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

Abstract

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...

The Unfolding Pathway and Conformational Stability of Potato Carboxypeptidase Inhibitor

“…The mechanism of "all-or-none" disulfide reduction has been shown in numerous single domain proteins (11). The dramatic difference in stability between the native and non-native (scrambled) disulfide bonds has also been demonstrated in other proteins.…”

“…Unlike the native disulfide bonds that are stabilized by noncovalent interactions, non-native disulfide bonds of scrambled proteins are considerably more fragile and can be readily reduced by 0.5-1 mM dithiothreitol within 5-10 min of incubation at room temperature. This phenomenon has been shown with scrambled isomers of RNase A (26), hirudin, and tick anticoagulant peptide (11).…”

“…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).…”

A Localized Specific Interaction Alters the Unfolding Pathways of Structural Homologues

“…[9][10][11][12] Reductive unfolding, the converse of oxidative folding, is the process by which a disulfidebond-containing protein loses its native structure (unfolds) through the (successive) reduction of its disulfide bonds. [13][14][15][16][17][18][19][20][21] In reductive unfolding, protein disulfide bonds scattered throughout the macromolecular architecture can serve as reporter groups for probing local fluctuations within the folded polypeptide; these fluctuations eventually lead to the unfolding of the whole molecule. 17 By assessing the relative susceptibilities of individual disulfide bonds to reduction by an extrinsic reducing agent such as dithiothreitol (DTT red ), it is possible to determine whether a particular disulfide bond is preferentially reduced; the region in which the easily reducible disulfide bond resides is the most flexible one and, therefore, prone to unfolding.…”

An Internal Cysteine Is Involved in the Thioredoxin-dependent Activation of Sorghum Leaf NADP-malate Dehydrogenase

“…Until now, isomerization of disulfides was shown to be an obligate step of the protein folding pathway (24), but recently it has been demonstrated that isomerization could also occur upon an unfolding triggered by disulfide reduction (25). Correct protein folding is considered to be catalyzed by protein-disulfide isomerase (26), which is a strong oxidant.…”