Controlling the speed of hirudin folding

Chang, Jui-Yao

doi:10.1042/bj3000643

Cited by 68 publications

(104 citation statements)

References 37 publications

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“…The reaction undergoes complex intermediates that consist of 2-and 1-disulfide species. Judging from the HPLC pattern, the composition of these unfolding intermediates are indistinguishable from those found along the pathway of oxidative folding of hirudin (25,31). Furthermore, the rate of reductive unfolding and the composition of unfolding intermediates are largely unaffected by the presence of 6 M GdmCl (Fig.…”

Section: Unfolding Of the Native Protein To The State Of Scrambledmentioning

confidence: 76%

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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Section: Unfolding Of the Native Protein To The State Of Scrambledmentioning

confidence: 76%

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

Chang

1997

Journal of Biological Chemistry

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“…The addition of arginine which suppresses aggregation [21,22], glycerol or sucrose which enhance protein stability in solution [23,24], also did not significantly increase the yields of biologically active E5/K5-coil-EGF after refolding (data not shown). It has been reported that cysteine has a higher redox potential than glutathione [25,26]. Results published by Chang et al [27] demonstrated that the use of cysteine increased not only the rate at which hEGF was refolded but also its yield [13,27].…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Lenferink

Pinard

et al. 2009

Protein Expression and Purification

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“…By comparison, the rate constant k NfII is greater than k IIfR by a factor of 250,000. The vast difference of these two rate constants reflects both the weak nature of the Cys 14 , is about 10-fold more susceptible to DTT reduction than non-native disulfides of scrambled hirudin and TAP.…”

Section: Diversity Of Disulfide Folding Pathwaysmentioning

confidence: 99%

“…They exist in high concentration along the folding pathways of hirudin, PCI, and TAP, as well as EGF. With the exception of EGF, accumulation of scrambled isomers as folding intermediates can be greatly enhanced by allowing the folding in the buffer containing oxidized glutathione or cystine (14). For instance, when folding of PCI was performed in the presence of 0.5 mM cystine, more than 98% of the total protein was trapped as scrambled species before trace amounts of the native PCI even appeared (10).…”

mentioning

confidence: 99%

The Underlying Mechanism for the Diversity of Disulfide Folding Pathways

Chang¹,

Li²,

Bulychev³

2000

Journal of Biological Chemistry

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The disulfide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) is characterized by the predominance of folding intermediates with native-like structures. Our laboratory has recently analyzed the folding pathway(s) of four 3-disulfide-containing proteins, including hirudin, potato carboxypeptidase inhibitor, epidermal growth factor, and tick anticoagulant peptide. Their folding mechanism(s) differ from that of BPTI by 1) a higher degree of heterogeneity of 1-and 2-disulfide intermediates and 2) the presence of 3-disulfide scrambled isomers as folding intermediates. To search for the underlying causes of these diversities, we conducted kinetic analyses of the reductive unfolding of these five proteins. The experiment of reductive unfolding was designed to evaluate the relative stability and interdependence of disulfide bonds in the native protein. It is demonstrated here that among these five proteins, there exists a striking correlation between the mechanism(s) of reductive unfolding and that of oxidative folding. Those proteins with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of heterogeneity of folding intermediates and the accumulation of scrambled isomers along the folding pathway. A sequential reduction of the native disulfide bonds is associated with the presence of predominant intermediates with native-like structures. -Cys 38 ) (1). Unfolded and fully reduced BPTI refolds spontaneously to form the native structure under selected redox conditions. The mechanism of BPTI unfolding and refolding has been a subject of intensive investigation and represents one of the best and most extensively characterized models (2-7). The folding pathway of BPTI was elucidated by trapping and structural characterization of disulfide bond intermediates. The original model of Creighton (2-5) identified seven predominant intermediates, two 1-disulfide species and five 2-disulfide species, with 75% of the disulfide bonds being native. The native disulfide of BPTI (Cys 30 -Cys 51 ) was found to be a major component of both 1-and 2-disulfide intermediates. In the revised model proposed by Weissman and Kim (6, 7), five well populated intermediates, two 1-disulfide and three 2-disulfide species, were described, and all of them were shown to contain only native disulfide bonds. Another important feature of the BPTI folding pathway is the kinetic role of a 2-disulfide intermediates that contains two of disulfide bonds of the native BPTI (Cys 30 -Cys 51 , Cys 5 -Cys 55 ) (N SH SH ) (3,5,6,8). N SH SH is the immediate precursor of the native BPTI. Formation of the third native disulfide, Cys14 -Cys38, completes the folding and accounts for the final step of the BPTI folding pathway. Prevalence of the native disulfide bonds along the folding pathway has major implications. It implies that non-covalent specific interactions that stabilize the native BPTI and local structures of BPTI play a crucial role in guiding the folding in its early stages and hence dictate the ...

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Controlling the speed of hirudin folding

Cited by 68 publications

References 37 publications

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

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

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

The Underlying Mechanism for the Diversity of Disulfide Folding Pathways

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