomers of EGF were further generated from denaturation of wild-type and mutant EGF via the method of disulfide scrambling. Properties of these diverse isomers of EGF, including their isomerization, stability, unfolding, refolding, and disulfide structures, are described in this paper.

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confidence: 69%

Isomers of Epidermal Growth Factor with Ser ⇒ Cys Mutation at the N-Terminal Sequence: Isomerization, Stability, Unfolding, Refolding, and Structure

Lu¹,

Jiang²,

Chang³

2005

Biochemistry

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“…All of them could be fully reduced by 0.5-1 mM DTT within 10 -20 min at room temperature (23°C). The stability of scrambled disulfides is also comparable with the inter-disulfide bond that cross-links two intact hirudin monomers (32).…”

Section: Reduction Of Scrambled Proteins (Stage Ii)-thementioning

confidence: 88%

A Two-Stage Mechanism for the Reductive Unfolding of Disulfide-containing Proteins

Chang

1997

Journal of Biological Chemistry

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Reductive unfolding of disulfide-containing proteins can be experimentally dissected into two distinct stages. In the presence of denaturant and thiol catalyst, native proteins unfold by reshuffling their native disulfides and convert to a mixture of scrambled structures. Subsequent reduction of the disulfide bonds of scrambled proteins requires only mild concentration of reductant (0.2-0.5 mM reduced dithiothreitol) and undergoes intermediates that consist of highly heterogeneous disulfide isomers. These properties have been characterized with three cystine-containing proteins, namely hirudin, tick anticoagulant peptide (TAP), and bovine ribonuclease A. In the cases of hirudin and TAP, most intermediates observed during the oxidative folding were found to exist along the pathway of reductive unfolding as well.Elucidation of the pathway of protein folding and unfolding has remained one of the most demanding tasks in protein chemistry (1-7). The challenge stems primarily from the difficulty of analyzing the transient intermediates involved in these pathways. For proteins containing disulfide bonds, unfolding and refolding are generally followed by reduction and oxidation of the native disulfides (8, 9). Since breaking and formation of disulfide bonds can be chemically trapped and characterized (10), the disulfide unfolding and folding pathways can thus be constructed on the basis of the heterogeneity and structures of the trapped intermediates (11-15).Unfolding of a disulfide-containing protein can be achieved conventionally by either reduction of disulfide bonds in the absence of denaturant (reductive unfolding) (11,(15)(16)(17) or by denaturation (e.g. GdmCl) in the absence of reductant (disulfide-intact unfolding) (18,19). In the latter case, the unfolded protein retains intact native disulfides. So far, the pathway of reductive unfolding has been investigated with limited numbers of proteins (11, 15, 20 -23). BPTI and RNase A are two most notable examples. These data, however, were largely obtained from the reductive unfolding performed in the absence of denaturant (11,15).During our recent analysis of the properties of scrambled hirudins (24), we have observed that denaturant and reductant can actually be applied in a two-step manner to unfold native protein. In an alkaline solution including strong denaturant and thiol catalyst, the native protein unfolds to form a mixture of scrambled species that admit mostly non-native disulfides but still retain the intact number of disulfide bonds. This is followed by reduction of the disulfides of scrambled proteins. This approach is distinguished from the conventional techniques described above. In this study, this new method was applied to characterize the unfolding pathways of three disulfide-containing proteins, namely hirudin (3 disulfides), tick anticoagulant peptide (TAP) 1 (3-disulfides), and ribonuclease A (RNase A) (4 disulfides). The disulfide folding pathways of hirudin and TAP have been analyzed in our laboratory (25,26). Many well populated folding inter...

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“…(a) The presence of an extra internal Cys, similar to that existing in the proBPTI sequence, serves as a This was also demonstrated in the folding of a hirudin variant with Asp 33 f Cys 33 mutation (Cys 33 -hirudin). 64 Because of the presence of an extra Cys, reduced Cys 33 -hirudin is capable of folding via X-3SS intermediates to form the native structure in the buffer without thiol catalyst. (b) The kinetic property of [proN 0 ] is not alone.…”

Section: ' the Pathway Of Oxidative Folding Of Pro-bpti: A Bpti-hirud...mentioning

confidence: 99%

Diverse Pathways of Oxidative Folding of Disulfide Proteins: Underlying Causes and Folding Models

Chang¹

2011

Biochemistry

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The pathway of oxidative folding of disulfide proteins exhibits a high degree of diversity, which is manifested mainly by distinct structural heterogeneity and diverse rearrangement pathways of folding intermediates. During the past two decades, the scope of this diversity has widened through studies of more than 30 disulfide-rich proteins by various laboratories. A more comprehensive landscape of the mechanism of protein oxidative folding has emerged. This review will cover three themes. (1) Elaboration of the scope of diversity of disulfide folding pathways, including the two opposite extreme models, represented by bovine pancreatic trypsin inhibitor (BPTI) and hirudin. (2) Demonstration of experimental evidence accounting for the underlying mechanism of the folding diversity. (3) Discussion of the convergence between the extreme models of oxidative folding and models of conventional conformational folding (framework model, hydrophobic collapse model).

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Production of disulfide‐linked hirudin dimer by in vitro folding

Cited by 9 publications

References 18 publications

Isomers of Epidermal Growth Factor with Ser ⇒ Cys Mutation at the N-Terminal Sequence: Isomerization, Stability, Unfolding, Refolding, and Structure

Isomers of Epidermal Growth Factor with Ser ⇒ Cys Mutation at the N-Terminal Sequence: Isomerization, Stability, Unfolding, Refolding, and Structure

A Two-Stage Mechanism for the Reductive Unfolding of Disulfide-containing Proteins

Diverse Pathways of Oxidative Folding of Disulfide Proteins: Underlying Causes and Folding Models

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