The Underlying Mechanism for the Diversity of Disulfide Folding Pathways

Chang, Jui‐Yoa; Li, Li; Bulychev, Alexey

doi:10.1074/jbc.275.12.8287

Cited by 58 publications

(57 citation statements)

References 23 publications

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“…In all conditions, the reduction of their three native disulfide bonds undergoes an apparent "all-or-none" mechanism in which only low amounts of partially reduced intermediates accumulate (25). Interestingly, a minor interconversion from IIIb to IIIa can be observed before the concerted reduction of its disulfide bonds.…”

Section: Resultsmentioning

confidence: 93%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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Tick carboxypeptidase inhibitor (TCI) is a small, disulfiderich protein that selectively inhibits metallocarboxypeptidases and strongly accelerates the fibrinolysis of blood clots. TCI consists of two domains that are structurally very similar, each containing three disulfide bonds arranged in an almost identical fashion. The oxidative folding and reductive unfolding pathways of TCI and its separated domains have been characterized by kinetic and structural analysis of the acid-trapped folding intermediates. TCI folding proceeds through a sequential formation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide species to reach the native form. Folding intermediates of TCI comprise two predominant 3-disulfide species (named IIIa and IIIb) and a major 6-disulfide scrambled isomer (Xa) that consecutively accumulate along the reaction and are strongly prevented by the presence of protein disulfide isomerase. This study demonstrates that IIIa and IIIb are 3-disulfide species containing the native disulfide pairings of the N-and C-terminal domains of TCI, respectively, and explains why the two domains of TCI fold sequentially and independently. Also, we show that the reductive unfolding of TCI undergoes two main independent unfolding events through the formation of IIIa and IIIb intermediates. Together, the comparison of the folding, stability, and inhibitory activity of TCI with those of the isolated domains reveals the reasons behind the two-domain nature of this protein: both domains contribute to the specificity and high affinity of its double-headed binding to carboxypeptidases. The results obtained herein provide valuable information for the design of more potent and selective TCI molecules.The mechanism by which an unfolded protein achieves its native state is one of the most complex problems in structural biology. Understanding the sequence of folding events in proteins may not only help to predict protein structures from amino acid sequences but also provide invaluable information to a variety of related fields, such as protein design or protein misfolding associated with pathological diseases (1, 2). A number of studies concerning folding have been focused on small, disulfide-rich proteins, given that the chemistry of disulfide bond formation allows the trapping and subsequent characterization of the folding intermediates that accumulate, something difficult to attain with disulfide-free proteins (3, 4). In oxidative folding, reduced and denatured proteins are allowed to recover both its native disulfide bonds and native structures in the absence or presence of redox agents. Bovine pancreatic trypsin inhibitor, ribonuclease A, lysozyme, ␣-lactalbumin, and hirudin have been extensively investigated using this methodology (5-9). The heterogeneity and structures of the intermediates that occur during the process define their respective folding landscapes (10).Although most of the analyzed proteins comprise a small, single-domain fold containing three or four disulfide bonds, a great diversity of folding mechanisms is observ...

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Section: Resultsmentioning

confidence: 93%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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“…The so-called "all-or-none" reductive unfolding mechanism observed for native AAI is shared by other small cystine-rich proteins, such as hirudin, potato carboxypeptidase inhibitor, and tick anticoagulant protein (9). The native disulfides of such proteins are stabilized in a concerted and interdependent manner because they do not allow for intermediate species when the native disulfide bonds are reduced.…”

Section: Discussionmentioning

confidence: 96%

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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“…The in vitro oxidative folding pathways of proteins are quite varied (35)(36)(37), and although fully oxidized intermediates with nonnative disulfides are known to occur (35,(38)(39)(40), the intermediates studied here (MFI, I-5, and I-6) appear to be the first such folding intermediates possessing a vicinal disulfide bond. The predominance of the vicinal disulfide bridge is conspicuous because of the 15 theoretically possible fully oxidized intermediates of AAI, the three that contain a vicinal bridge were all detected experimentally (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Cemazar

Zahariev

Lopez

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The oxidative folding of the Amaranthus ␣-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by 1 H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.T he cystine knot, or the knottin fold, is a compact structure consisting of a short triple-stranded antiparallel ␤-sheet reinforced by three disulfide bridges that form a topological knot [see Amaranthus ␣-amylase inhibitor (AAI) structure, Fig. 1A Inset]. It is found both in small peptides and as a domain of larger proteins (1) and in addition, it is a recurrent substructure of larger cysteine-rich motifs such as the well known conotoxin fold (2). Despite their small size, cystine knots can fulfill a large variety of functions ranging from ion-channel blocking to enzyme inhibition, which makes them ideal protein engineering scaffolds for industrial applications (3, 4). Because cystine knots occur in many unrelated species (fungi, plants, snails, and spiders, etc.), it is presumed that this fold has emerged via convergent evolution (2). The compactness and versatility of this fold are best illustrated by the fact that both the shortest peptide inhibitor of ␣-amylases, AAI [32 amino acids (Fig. 1 A)] (5-8) and the shortest lectin molecule known so far (9), are members of this family.Cystine knot peptides are known to form readily in vitro under the conditions of oxidative folding (4). In this work, we describe an analysis of the oxidative folding intermediates of AAI, which shows that the native protein (N) forms via several fully oxidized intermediates with nonnative disulfide bonds, including the vicinal disulfide 17-18. NMR spectroscopy revealed that the main folding intermediate has a structure as compact as the completely folded peptide, comprising a number of slowly interconverting backbone conformations. On the basis of the experimental data, we suggest that the compactness, a prerequisi...

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The Underlying Mechanism for the Diversity of Disulfide Folding Pathways

Cited by 58 publications

References 23 publications

Characterizing the Tick Carboxypeptidase Inhibitor

Characterizing the Tick Carboxypeptidase Inhibitor

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

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