DOI: 10.1002/ijch.201100105

Selenoglutathione-Mediated Rescue of Kinetically TrappedIntermediates in Oxidative Protein FoldingNorman Metanis,[a] Carlotta Foletti,[a] Joris Beld ,[a, b] and Donald Hilvert*[a]

1. Introduction

Disulfide bonds influence the folding and stability ofmany secreted proteins. These covalent cross-links areformed by the oxidation of cysteine thiols. If a proteincontains multiple cysteine residues, many different disul-fide-bonded species are possible, and these must rear-range via thiol/disulfide exchange reactions to reach thenative state. As a consequence, oxidative protein foldingis often slow.

In vivo, specialized oxidoreductase enzymes possessinga characteristic CXXC motif are recruited to catalyzethiol oxidation and disulfide isomerization in proteins.[1]

In contrast, oxidative folding of recombinant proteins invitro is usually accomplished with redox buffers, whichtypically consist of mixtures of oxidized and reduced glu-tathione (GSSG and GSH, respectively).[2,3] The GSSGdisulfide serves as a stoichiometric oxidant of the reducedprotein, whereas the GSH thiol promotes subsequent di-sulfide bond isomerization.

Redox buffers are not restricted to disulfide deriva-tives.[4,5] For example, we recently discovered that redoxbuffers containing selenoglutathione (GSeSeG), a seleni-um-containing analogue of GSSG, have significant advan-tages over GSSG for the oxidative folding of many pro-teins.[6–8] Although thermodynamically more stable thandisulfides,[9] diselenides are kinetically superior as oxi-dants for cysteine thiols. The greater polarizability of sele-nium makes the diselenide bond a particularly effectiveelectrophile in nucleophilic exchange reactions.[10] The po-tential magnitude of this effect is indicated by kineticstudies showing that thiol attack on a diselenide bond canbe up to four orders of magnitude faster than attack onan analogous disulfide bond.[11] The nucleophilic selenolthat is produced upon reduction of the diselenide mayfurther contribute to the efficacy of oxidative proteinfolding by catalyzing thiol/disulfide exchange reac-

tions.[11,12] Because selenols are more acidic than thiols(DpKa � 3),[13] they are typically ionized at physiologicalpH, enhancing their intrinsic reactivity. As a result ofthese properties, redox buffers containing small moleculediselenides afford the same rates and yields for the fold-ing of a variety of proteins as standard glutathione redoxbuffers, but at substantially lower concentrations.[8] Iffolding is carried out under aerobic conditions, the overallprocess can even proceed catalytically in diselenide be-cause selenols are rapidly oxidized by atmosphericoxygen.[7]

The utility of GSeSeG is particularly striking for pro-teins like bovine pancreatic trypsin inhibitor (BPTI), lyso-zyme, and human epidermal growth factor (hEGF),[8]

whose folding mechanisms are characterized by the accu-mulation of kinetically stable intermediates. To investi-gate how the diselenide contributes to folding efficiencyin these cases, we have revisited the well-studied foldingpathway of BPTI.[14–17] By identifying the nature and fateof the species that build up during anaerobic folding, par-ticularly the two-disulfide intermediates, we have deter-mined which steps in the folding pathway are sensitive tothe selenium-containing redox buffer.

Abstract : Selenoglutathione has been shown to have consid-erable potential as a catalyst of oxidative protein folding.Here we examine how this reagent modulates the foldingpathway of bovine pancreatic trypsin inhibitor (BPTI) and

show that the diselenide increases the efficiency of this pro-cess primarily by accelerating the conversion of a kineticallytrapped folding intermediate.

Keywords: bovine pancreatic trypsin inhibitor · protein folding · redox chemistry · selenoglutathione · thiol/disulfide exchange

[a] N. Metanis, C. Foletti, J. Beld , D. HilvertLaboratory of Organic Chemistry, ETH Z�rich,8093 Z�rich, Switzerlandphone:+41 (0)44 632-3176fax:+41 (0)44 632-1486e-mail: hilvert@org.chem.ethz.ch

[b] J. BeldPresent address: Department of Chemistry and BiochemistryUniversity of California–San DiegoLa Jolla, California 92093-0358, USA

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2. Materials and Methods

Materials : Buffers were prepared with ultrapure water. Allchemicals were purchased from Sigma–Aldrich, Fluka, or Acros.Bovine pancreatic trypsin inhibitor (BPTI) was a generous gift ofBayer AG. Selenoglutathione was synthesized by Boc/tBu solid-phase peptide synthesis as previously described[6] and purified bypreparative HPLC.

Reduced BPTI : Native folded BPTI was dissolved in degassed200 mm sodium phosphate buffer (pH 8.3) containing 6m

GdmCl, and treated with a 150-fold excess of dithiothreitol(DTT). After 2 h, the sample was acidified to pH 2 by additionof 1m HCl, and the denatured and reduced BPTI purified bypreparative HPLC. Protein solutions were injected onto aWaters XBridge C18 column (5 mm, 19� 150 mm) and elutedwith a 20 : 80 to 50 : 50 gradient (acetonitrile–0.08% TFA :water–0.1 % TFA) over 60 min. The fractions containing reduced pro-tein were lyophilized and subsequently dissolved in 10 mm HClto a concentration of 680 mm (e280 5400m

�1 cm�1).

Oxidative folding of reduced BPTI : The oxidative renaturationof BPTI was monitored based on the method of Weissman andKim.[16] The folding reactions were carried out in degassed bufferin a glove-bag under a mixture of argon and nitrogen. OxidizedGSSG or GSeSeG (final concentration 150 mm) was added to re-duced BPTI (30 mm) either in phosphate buffer (100 mm sodiumphosphate, 150 mm NaCl, 1 mm EDTA, pH 7.3) or Tris-HClbuffer (100 mm Tris-HCl, 200 mm KCl, 1 mm EDTA, pH 8.7). Atvarious time intervals, 85 mL aliquots were removed, quenchedwith 15 mL 3m HCl, and stored at �20 8C prior to separation byanalytical HPLC. The samples were injected onto an Atlantis T3column (3 mm 4.6 �150 mm), heated at 40 8C, and eluted with a10 :90 to 25 : 75 gradient (acetonitrile-0.08% TFA:water-0.1 %TFA) over 8 min, increasing to 40 : 60 (acetonitrile–0.08% TFA :water–0.1% TFA) over 50 min. The chromatograms were moni-tored at 220 and 280 nm. For kinetic analysis, all peak areas weresummed and the total was assigned a value of 100%. The peakassignment for native BPTI was confirmed by co-injection of theoxidative folding product with an authentic standard of nativeBPTI; the peaks of the intermediates that accumulate duringfolding were assigned according to the literature.[16,18–20]

Isolation of N� and N* intermediates : Reduced BPTI (1.7 mg,0.261 mmol) was dissolved in 10 mm HCl (930 mm) and mixedwith phosphate buffer (100 mm sodium phosphate, 150 mm NaCl,1 mm EDTA, pH 7.3) containing 150 mm GSSG to a final concen-tration of 30 mm. The reaction was quenched with acid after90 min when N’ and N* constitute the major species. N’ and N*were separated by analytical HPLC as described above.

Oxidative folding of N� and N*: Purified N’ or N* were dissolvedin degassed 100 mm Tris-HCl buffer (pH 8.7) containing 200 mm

KCl and 1 mm EDTA to give a final concentration of 15 mm in atotal volume of 500 mL. Folding reactions were initiated by addi-tion of GSSG or GSeSeG (37.5, 75, or 150 mm final concentra-tion) to the sample in a glove-bag under a mixture of argon andnitrogen. At periodic time intervals, 80 mL aliquots were re-moved from the reaction mixture and quenched with 35 mL 1m

HCl. The samples were stored at �20 8C until subjected to ana-lytical HPLC analysis as described above.

3. Results

3.1. Oxidative Folding of BPTI at Neutral pH

BPTI is a 58 amino acid long protein that contains sixcysteines in three disulfide bonds designated as [5–55; 14–38; 30–51]. Its folding mechanism has been extensivelystudied and proceeds via well-characterized one- andtwo-disulfide-bonded intermediates (Scheme 1).[14–17]

Under anaerobic conditions in the presence of gluta-thione, one fast and one slow route to the native state areobserved, with roughly half of the reduced protein form-ing the native state via the N’ intermediate lacking the 5–55 disulfide (i.e., [14–38; 30–51]). The other half istrapped as a dead-end intermediate called N* which lacksthe 30–51 disulfide (i.e., [5–55; 14–38]) and is stable forweeks at neutral pH. Accumulation of this non-produc-tive species is a distinctive feature of the BPTI foldingpathway.

Our results on the oxidative folding of BPTI withGSSG at pH 7.3 agree well with published data reportedby Weissman and Kim.[16] As shown in Figure 1 A, we ob-serve initial accumulation of the one-disulfide intermedi-ates [30–51] and [5–55], which are believed to have sub-stantial native-like structure.[21,22] Because Cys14 andCys38 are solvent-exposed, subsequent reaction withGSSG to form the two-disulfide intermediates N’ and N*is relatively rapid. N’ and N* also have essentially native-like structures but their free cysteines (Cys5 and Cys55 inN’; Cys30 and Cys51 in N*) are buried,[23–26] which hindersspontaneous formation of the third and last disulfidebond. Instead, they undergo a rate-limiting intramolecu-lar rearrangement to a third two-disulfide intermediate,[5–55; 30–51], also known as NSH

SH, which rapidly oxidizesto native protein.[14–16] At neutral pH, this species is notsignificantly populated, however. In contrast to N’, which

Scheme 1. Kinetically favored BPTI folding pathway at pH 7.3 ac-cording to reference 16. R and N are reduced and native BPTI, re-spectively; intermediates that accumulate during folding are desig-nated by the disulfide bonds they contain. Initial oxidation of thereduced proteins affords a broad distribution of single disulfide in-termediates that rearrange (dotted lines) to give [30–51] and [5–55]. Qualitative estimates of the relative rates of the other stepsare indicated above or below the arrows.

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rearranges relatively rapidly to the more stable NSHSH and

N* in the absence of oxidant, N* is a long-lived speciesthat persists on the time scale of the experiment (Fig-ure 1A). As a consequence of this forked folding path-way, the yield of native BPTI is only ~50% after 24 h.

When GSeSeG is used as an oxidant instead of GSSG,the folding mechanism is qualitatively similar. However,initial oxidation is considerably faster and the distributionof intermediates somewhat different. For example, a sig-nificant population of one- and two-disulfide intermedi-ates is already seen after one minute, whereas negligiblereaction is evident at the same time point with GSSG(Figure 1 A versus B). The native [30–51] intermediatepredominates at the single-disulfide stage, leading to pref-erential accumulation of N’. Although partitioning of N’to NSH

SH and N* appears to be independent of the oxidant,N* is converted to the fully oxidized protein significantlymore rapidly in the presence of GSeSeG than GSSG.Thus, after 24 h, oxidative folding of BPTI is essentiallycomplete (Figure 1 A and B).

3.2. Oxidative Folding of BPTI at pH 8.7

Because deprotonation of cysteine side chains facilitatesthiol/disulfide exchange reactions, oxidative folding ofBPTI is considerably faster under more basic condi-tions.[14,16] For example, at pH 8.7, GSSG-mediated fold-ing of this protein is complete within approximately 24 h

(Figure 2 A). Although greater heterogeneity is observedin the population of one-disulfide intermediates, the fold-ing pathway is qualitatively similar to that observed atpH 7.3. Upon initiation of the reaction, significantamounts of N’ and N* accumulate within a few minutesand then undergo rearrangement and further oxidation(Figure 2 A).

The beneficial effects of GSeSeG seen at neutral pHare also evident at pH 8.7. Specifically, the folding reac-tion with GSeSeG is substantially faster than the reactionwith GSSG. Initial oxidation of reduced protein (R) byGSeSeG is very rapid, and after only 30 seconds, the two-disulfide bond intermediates N’ and N* predominate.Within 1 h essentially all the protein treated withGSeSeG is converted to the N state (Figure 2 B), whereastraces of N* are still evident in the analogous GSSG reac-tion at 21 h. These data suggest that GSeSeG significantlyaccelerates the conversion of N* to N.

3.3. Reactions of Purified N’ and N* Intermediates

To examine the rearrangement and further oxidation ofN’ and N* in greater detail, we isolated and purified bothspecies. A large scale folding experiment (pH 7.3) wasquenched with hydrochloric acid after 90 min when N*and N’ are the two major species (Figure 1 A). The twointermediates were separated by HPLC and lyophilized.The purified proteins (15 mm) were then folded individu-

Figure 1. Oxidative folding of reduced BPTI at pH 7.3 using (A)GSSG or (B) GSeSeG. [BPTI] = 30 mm ; [oxidant] = 150 mm.

Figure 2. Oxidative folding of reduced BPTI at pH 8.7 using (A)GSSG or (B) GSeSeG. [BPTI] = 30 mm ; [oxidant] = 150 mm.

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Selenoglutathione-Mediated Rescue of Kinetically Trapped Intermediates in Oxidative Protein Folding

ally under anaerobic conditions by adjusting the pH to8.7 and adding GSSG or GSeSeG.

As previously reported,[14] the reaction of N’ is inde-pendent of GSSG concentration. Instead of reacting di-rectly with the oxidant, this intermediate partitions to N*and NSH

SH via intramolecular disulfide bond rearrange-ments (Figure 3 A). The free thiols of NSH

SH are relativelyaccessible to GSSG, which facilitates formation of thethird disulfide bond. Folding of N’ in the presence of lowconcentrations of GSeSeG is qualitatively similar, but the

distribution of intermediates again differs somewhat,leading to slightly faster formation of native BPTI (Fig-ure 3B). Although the long-lived N* is still produced,NSH

SH never accumulates, presumably because it reactsmore rapidly with GSeSeG than with GSSG. Instead, sig-nificant amounts of a new reaction intermediate are ob-served at short reaction times (t=1 min). Based on its re-tention time of 19.6 min (Figure 4 C), this species is prob-ably a mixed selenosulfide, i.e. , N’(SeG), between seleno-glutathione and Cys5 or Cys55.[20] Consistent with this in-terpretation, this new species accumulates to a greaterextent when the GSeSeG concentration is doubled, asdoes the rate at which N’ disappears (Figure 4A and B).At 75 mm GSeSeG, N* and NSH

SH do not form in significantamounts but a large number of other unidentified species

are observed, suggesting that the folding population ishighly dynamic. Although N’(SeG) can be converted di-rectly to N by intramolecular attack of the free thiol onthe mixed selenosulfide, it appears to scramble throughreaction with the reduced selenoglutathione present in so-lution. Because the rate of native BPTI formation doesnot increase substantially compared to the analogous re-action with 75 mm GSSG, many of these species may bekinetic or oxidative traps.[27]

The effect of GSeSeG on the conversion of the kineti-cally trapped N* intermediate is even more striking.Under standard folding conditions with GSSG, this trans-formation is normally much slower than the oxidation ofN’ to N (Figure 5 A).[15,16] In this case, no intermediatesare observed, although formation of native BPTI is also

Figure 3. Oxidative folding of N’ at pH 8.7 using (A) GSSG or (B)GSeSeG. [N’] = 15 mm ; [oxidant] = 37.5 mm.

Figure 4. Time course for the conversion of N’ to N as a functionof oxidant concentration. (A) [N’] = 15 mm, [GXXG] = 37.5 mm,pH 8.7; (B) [N’] = 15 mm, [GXXG] = 75 mm, pH 8.7. (C) HPLC chroma-tograph obtained with 75 mm GSeSeG at t = 1 min, showing N’ at22 min and N’(SeG) at 19.6 min. Solid lines, filled symbols: GSeSeG;dashed lines, open symbols: GSSG. N’ (red), N* (green), NSH

SH (pink),N’(SeG) (black), and N (blue).

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believed to proceed via intramolecular rearrangement toNSH

SH.[16] As shown in Figure 5B, however, the diselenidedramatically increases the rate of the folding reaction, ac-celerating both the disappearance of N* and the forma-tion of N by nearly two orders of magnitude compared tothe reaction with GSSG. As a result, the conversion ofN* to N occurs on roughly the same time scale as theconversion of N’ to N.

Increasing the concentration of GSeSeG leads to pro-portional increases in reaction rate (Figure 6 A-C), sug-gesting that N* reacts directly with the oxidant. This con-clusion is supported by the detection of a selenogluta-thione adduct with a retention time of 18.4 min (Fig-ure 6D). This species, which accumulates to a significantextent at 150 mm GSeSeG (�40% of the population after12 seconds), was isolated and its identity confirmed byelectrospray mass spectrometry (calculated, 6866.5 Da;observed, 6866.4 Da). Intramolecular attack of the re-maining free thiol on the mixed selenosulfide would pro-duce N, circumventing slow intramolecular rearrangementto NSH

SH. At high GSeSeG concentration, some overoxida-tion of N*(SeG) to produce a double mixed selenosulfide,N*(SeG)2, may compete to some extent with the forma-tion of N, as judged by the appearance of another newpeak at 17.0 min (Figure 6 D). Because it lacks free thiols,the double mixed diselenosulfide is a nonproductive de-rivative that must be reduced again by free GSeH toform native BPTI.

Figure 5. Oxidative folding of N* at pH 8.7 using (A) GSSG or (B)GSeSeG. [N*] = 15 mm ; [oxidant] = 37.5 mm.

Figure 6. Time course for the conversion of N* to N as a function of oxidant concentration. (A) [N*] = 15 mm, [GXXG] = 37.5 mm, pH 8.7; (B)[N*] = 15 mm, [GXXG] = 75 mm, pH 8.7; (C) [N*] = 15 mm, [GXXG] = 150 mm, pH 8.7; (D) HPLC chromatograph obtained in (C) at t= 12 s, show-ing N* at 23 min, N*(SeG) at 18.4 min, and N*(SeG)2 at 17.0 min. Solid lines, filled symbols: GSeSeG; dashed lines, open symbols: GSSG. N’(red), N* (green), N*(SeG) (black), and N (blue).

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Selenoglutathione-Mediated Rescue of Kinetically Trapped Intermediates in Oxidative Protein Folding

4. Discussion

Redox buffers containing selenoglutathione significantlyenhance oxidative folding of BPTI. Although the samenative disulfide-bonded intermediates accumulate in thepresence of this oxidant as seen under standard foldingconditions with GSSG, our kinetic analysis shows thattheir distribution and relative rates of formation anddecay differ. The two order of magnitude acceleration ofthe conversion of the “nonproductive” N* species tonative BPTI is particularly striking in this context.

The major two-disulfide intermediates that accumulateduring oxidative folding of BPTI, N’ and N*, are confor-mational kinetic traps.[15,16] Their conversion to fully oxi-dized and folded BPTI is slow because native structureburies the free thiols, rendering them inaccessible to oxi-dant and thus impeding direct oxidation. Because forma-tion of the last disulfide bond is difficult, intramolecularthiol/disulfide exchange to generate NSH

SH, another native-like two-disulfide species, is the rate-limiting step understandard folding conditions (Scheme 1). Various lines ofevidence suggest that these transformations require con-siderable loss of native structure.[16,28,29] Rearrangementof the thermodynamically more stable N* is particularlyslow, which is why it has been traditionally considered tobe a nonproductive state. In contrast, in NSH

SH, the Cys14and Cys38 thiols are relatively solvent-accessible, so oxi-dation to native BPTI proceeds comparatively rapidly.

Our kinetic data suggest that selenoglutathione increas-es the efficiency of BPTI folding in several ways. Consis-tent with the greater kinetic reactivity of diselenides com-pared to disulfides,[11,12] GSeSeG accelerates the oxidationof the reduced protein to afford one and two-disulfidebonded intermediates. Notably, however, less N* accumu-lates with this oxidant, and since N* is a major bottleneckin the BPTI folding pathway, reducing its concentrationimproves overall folding efficiency. Selenoglutathione ap-parently influences both the production and subsequentdisappearance of this intermediate by reacting directlywith N’ and N* to form the mixed selenosulfide adductsN’(SeG) and N*(SeG). Thiol attack on a diselenide hasbeen shown to be four orders of magnitude faster thanthiol attack on a disulfide.[11] As a consequence, these bi-molecular reactions can compete effectively with thenormal intramolecular rearrangements, especially at highconcentrations of oxidant, opening alternative routes tonative folded BPTI. Analogous glutathione adducts ofBPTI have been previously observed, albeit only at veryhigh concentrations of oxidant.[20,27]

In the case of N’, N’(SeG) is formed at the expense ofboth NSH

SH and N*. In principle, it can be converted direct-ly to N by intramolecular attack of the remaining freethiol on the selenosulfide bond. Nevertheless, onlymodest increases in the yield of BPTI were observedwhen isolated N’ was treated with GSeSeG versus GSSG.A large number of other species are also formed during

the reaction (Figure 4 C), probably initiated by selenol/di-sulfide exchange reactions with the free GSeH that wasreleased upon formation of the mixed selenosulfide. Eventhough less N* is produced, many of these species may benonproductive kinetic or oxidative traps that detract fromfolding efficiency.[27]

In contrast, selenoglutathione dramatically enhancesthe conversion of N* to N. As with N’, formation of asingle mixed selenosulfide appears to be quite efficient.Structural data suggest that Cys30 is more solvent-ex-posed than Cys51,[30] so it probably reacts first withGSeSeG to form N*(SeG). Intramolecular attack of theCys51 thiol on this mixed selenosulfide would then formthe native 30–51 disulfide bond, providing a direct routeto the native state. The rate constant for conversion ofthe corresponding mixed disulfide with glutathione hasbeen reported to be ca. 2� 10�4 s�1 at pH 7.3.[27] Since se-lenides and sulfides are roughly comparable as leavinggroups in this type of exchange reaction,[11] we can esti-mate that the half-life of N*(SeG) would be substantiallyless than an hour under more alkaline conditions. Otherpathways to product are also conceivable. For example,transient cleavage of the exposed and easily reduced 14–38 disulfide in N*(SeG) by GSeH present in the reactionmixture could facilitate partial denaturation of the pro-tein and thus circumvent the conformational barrier nor-mally associated with formation of the 30–51 disulfidebond. The enhanced nucleophilicity of selenols comparedto thiols in thiol/disulfide exchange reactions would cer-tainly favor such a reaction.[11, 12] This reactivity presuma-bly also minimizes potential problems associated withoveroxidation of N* (and N’) to give dead-end doublemixed selenosulfide adducts. Although we detect smallamounts of N*(SeG)2 at high GSeSeG concentrations(Figure 6 D), these species do not persist. As a conse-quence, the selenoglutathione-catalyzed conversion of N*to native BPTI is roughly comparable in rate to the con-version of N’. It is also worth noting here that the rate ac-celeration for this reaction is only 60-fold lower than thatachieved by protein disulfide isomerase (PDI),[18] a eu-karyotic enzyme that catalyzes disulfide bond formationand isomerization during protein biosynthesis.

Oxidative folding of many disulfide-containing proteinsis determined by a balance between productive and non-productive routes. Our results show that GSeSeG can fa-vorably influence this balance by virtue of its rapid reac-tion with thiols and its ability to provide energetically fa-vorable routes to the native state that compete with oth-erwise rate limiting protein disulfide bond isomerization.As has been shown for standard GSSG/GSH redox buf-fers,[27] even larger effects may be achievable through sys-tematic optimization of the reaction conditions. The abili-ty of GSeSeG to rescue kinetically trapped folding inter-mediates is of potentially broad applicability for the fold-ing of disulfide-containing proteins.

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Acknowledgments

We would like to thank Dr. Cindy Schulenburg and Dr.An Vandemeulebroucke for helpful discussions. Thisstudy was supported by the ETH Zurich. N.M. thanks theIsrael Science Foundation for financial support.

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Received: September 11, 2011Accepted: September 14, 2011

Published online: October 26, 2011

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Selenoglutathione-Mediated Rescue of Kinetically Trapped Intermediates in Oxidative Protein Folding