Unfolding of hirudin characterized by the composition of denatured scrambled isomers

The native core structure of hirudin, a thrombin specific inhibitor, contains 24 hydrogen bonds, two stretches of -sheet and three disulfide bonds. Hirudin unfolds in the presence of denaturant and thiol catalyst by shuffling its native disulfide bonds and converting to scrambled structures that consist of 11 identified isomers. The composition of scrambled isomers, which characterizes the structure of denatured hirudin, varies as a function of denaturing conditions. The unfolding pathway of hirudin has been constructed by quantitative analysis of scrambled isomers unfolded under increasing concentrations of various denaturants. The results demonstrate a progressive expansion of the polypeptide chain and the existence of a structurally defined stable intermediate along the pathway of unfolding.     

The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers

The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys⁶-Cys¹⁴, Cys²⁰-Cys³¹, and Cys³³-Cys⁴². The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4–5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.     

The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin

The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.     

The folding pathway of alpha-lactalbumin elucidated by the technique of disulfide scrambling. Isolation of on-pathway and off-pathway intermediates

The technique of disulfide scrambling permits reversible conversion of the native and denatured (scrambled) proteins via shuffling and reshuffling of disulfide bonds. Under strong denaturing conditions (e.g. 6 m guanidinium chloride) and in the presence of a thiol initiator, alpha-lactalbumin (alphaLA) denatures by shuffling its four native disulfide bonds and converts to an assembly of 45 species of scrambled isomers. Among them, two predominant isomers, designated as X-alphaLA-a and X-alphaLA-d, account for about 50% of the total denatured structure of alphaLA. X-alphaLA-a and X-alphaLA-d, which adopt the disulfide patterns of (1-2,3-4,5-6,7-8) and (1-2,3-6,4-5,7-8), respectively, represent the most unfolded structures among the 104 possible scrambled isomers (Chang, J.-Y., and Li, L. (2001) J. Biol. Chem. 276, 9705-9712). In this study, X-alphaLA-a and X-alphaLA-d were purified and allowed to refold through disulfide scrambling to form the native alphaLA. Folding intermediates were trapped kinetically by acid quenching and analyzed quantitatively by reversed phase high pressure liquid chromatography. The results revealed two major on-pathway productive intermediates, two major off-pathway kinetic traps, and at least 30 additional minor transient intermediates. Of the two major on-pathway intermediates, one takes on a native-like alpha-helical domain, and the other comprises a structured beta-sheet, calcium binding domain. The two major kinetic traps are apparently stabilized by locally formed non-native-like structures. Overall, the folding mechanism of alphaLA is essentially congruent with the model of "folding funnel" furnished with a rather intricate energy landscape.     

The unfolding pathway of leech carboxypeptidase inhibitor

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.     

The unfolding pathway and conformational stability of potato carboxypeptidase inhibitor

The unfolding and denaturation curves of potato carboxypeptidase inhibitor (PCI) were investigated using the technique of disulfide scrambling. In the presence of denaturant and thiol initiator, the native PCI denatures by shuffling its native disulfide bonds and converts to form a mixture of scrambled PCI that consists of 9 out of a possible 14 isomers. The denaturation curve is determined by the fraction of native PCI converted to scrambled isomers under increasing concentrations of denaturant. The concentration of guanidine thiocyanate, guanidine hydrochloride, and urea required to denature 50% of the native PCI was found to be 0.7, 1.45, and 8 m, respectively. The PCI unfolding curve was constructed through the analysis of structures of scrambled isomers that were denatured under increasing concentrations of denaturant. These results reveal the existence of structurally defined unfolding intermediates and a progressive expansion of the polypeptide chain. The yield of the beads-form isomer (Cys(8)-Cys(12), Cys(18)-Cys(24), and Cys(27)-Cys(34)) as a fraction of total denatured PCI was shown to be directly proportional to the strength of the denaturing condition. Furthermore, the PCI sequence was unable to fold quantitatively into a single native structure. Under physiological conditions, the scrambled isomers of PCI that constitute about 4% of the protein were in equilibrium with native PCI.     

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.     

The underlying mechanism for the diversity of disulfide folding pathways

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.     

1.42 Å crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.     

1.42A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.     

Major kinetic traps for the oxidative folding of leech carboxypeptidase inhibitor

The leech carboxypeptidase inhibitor (LCI) is a 66-amino acid protein, containing four disulfides that stabilize its structure. This polypeptide represents an excellent model for the study and understanding of the diversity of folding pathways in small, cysteine-rich proteins. The pathway of oxidative folding of LCI has been elucidated in this work, using structural and kinetic analysis of the folding intermediates trapped by acid quenching. Reduced and denatured LCI refolds through a rapid, sequential flow of one- and two-disulfide intermediates and reaches a rate-limiting step in which a mixture of three major three-disulfide species and a heterogeneous population of non-native four-disulfide (scrambled) isomers coexist. The three three-disulfide intermediates have been identified as major kinetic traps along the folding pathway of LCI, and their disulfide structures have been elucidated in this work. Two of them contain only native disulfide pairings, and one contains one native and two non-native disulfide bonds. The coexistence of three-disulfide kinetic traps adopting native disulfide bonds together with a significant proportion of fully oxidized scrambled isomers shows that the folding pathway of LCI features properties exhibited by both the bovine pancreatic trypsin inhibitor and hirudin, two diverse models with extreme folding characteristics. The results further demonstrate the large diversity of disulfide folding pathways.     

Quantitative analysis of the composition of the native and scrambled ribonuclease A

Reversible conversion between the native and scrambled proteins can be applied to analyze the denaturation curve of a disulfide-containing protein. In the case of RNase A, scrambled species could not be well separated from the native species by HPLC to permit precise quantitative analysis of the extent of denaturation. Methods are developed here to overcome this problem. The methods exploit the difference of conformational stability between the native and scrambled RNase A. When a sample of partially denatured RNase A was placed under mild reducing conditions (0.2-1 mM dithiothreitol for 10 min), the disulfide bonds of the native RNase A remain intact, whereas those of scrambled isomers become fully reduced. The native and fully reduced species of RNase A can be completely separated by HPLC. Alternatively, a mixture of partially denatured RNase A can be treated with mild concentration of proteolytic enzymes (trypsin or thermolysin). In this approach, scrambled isomers of RNase A were totally fragmented and readily separated from the native RNase A. These methods allow analysis and construction of the denaturation curves of RNase A in the presence of urea, GdmCl and GdmSCN.     

Sequences of B-chain/domain 1-10/1-9 of insulin and insulin-like growth factor 1 determine their different folding behavior

Although insulin and insulin-like growth factor-1 (IGF-1) belong to one family, insulin folds into one thermodynamically stable structure, while IGF-1-folds into two thermodynamically stable structures (native and swap forms). We have demonstrated previously that the bifurcating folding behavior of IGF-1 is mainly controlled by its B-domain. To further elucidate which parts of the sequences determine their different folding behavior, by exchanging the N-terminal sequences of mini-IGF-1 and recombinant porcine insulin precursor (PIP), we prepared four peptide models: [1-9]PIP, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1 by means of protein engineering, and their disulfide rearrangement, V8 digestion, circular dichroic spectra, disulfide stability, and in vitro refolding were investigated. Among them only [1-9]PIP, like mini-IGF-1/IGF-1, was expressed in yeast as two isomers: isomer 1 (corresponding to swap IGF-1) and isomer 2 (corresponding to native IGF-1), which are supported by the experimental results of disulfide rearrangements, peptide mapping of V8 endoprotenase digests, circular dichroic analysis, in vitro refolding, and disulfide stability analysis. The other peptide models, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1, fold into one stable structure as PIP does, which indicates that sequence 1-4 of mini-IGF-1 is important for the folding behavior of mini-IGF-1/IGF-1 but not sufficient to lead to a bifurcating folding. The results demonstrated that the folding information, by which mini-IGF-1/IGF-1-folds into two thermodynamically structures, is encoded/written in its sequence 1-9, while sequences 1-10 of B chain in insulin/PIP play an important role in the guide of its unique disulfide pairing during the folding process.     

Distinct Unfolding and Refolding Pathways of Lipid Transfer Proteins LTP1 and LTP2

Plant non-specific lipid transfer proteins (ns-LTPs) comprise two families, LTP1s and LTP2s, all structurally stabilized by four native disulfide bonds. Solution and crystal structures of both LTP1s and LTP2s from various plants have been determined. Despite the similarities of their biological function and backbone folds, the biophysical properties of LTP1s and LTP2s differ significantly. In this report, the mechanisms of unfolding and refolding of rice LTP1 and LTP2 have been investigated using the technique of disulfide bonds scrambling. LTP1 is shown to unfold and refold via predominant species of partially structured intermediates. Four isomers of partly unfolded and extensively unfolded LTP1 were identified, isolated and their disulfide structures were determined. By contrast, unfolding and refolding of LTP2 adopt a (close to) two-state mechanism, and undergo a reversible conversion between the native and a single extensively unfolded isomer without accumulation of any significant intermediate.     

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

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.     

The structure of denatured bovine pancreatic trypsin inhibitor (BPTI)

In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3-4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead-form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5-Cys14, Cys30-Cys38 and Cys51-Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.     

Purification and refolding of recombinant human IGF II from silkworms infected with recombinant Bombyx mori nuclear polyhedrosis virus.

We have reported that insulin-like growth factor II (IGF II) was produced as a fusion protein in Bombyx mori (silkworm) larval bodies infected with recombinant B. mori nuclear polyhedrosis virus [J. Gen. Virol., 68, 2599-2606 (1987)]. In this study, the purification of IGF II from the infected silkworms is reported. The fusion protein was extracted with 6.0 M guanidine-HCl from the infected larval bodies homogenized in water. The use of organic solvents to remove the impurities, such as lipid derived from the larval bodies, was a very effective method of purification. IGF II was released from the partially purified fusion protein by treatment with CNBr, purified by HPLC, and refolded by air-oxidization. Refolded IGF II had an identical primary structure including disulfide bonds and showed identical thymidine uptake stimulation activity with human IGF II. Furthermore, protein disulfide-isomerase was shown to be able to refold scrambled IGF II rapidly.     

Pathway of oxidative folding of alpha-lactalbumin: a model for illustrating the diversity of disulfide folding pathways

The pathway of oxidative folding of alpha-lactalbumin (alpha LA) (four disulfide bonds) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. In the absence of calcium, oxidative folding of alpha LA proceeds through highly heterogeneous species of one-, two-, three-, and four-disulfide (scrambled) intermediates to reach the native structure. In the presence of calcium, the folding intermediates of alpha LA comprise two predominant isomers (alpha LA-IIA and alpha LA-IIIA) adopting exclusively native disulfide bonds, including the two disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) located within the beta-sheet calcium binding domain. alpha LA-IIA is a two-disulfide species consisting of Cys(61)-Cys(77) and Cys(73)-Cys(91) disulfide bonds. alpha LA-IIIA contains Cys(61)-Cys(77), Cys(73)-Cys(91), and Cys(28)-Cys(111) disulfide bonds. The underlying mechanism of the contrasting folding pathways of calcium-bound and calcium-depleted alpha LA is congruent with the cause of diversity of disulfide folding pathways observed among many well-characterized three-disulfide proteins, including bovine pancreatic trypsin inhibitor and hirudin. Our study also reveals novel aspects of the folding mechanism of alpha LA that have not been described previously.     

Evidence for the underlying cause of diversity of the disulfide folding pathway

The pathways of oxidative folding of disulfide proteins exhibit a high degree of diversity, which is illustrated by the varied extent of (a) the heterogeneity of folding intermediates, (b) the predominance of intermediates containing native disulfide bonds, and (c) the level of accumulation of fully oxidized scrambled isomers as intermediates. BPTI and hirudin exemplify two extreme cases of such divergent folding pathways. We previously proposed that the underlying cause of this diversity is associated with the degree of stability of protein subdomains. Here we present compelling evidence that substantiates this hypothesis by studying the folding pathway of alphaLA-IIA. alphaLA-IIA is a partially folded intermediate of alpha-lactalbumin (alphaLA). It comprises a structured beta-sheet (calcium-binding) domain linked by two native disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) and a disordered alpha-helical domain with four free cysteines (Cys(6), Cys(28), Cys(111), and Cys(120)). Purified alphaLA-IIA was allowed to refold without and with stabilization of its structured beta-sheet domain by calcium. In the absence of calcium, the folding pathway of alphaLA-IIA resembles that of hirudin, displaying a highly heterogeneous population of folding intermediates, including fully oxidized scrambled species. Upon stabilization of its beta-sheet domain by bound calcium, oxidative folding of alphaLA-IIA undergoes a pathway conspicuously similar to that of BPTI, exhibiting limited species of folding intermediates containing mostly native disulfide bonds.     

Unfolding and refolding pathways of a major kinetic trap in the oxidative folding of alpha-lactalbumin

Alpha-lactalbumin (alphaLA)-IIIA is a major kinetic intermediate present along the pathways of reductive unfolding and oxidative folding of bovine alpha-lactalbumin (alphaLA). It is a three-disulfide variant of native alphaLA lacking Cys(6)-Cys(120) at the alpha-helical domain. Stability and the unfolding/refolding mechanism of carboxymethylated alphaLA-IIIA have been investigated previously by stop-flow circular dichroism (CD) and fluorescence spectroscopy. A stable intermediate compatible with molten globule was shown to exist along the pathways of unfolding-refolding of alphaLA-IIIA [Ikeguchi et al. (1992) Biochemistry 31, 16695-12700; Horng et al. (2003) Proteins 52, 193-202]. We investigate here the unfolding-refolding pathways and conformational stability of alphaLA-IIIA using the method of disulfide scrambling with the following specific aims: (a) to isolate and characterize the observed stable molten globule, (b) to analyze the heterogeneity of folding-unfolding intermediates, (c) to elucidate the disulfide structure of extensively unfolded isomer of alphaLA-IIIA, and (d) to clarify the relative conformational stability between alphaLA-IIIA and alphaLA. Two scrambled isomers, designated as X-alphaLA-IIIA-c and X-alphaLA-IIIA-a (X stands for scrambled), were isolated under mild and strong denaturing conditions. Their disulfide structures, CD spectra, and manners of refolding to form the native alphaLA-IIIA were analyzed in this report. The results are consistent with the notion that X-alphaLA-IIIA-c and X-alphaLA-IIIA-a represent a partially unfolded and an extensively unfolded isomers of native alphaLA-IIIA, respectively. The unfolding-refolding pathways of alphaLA-IIIA are elaborated and compared with that of intact alphaLA. These results display new insight into one of the most extensively studied molecules in the field of protein folding and unfolding.