The Disulfide Structures of Scrambled Hirudins

Chang, Jui‐Yoa; Schindler, Patrick; Chatrenet, B.

doi:10.1074/jbc.270.20.11992

Cited by 47 publications

(54 citation statements)

References 25 publications

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“…For hirudin and TAP that contain 3 disulfides, there exists 14 possible scrambled isomers. Eleven species of scrambled hirudins have been isolated and structurally characterized (27). Similar numbers of scrambled TAP were identified as well (26).…”

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

confidence: 94%

“…Scrambled species are marked alphabetically (a-i) in both examples. The disulfide structures of all marked scrambled hirudins (27) and four well populated scrambled TAP (a, d, f, and g) (26) N, III, II, I, and R stand for the native, 3-disulfide scrambled, 2-disulfide, 1-disulfide, and fully reduced (0-disulfide) species, respectively. III, II, and I all consist of equilibrated isomers.…”

Section: Discussionmentioning

confidence: 99%

“…The disulfide folding pathways of hirudin and TAP have been analyzed in our laboratory (25,26). Many well populated folding intermediates of hirudin and TAP were isolated and structurally characterized (26,27). Demonstration of whether those folding intermediates exist as well along the unfolding pathway will be crucial to our understanding of the relative mechanism of their folding/unfolding.…”

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confidence: 99%

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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: 94%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Chang

1997

Journal of Biological Chemistry

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“…The protein does not lose the preference of the primary sequence for the formation of the native structure with native disulfide bridges, which remains the most abundant species. Additionally, when some secondary and tertiary interactions are disrupted, the bead-like structure, in which disulfide bridges are formed from the cysteines nearest to each other within the sequence, has the highest probability of formation (12,52). MFI is statistically the most probable disulfide conformation when reduced AAI protein is allowed to refold, and this is why its abundance increases as secondary and tertiary interactions are disrupted.…”

Section: Discussionmentioning

confidence: 99%

“…Chang and co-workers (9 -18) have studied extensively the oxidative folding mechanisms of various three-and four-disulfide proteins and have found different mechanisms, which reflect the variety of ways in which the native disulfide bonds in a protein are stabilized. Hirudin (11,12), tick anticoagulant protein (18), and potato carboxypeptidase inhibitor (10,14) share a common mechanism with a high heterogeneity of one-, two-, and three-disulfide intermediates, some of which have non-native disulfides. Epidermal growth factor (EGF) (13) forms both non-native three-disulfide isomers as well as a predominant species with two native disulfides (EGF-II).…”

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confidence: 99%

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Cemazar

Zahariev

Pongor

et al. 2004

Journal of Biological Chemistry

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Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus ␣-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.Cracking the code for folding a string of amino acid residues into a biologically active three-dimensional structure is still a major challenge of both chemical and biological interest. The information that determines the folding of a protein is contained solely in its amino acid residues (1). In proteins that contain cysteine residues the formation of disulfide bonds is an additional degree of freedom in the folding process. In these proteins a fusion between the recovery of the native tertiary structure (conformational folding) and the regeneration of native disulfide bonds is coupled in the oxidative folding process (2).Disulfide formation is important for in vivo folding of a considerable number of eukaryotic proteins and is a key posttranslational modification for the stabilization of folded structures. The study of oxidative folding in vitro can provide a detailed structural description in terms of the disulfide intermediate species present along the pathway of the complex folding process. A thorough account of an oxidative folding reaction should combine a study of the formation of covalently stable intermediate species and their disulfide bond connectivity and an analysis of the conformational assembly of these species. An established method for elucidation of the exact role that disulfides play in the process of achieving...

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Structures of scrambled disulfide forms of the potato carboxypeptidase inhibitor predicted by molecular dynamics simulations with constraints

et al. 2000

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The structures of two species of potato carboxypeptidase inhibitor with nonnative disulfide bonds were determined by molecular dynamics simulations in explicit solvent using disulfide bond constraints that have been shown to work for the native species. Ten structures were determined; five for scrambled A (disulfide bonds between Cys8–Cys27, Cys12–Cys18, and Cys24–Cys34) and five for the scrambled C (disulfide bonds Cys8–Cys24, Cys12–Cys18, and Cys27–Cys34). The two scrambled species were both more solvent exposed than the native structure; the scrambled C species was more solvent exposed and less compact than the scrambled A species. Analysis of the loop regions indicates that certain loops in scrambled C are more nativelike than in scrambled A. These factors, combined with the fact that scrambled C has one native disulfide bond, may contribute to the observed faster conversion to the native structure from scrambled C than from scrambled A. Results from the PROCHECK program using the standard parameter database and a database specially constructed for small, disulfide‐rich proteins indicate that the 10 scrambled structures have correct stereochemistry. Further, the results show that a characteristic feature of small, disulfide‐rich proteins is that they score poorly using the standard PROCHECK parameter database. Proteins 2000;40:482–493. © 2000 Wiley‐Liss, Inc.

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The Disulfide Structures of Scrambled Hirudins

Cited by 47 publications

References 25 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

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Structures of scrambled disulfide forms of the potato carboxypeptidase inhibitor predicted by molecular dynamics simulations with constraints

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