Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically

Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX3C Zn2+-finger motif. While disulphide bond formation between the Cys residues of this motif is essential for complex formation by the small Tim proteins, the specific role of Zn2+-binding during the import and assembly of these proteins is not clear. In this study, we investigated the effects of the biologically relevant thiol-disulphide redox molecule, glutathione, and Zn2+-binding on the oxidative folding of yeast mitochondrial Tim10 using both biochemical and biophysical methods in vitro. We show that, whilst oxidized Tim10 cannot be reduced by reduced glutathione, reduced Tim10 is effectively oxidized at levels of glutathione comparable to those found in the cytosol. The oxidized Tim10 generated in the presence of glutathione is competent for complex formation with its partner protein Tim9, confirming it has a native fold. The standard redox potential of Tim10 at pH 7.4 was determined to be -0.32 V, confirming that Tim10 is a much stronger reductant than glutathione (-0.26 V, at pH 7.4) and could therefore be oxidized rapidly by oxidized glutathione in the cytosol. However, we found that Zn2+-binding can stabilize the reduced Tim10, decreasing the rate of the oxidative folding more than tenfold. In addition, we show that protein disulphide isomerase can catalyse the oxidative folding of Tim10 provided that Zn2+ was removed. We propose that Zn2+-binding is essential to maintain the protein in a reduced and import-competent state in the cytosol, and that zinc has to be removed after the protein is imported into mitochondria to initiate protein oxidative folding and assembly.     

Intermediates in the refolding of reduced ribonuclease A.

The intermediates with one, two, three or four disulphide bonds which accumulate during unfolding of native ribonuclease and refolding of the reduced protein have been trapped by rapid alkylation with iodoacetate and separated by ionexchange chromatography. They have been characterized to varying extents by their enzymic activity, electrophoretic mobility through polyacrylamide gels, disulphide bonds between cysteine residues, the environments of the six tyrosine residues as indicated by ultraviolet absorption and fluorescence spectra, interaction with antibodies directed against either the trapped unfolded reduced protein or the native folded protein, and for the disruption by urea of any stable conformation producing a change in molecular shape.Correctly refolded ribonuclease was indistinguishable from the original native protein, but virtually all the intermediates with up to four disulphide bonds formed directly from the reduced protein were enzymically inactive and unfolded by these criteria. Unfolding of native ribonuclease was an all-or-none transition to the fully reduced protein, with no accumulation of disulphide intermediates. The intermediates in refolding are separated from the fully folded state by the highest energy barrier in the folding transition; they may be considered rapidly interconvertible, relatively unstable microstates of the unfolded protein. The measured elements of the final conformation are not acquired during formation of the first three disulphide bonds, but appear simultaneously with formation of the fourth native disulphide bond.These observations with ribonuclease are qualitatively similar to those made previously in greater detail with pancreatic trypsin inhibitor and suggest a possible general pattern for the kinetic process of protein unfolding and refolding.     

Direct observation of conformational folding coupled with disulphide rearrangement by using a water-soluble selenoxide reagent--a case of oxidative regeneration of ribonuclease A under weakly basic conditions

Oxidative regeneration pathways of bovine pancreatic ribonuclease A (RNase A), which has four SS linkages, were studied at 25 degrees C and pH 8.0 by using trans-3,4-dihydroxy-1-selenolane oxide (DHS(ox)), a new selenoxide reagent with strong oxidation power. The short-term folding study using a quench-flow instrument ( approximately 1 min) revealed that early intermediates (1S, 2S, 3S and 4S) are formed stochastically and irreversibly from the reduced protein (R) and do not have any stable structures. In the long-term folding study ( approximately 300 min), on the other hand, slow generation of the key intermediates (des[65-72] and des[40-95]) through SS rearrangement from the 3S intermediate ensemble was observed, followed by slight formation of native RNase A (N). The parallel UV and CD measurements demonstrated that formation of the key intermediates is accompanied with the formation of the native-like structures. Thus, DHS(ox) allowed facile identification of the conformational folding steps coupled with SS rearrangement on the major oxidative folding pathways.     

Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.     

Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.     

Substitution of serine for non-disulphide-bond-forming cysteine in grass carp (Ctenopharygodon idellus) growth hormone improves in vitro oxidative renaturation

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.     

Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme. The effects on folding, stability and enzymatic properties.

Secretory phospholipases A2 (PLA2s) are small homologous proteins rich in disulphide bridges. These PLA2s have been classified into several groups based on the disulphide bond patterns found [Dennis, E. A. (1997) Trends Biochem. Sci. 22, 1-2]. To probe the effect of the various disulphide bond patterns on folding, stability and enzymatic properties, analogues of the secretory PLA2s were produced by protein engineering of porcine pancreatic PLA2. Refolding experiments indicate that small structural variations play an important role in the folding of newly made PLA2 analogues. Introduction of a C-terminal extension together with disulphide bridge 50-131 gives rise to an enzyme that displays full enzymatic activity having increased conformational stability. In contrast, introduction of a small insertion between positions 88 and 89 together with disulphide bridge 86-89 decreases the catalytic activity significantly, but does not change the stability. Both disulphide bridges 11-77 and 61-91 are important for the kinetic properties and stability of the enzyme. Disulphide bridge 11-77, but not 61-91, was found to be essential to resist tryptic breakdown of native porcine pancreatic PLA2.     

Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) by bis-cysteinyl modification and mass spectrometry

Melarsen oxide [p-(4,6-diamino-1,3,5-triazin-2-yl)aminophenylarsonous acid (MEL)], which selectively bridges spatially neighboring bis-cysteinyl residues in (reduced) proteins, was used to trap folding intermediates chemically during 1) time-dependent renaturation of recombinant human macrophage colony-stimulating factor (rhM-CSF); by redox refolding in vitro; 2) reductive unfolding in the presence of the trapping reagent; and 3) denaturing unfolding reactions in urea and guanidinium hydrochloride. Characterization of intermediates from folding and unfolding reactions was performed by electrospray ionization mass spectometry (ESI-MS). In all folding and unfolding reactions a characteristic dimeric intermediate with two attached melarsen oxide (MEL) groups was observed, suggesting that these rhM-CSF β species were important refolding intermediates. These intermediates presented a characteristic “charge structure” in ESI spectra with a most abundant 26+ charged molecular ion whereas the mature homodimeric rhM-CSF β showed a most abundant 23+ molecular ion, indicating that the final product was more compact. The major locations of the two MEL groups were identified by mass spectrometric peptide mapping at cysteine residues C157 and C159 from each monomer. Cysteine residues C7 and C90 were minor modification sites. The mass spectrometric results from the in vitro folding reactions of rhM-CSF β are in agreement with intrinsic tryptophan fluorescence measurements and are consistent with the folding pathway that starts with a fully reduced monomer (R), includes partially folded monomeric intermediates (M) and dimeric intermediates (D), and yields a final product with the native tertiary structure (N): 2R ⇒ 2M ⇒ D ⇒ N. Our results show that selective chemical trapping of bis-thiol groups of proteins with MEL permits study of folding pathways by mass spectrometric structure characterization of intermediates with otherwise transient conformations. Proteins Suppl. 2:50–62, 1998. © 1998 Wiley-Liss, Inc.     

Intrachain disulphide bridges in immunoglobulin G heavy chains. The Fc fragment

The disulphide bridges of the Fc fragment (C-terminal half of the heavy chain) have been studied in several human immunoglobulins, containing heavy chains of different antigenic types (gamma1, gamma2, gamma3 and gamma4), and in heavy-chain-disease proteins. Two intrachain disulphide bridges were found to be present. The sequences appear to be identical in the Fc fragments of two types of chain studied (gamma1 and gamma3), and very similar to corresponding sequences of the Fc fragment in rabbit. These results suggest that the C-terminal half of the heavy chains is covalently folded (in a similar fashion to the light chains) with a C-terminal loop and an N-terminal loop. The similarity is emphasized by comparison of the sequence and location of the disulphide-bridged peptides of the C-terminal loop of heavy and light chains. The N-terminal loop, on the other hand, appears to be very different in Fc fragments and light chains. The C-terminal loop is the only one present in the F'c fragment.     

Oxidative refolding of amyloidogenic variants of human lysozyme

The oxidative refolding of human lysozyme and its two best characterised amyloidogenic variants, Ile56Thr and Asp67His, has been investigated in vitro by means of the concerted application of a range of biophysical techniques. The results show that in each case the ensemble of reduced denatured conformers initially collapses into a large number of unstructured intermediates with one or two disulphide bonds, the majority of which then fold to form the native-like three-disulphide intermediate, des-[77-95]. The slow step in the overall folding reaction involves the rearrangement of the latter to the fully oxidised native protein containing four disulphide bonds. The Ile56Thr and Asp67His variants were found to fold faster than the wild-type protein by a factor of 2 and 3 respectively, an observation that can be attributed primarily to the reduction in the barriers to conformational rearrangements that results from both the mutations. The efficient folding of these variants despite their enhanced propensities to aggregate when compared to the wild-type protein is consistent with their ability to be secreted in sufficient quantities to give rise to the systemic amyloidoses with which they are associated.     

Characterisation of the dominant oxidative folding intermediate of hen lysozyme.

Reduced denatured lysozyme has been oxidised and refolded at pH values close to neutral in an efficient way by dilution from buffers containing 8.0 M urea, and refolding intermediates were separated by reverse-phase HPLC at pH 2. By using peptic digestion in combination with high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem MS/MS the dominant intermediate was identified to be des-[76-94]. This species has three of the four native disulphide bonds, but lacks the Cys76-Cys94 disulphide bond which connects the two folding domains in the native protein. Characterisation of des-[76-94] by 2D1H NMR shows that it has a highly native-like structure. This provides an explanation for the accumulation of this species during refolding as direct oxidation to the fully native protein will be restricted by the burial of Cys94 in the protein interior.     

Shifting the competition between the intramolecular Reshuffling reaction and the direct oxidation reaction during the oxidative folding of kinetically trapped disulfide-insecure intermediates

The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.     

In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient Ⅰ was formed from P3A through thiol/disulfide exchange reaction; then, transients Ⅱ and Ⅲ, each containing two native disulfides, were formed through the recognition and interaction of transient Ⅰ with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients Ⅱ and Ⅲ, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

Role of kinetic intermediates in the folding of leech carboxypeptidase inhibitor

The oxidative folding and reductive unfolding pathways of leech carboxypeptidase inhibitor (LCI; four disulfides) have been characterized in this work by structural and kinetic analysis of the acid-trapped folding intermediates. The oxidative folding of reduced and denatured LCI proceeds rapidly through a sequential flow of 1-, 2-, 3-, and 4-disulfide (scrambled) species to reach the native form. Folding intermediates of LCI comprise two predominant 3-disulfide species (designated as III-A and III-B) and a heterogeneous population of scrambled isomers that consecutively accumulate along the folding reaction. Our study reveals that forms III-A and III-B exclusively contain native disulfide bonds and correspond to stable and partially structured species that interconvert, reaching an equilibrium prior to the formation of the scrambled isomers. Given that these intermediates act as kinetic traps during the oxidative folding, their accumulation is prevented when they are destabilized, thus leading to a significant acceleration of the folding kinetics. III-A and III-B forms appear to have both native disulfides bonds and free thiols similarly protected from the solvent; major structural rearrangements through the formation of scrambled isomers are required to render native LCI. The reductive unfolding pathway of LCI undergoes an apparent all-or-none mechanism, although low amounts of intermediates III-A and III-B can be detected, suggesting differences in protection against reduction among the disulfide bonds. The characterization of III-A and III-B forms shows that the former intermediate structurally and functionally resembles native LCI, whereas the III-B form bears more resemblance to scrambled isomers.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

<Emphasis Type="Italic">In vitro</Emphasis> insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

Characterization of kinetic and thermodynamic phases in the prefolding process of bovine pancreatic ribonuclease A coupled with fast SS formation and SS reshuffling

In the redox-coupled oxidative folding of a protein having several SS bonds, two folding phases are usually observed, corresponding to SS formation (oxidation) with generation of weakly stabilized heterogeneous structures (a chain-entropy losing phase) and the subsequent intramolecular SS rearrangement to search for the native SS linkages (a conformational folding phase). By taking advantage of DHS(ox) as a highly strong and selective oxidant, the former SS formation phase was investigated in detail in the oxidative folding of RNase A. The folding intermediates obtained at 25 °C and pH 4.0 within 1 min (1S°-4S°) showed different profiles in the HPLC chromatograms from those of the intermediates obtained at pH 7.0 and 10.0 (1S-4S). However, upon prolonged incubation at pH 4.0 the profiles of 1S°-3S° transformed slowly to those similar to 1S-3S intermediate ensembles via intramolecular SS reshuffling, accompanying significant changes in the UV and fluorescence spectra but not in the CD spectrum. Similar conversion of the intermediates was observed by pH jump from 4.0 to 8.0, while the opposite conversion from 1S-4S was observed by addition of guanidine hydrochloride to the folding solution at pH 8.0. The results demonstrated that the preconformational folding phase coupled with SS formation can be divided into two distinct subphases, a kinetic (or stochastic) SS formation phase and a thermodynamic SS reshuffling phase. The transition from kinetically formed to thermodynamically stabilized SS intermediates would be induced by hydrophobic nucleation as well as generation of the native interactions.     

Pathway of oxidative folding of secretory leucocyte protease inhibitor: an 8-disulfide protein exhibits a unique mechanism of folding

Secretory leucocyte protease inhibitor (SLPI) is a 107-amino acid protein with a high density of disulfide pairing (eight). The mechanism of oxidative folding of reduced and denatured SLPI has been investigated here. Despite an exceedingly large number of possible folding intermediates ( approximately 46 million disulfide isomers) and their potential to complicate the refolding process, oxidative folding of SLPI turns out to be surprisingly simple and efficient. Complete oxidative folding and a near-quantitative recovery of the native SLPI can be achieved in a simple buffer solution using air oxidation without any supplementing thiol catalyst or redox agent, a phenomenon that has not yet been observed with other disulfide proteins. Because of the heterogeneity and extensive overlapping of folding intermediates, identification of the predominant intermediate was unfeasible. Nonetheless, studies of reductive unfolding of native SLPI and oxidative folding of a six-disulfide variant of SLPI enable us to propose an underlying mechanism accounting for the unique folding efficiency of SLPI in the absence of a redox agent. Our studies indicate that oxidative folding of SLPI undergoes heterogeneous populations of one-, two-, three-, four-, five-, six-, and seven-disulfide isomers, including two nativelike isomers, SLPI-6A and SLPI-7A, as transient intermediates. Formation of the last two native disulfide bonds leading to the conversion of SLPI-6A --> SLPI-7A --> N-SLPI is relatively slow and represents the final stage of oxidative folding. Most importantly, free cysteines of SLPI-6A and SLPI-7A also act as a thiol catalyst in promoting the disulfide shuffling of diverse non-native intermediates accumulated along the folding pathway. This explains why a near-quantitative recovery of N-SLPI can be achieved in the absence of any thiol catalyst and redox agent. Properties of SLPI-6A and SLPI-7A were investigated and compared to those of other documented kinetic intermediates of oxidative folding. The correlation between the mechanism of SLPI folding and the three-dimensional structure of SLPI is also elaborated.     

Oxidative folding of Amaranthus alpha-amylase inhibitor: disulfide bond formation and conformational folding

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 alpha-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.