influence of protein conformation on disulfide bond formation in the oxidative folding of...

JMB—MS 651 Cust. Ref. No. RH027/95 [SGML]

J. Mol. Biol. (1995) 251, 135–149

Influence of Protein Conformation on DisulfideBond Formation in the Oxidative Folding ofRibonuclease T 1

†

Christian Frech and Franz X. Schmid*

Laboratorium fur Biochemie In oxidative protein folding the interdependence between the acquisitionof an ordered native-like conformation and disulfide bond formation wasUniversitat Bayreuth

D-95440 Bayreuth, Germany investigated by using the C2S/C10N variant of ribonuclease T1 as a model.This protein of 104 residues has a single disulfide bond between Cys6 andCys103. In the reduced form it is unfolded in the presence of urea, butnative-like folded when e1.5 M NaCl is present. The influence of a foldedconformation on the individual thiol/disulfide exchange reactions betweenthe protein and glutathione could thus be studied in oxidative folding byvarying the urea and NaCl concentrations. When the reduced protein wasfolded native-like the initial formation of the mixed disulfide between theprotein and glutathione was decelerated about fourfold. The attachment ofa glutathionyl moiety in this step destabilizes the protein by about 5 kJ mol−1

and led to a local unfolding near the two Cys residues. The reacting thiolgroups still remained in close proximity for the subsequent intramolecularthiol/disulfide exchange reaction, but an increase in the energy of thetransition state (e.g. by a hydrophobic environment or by steric strain) couldbe avoided. As a consequence the formation of the protein disulfide in thisreaction was 100-fold faster when the mixed-disulfide species was in thisordered conformation. These results illustrate the importance of a lowstability and a high flexibility of folding intermediates.

7 1995 Academic Press Limited

Keywords: protein folding; disulfide bonds; ribonuclease T1; folding*Corresponding author intermediates; glutathione

Introduction

Disulfide bonds are essential for the confor-mational stability of many proteins. Often they areburied in the interior of the folded protein andconnect cysteine residues that are remote from each

other in sequence. A close interrelationship shouldtherefore exist between the formation of thesecovalent crosslinks and the acquisition of the orderednative-like conformation during protein folding(Creighton, 1986, 1992; Weissman & Kim, 1991;Goldenberg, 1992; Darby & Creighton, 1993). Anative-like protein conformation brings the respect-ive cysteine residues into close proximity, and theformation of the correct disulfide bonds should thusbe facilitated. On the other hand, intermolecularthiol/disulfide exchange reactions with oxidants,such as glutathione or thiol disulfide oxidoreductases(Bardwell et al., 1991; Bardwell & Beckwith, 1993;Freedman, 1992; Freedman et al., 1994; Zapun et al.,1993; Wunderlich & Glockshuber, 1993) are requiredfor forming a protein disulfide bond, and thereforethe thiol groups should not become inaccessible bya premature formation of ordered structure.

We investigate here the interrelationship betweenprotein conformation and disulfide bond formation.In particular we ask how the individual steps in theformation of a disulfide bond are affected whenthe two cysteine residues are brought together by

Abbreviations used: RNase T1, ribonuclease T1;C2S/C10N-RNase T1, variant of RNase T1 withsubstitution of the 2–10 disulfide bond by a serine andan asparagine residue, respectively; RCM, form inwhich the 6–103 disulfide bond is reduced and thecysteine residues are carboxymethylated; RCAM, formin which the 6–103 disulfide bond is reduced and thecysteine residues are carbamidomethylated; Ceff,effective concentration; Ceff

k , Ceff measured from the ratioof the rate constants for comparable intra- andintermolecular reactions; GSH, glutathione; GSSG,oxidized glutathione; GdmCl, guanidinium chloride;PU, peptide acceleration unit; rate constants areindicated by k, equilibrium constants by K; U, Nunfolded and folded forms of C2S/C10N-RNase T1.

† This paper is dedicated to Professor Rainer Jaenickeon the occasion of his 65th birthday.

0022–2836/95/310135–15 $08.00/0 7 1995 Academic Press Limited

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Disulfide Bond Formation in RNase T1 Folding136

Scheme (I). Kinetic mechanism for the reactionsbetween C2S/C10N-RNase T1 and oxidized and reducedglutathione.

and Cys6 with Cys103 (Heinemann & Saenger, 1982;Martinez-Oyanedel et al., 1991; Pace et al., 1991). Inthe C2S/C10N variant only the Cys6–Cys103 disul-fide bond is present. Its formation can thus be studiedand analyzed in the absence of disulfide rearrange-ments on the basis of Scheme (I). Cys6 and Cys103form a large loop with 96 intervening amino acidsand link the amino- and carboxy-terminal regions ofRNase T1. The Cys6–Cys103 disulfide bond is in-accessible to solvent in the oxidized wild type protein(Martinez-Oyanedel et al., 1991), and its reductionis reversible (Pace & Creighton, 1986; Schonbrunner& Schmid, 1992; Frech & Schmid, 1995). OxidizedC2S/C10N-RNase T1 is unfolded in 6 M urea (pH 7.0,25°C) and folded below 2 M urea. The reducedprotein is unfolded at pH 7.0, 25°C in the absence ofdenaturants, but native-like folded in e1.5 M NaCl.Thus, the conformations of these forms can bechanged at will by changing the solvent conditions,and C2S/C10N-RNase T1 is therefore a good modelprotein to study the formation of a single disulfidebond during oxidative folding and to elucidate theinfluence of a folded protein conformation on theindividual steps of thiol/disulfide exchange.

We find that ordered structure in the reducedprotein has indeed a major effect on the thiol/disulfide exchange reactions. The initial formation ofthe mixed disulfide with glutathione (PSSG

SH ) is slightlydecelerated, but the intramolecular disulfide bondforms very rapidly when the reduced protein PSH

SH andthe mixed disulfide with glutathione PSSG

SH are foldednative-like.

Results

Conformational stability of C2S/C10N-RNase T 1

in the presence and in the absence of theCys6–Cys103 disulfide bond

To find appropriate solvent conditions for theoxidative folding experiments we first determinedthe conformational stabilities of the species that areinvolved in the thiol/disulfide exchange reactionsin Scheme (I). The folding transitions of PSH

SH, PSSGSSG,

and PSS could be measured at equilibrium. PSSG

SH canundergo intramolecular disulfide rearrangements,and its stability could thus not be determineddirectly. As will be shown later, it could be inferred,however, from the analysis of the oxidation kineticsas a function of the NaCl concentration.

The folding equilibria of PSHSH, PSSG

SSG, PSS and also of the

RCM and RCAM forms were measured at pH 7.0,25°C as a function of urea and NaCl concentrationby tryptophan fluorescence (Figure 1). The oxidizedform displays the highest stability, and its foldingtransition shows a midpoint at 3.0 M urea. The dis-ruption of the Cys6–Cys103 disulfide bond stronglydestabilizes the protein, and molecules without thiscrosslink are unfolded in aqueous buffer. A native-like folded structure is induced, however, in all theseforms when NaCl is added, and the respectivefolding transition of the reduced protein PSH

SH shows amidpoint near 0.7 M NaCl. The stability of the folded

inducing a native-like conformation in the reducedprotein. The introduction of a single disulfide bondinto a protein by reaction with a disulfide reagent,such as GSSG involves at least four species and threechemical equilibria, as shown in the minimalreaction mechanism in Scheme (I).

In the initial reaction either one of the two thiolgroups of the reduced protein PSH

SH can form a mixeddisulfide with glutathione. These two alternativereactions are represented by a single step (PSH

SH_PSSGSH )

in Scheme (I). The mixed disulfides PSSGSH which are

formed in this reaction can proceed in two directions.The remaining free protein thiol of PSSG

SH can eitherreact with another molecule of GSSG to form asecond mixed disulfide with glutathione in thePSSG

SH _PSSGSSG reaction, or else it can attack the mixed

disulfide between the protein and glutathione in theintramolecular PSSG

SH _PSS reaction to form the protein

disulfide in PSS. Kinetic mechanisms as in Scheme (I)

were used previously to analyze the introductionand breaking of single disulfides in peptides and inprotein fragments (Goto & Hamaguchi, 1981; Snyder,1987; Creighton, 1992; Darby & Creighton, 1993).

In our analysis we concentrate on the formation ofthe mixed disulfide with glutathione (PSH

SH:PSSGSH ) and

on the intramolecular disulfide exchange reaction(PSSG

SH :PSS). These two steps are obligatory for form-

ing the protein disulfide, and they should be sensitiveto the presence of a folded conformation in the pro-tein. The rate constant of the first step k1MD dependson the accessibility of the thiol groups in the reducedprotein, the rate constant of the second step kintra

depends on the effective concentration of the freeprotein thiol relative to the mixed disufide withglutathione and on the energy of the transition statefor disulfide exchange. The presence of a foldedprotein conformation could change the energy of thistransition state significantly by steric constraints orby effects on the pK values of the three thiol groupsthat are aligned in the transition state for thiol/disulfide exchange (Szajewski & Whitesides, 1980).Thus, both reactions should be sensitive to the con-formational states of the PSH

SH and of the PSSGSH species.

In our experiments we use a variant of ribo-nuclease T1 (RNase T1) from Aspergillus oryzae as amodel protein. RNase T1 is a small protein of 104amino acid residues, and the wild-type form containstwo disulfide bonds, which connect Cys2 with Cys10

JMB—MS 651

Disulfide Bond Formation in RNase T1 Folding 137

Figure 1. Comparison of the folding transitions ofvarious forms of C2S/C10N-RNase T1 at pH 7.0, 25°C. Thetransitions were followed as a function of the urea (leftpanel) and NaCl (right panel) concentration by fluor-escence at 320 nm after excitation at 268 nm. The proteinconcentrations were 0.5 mM in 0.1 M Hepes, 2 mM EDTA(pH 7.0). The solution of the reduced protein additionallycontained 4 mM DTT. A linear extrapolation of thebaselines in the pre- and posttransitional regions was usedto determine the fraction of folded protein f(N) within thetransition region by assuming a two-state model forfolding. The lines represent an analysis of the data by usingthe method of Santoro & Bolen (1988). (r) Oxidized pro-tein (PS

S); (w) reduced protein (PSHSH); (Q) RCAM-protein;

(q) double mixed disulfide (PSSGSSG); (W) RCM-protein.

acetamide. The trapped species PSHSH, PSSG

SH , PSSGSSG, and PS

S

were resolved by polyacrylamide gel electrophoresisin the absence of denaturants, and their relativeconcentrations were determined by laser densito-metry. The microscopic rate constants were derivedfrom a joint fit of the time courses of the individualspecies by using the differential rate equations which(on the basis of Scheme (I)) describe the concen-trations of these species as a function of time.

Under the conditions of the electrophoresis(pH 9.5, 5°C) only the native protein with the intactdisulfide bond is in a folded compact conformationand thus shows the highest electrophoretic mobility.All other species are unfolded and migrate moreslowly. Their mobilities are determined by thenumber of glutathione and CAM residues that areattached to the protein. The CAM moiety isuncharged, but each glutathione increases the netnegative charge of the protein by about 1.5 units. Asa consequence, the PSH

SH form with two CAM residuesshowed the lowest mobility, followed by the PSSG

SH

species with a single glutathione and a single CAMresidue, and by PSSG

SSG with two glutathione residues.The two alternative PSSG

SH species could not be resolvedby gel electrophoresis.

Time courses of reduction and oxidation

The kinetics of reduction of oxidized C2S/C10N-RNase T1 by 5 mM GSH in the presence of 6.0 M ureaare shown in Figure 2A. Under these unfoldingconditions reduction is an irreversible sequentialreaction. The concentration of the oxidized protein PS

S

decreases in a monoexponential process, which isgoverned by the rate constant kr (cf. Scheme (I)). Themixed disulfide PSSG

SH accumulates transiently, and thereduced protein PSH

SH is formed with a lag at the begin-ning. The double mixed-disulfide PSSG

SSG is not formedin this experiment, because GSSG is not present.

The kinetics of oxidation of the reduced andunfolded protein in a mixture of 2 mM GSSG and2 mM GSH at 0 M urea/0 M NaCl are shown inFigure 2B. Under these conditions both oxidation andreduction reactions occur and the analysis of thekinetic curves in Figure 2B can be used to determinethe rate constants of all steps in Scheme (I). Specieswithout the disulfide bond are unfolded in theabsence of NaCl. Oxidative folding is very slow, andthe time courses of all species (PSH

SH, PSSGSH , PSSG

SSG, and PSS)

could be followed. In the first step the reducedprotein PSH

SH reacts with a molecule of GSSG to formthe mixed disulfide PSSG

SH , and after 2000 secondsabout 50% of all molecules are in the PSSG

SH state. Thereaction with a second molecule of GSSG to form PSSG

SSG

is slightly favored over the intramolecular disulfideexchange reaction to PS

S, and, as a consequence,the concentration of the PSSG

SSG species increases morerapidly than the concentration of PS

S between 0 and8000 seconds. Under the conditions of the exper-iment, however, PS

S is the most stable species(cf. Figure 1) and is thus formed very slowly fromthe other species after extended times of oxidation(Figure 2B).

conformation decreases, and transition midpointsbetween 0.9 and 1.1 M NaCl were observed whenthe thiol groups were chemically modified with twomolecules of glutathione, iodoacetate or iodoacet-amide. Similar results were obtained by Ruoppolo &Freedman (1994) by a different method.

The stability curves in Figure 1 define three dis-tinct sets of conditions. In the presence of 6 M ureaall species are unfolded, irrespective of their stateof oxidation. In the absence of urea and NaCl,the protein is folded in the presence, but not in theabsence of the disulfide bond, and in the presence of2.5 M NaCl all species with and without the disulfidebond are in native-like folded conformations. In thereoxidation experiments we will use these threeconditions to investigate the interrelation betweenthe acquisition of folded structure and the formationof the disulfide bond.

Electrophoretic analysis ofthiol/disulfide exchange

The kinetics of forming and breaking of the disul-fide bond in C2S/C10N-RNase T1 were measured atpH 7.0, 25°C. Thiol/disulfide exchange was initiatedby diluting the reduced or the oxidized protein intosolutions that contained reduced (GSH) and/or oxi-dized (GSSG) glutathione in varying concentrations.Aliquots were then taken after different times, andfurther exchange was stopped by reaction with iodo-

JMB—MS 651


Oxidation under varying solvent conditions

The kinetics of oxidation of the reduced protein byGSSG under different solvent conditions are shownin Figure 3. Only oxidation reactions occur at anappreciable rate when GSSG, but not GSH, is present,and the reverse reactions PS

S:PSSGSH and PSSG

SSG:PSSGSH

(Scheme (I)) do not contribute to the measuredkinetics. The species PS

S and PSSGSSG are formed from the

mixed disulfide PSSGSH with the same apparent rate

constant, which is equal to the sum of the rate con-stant kintra for intramolecular disulfide bond formation(PSSG

SH :PSS) and k2MD × [GSSG] for intermolecular di-

sulfide formation with a second molecule of GSSG(PSSG

SH :PSSGSSG). The ratio of the products [PS

S]/[PSSGSSG] is

determined by the ratio of these rate constants,kintra/(k2MD × [GSSG]).

Oxidation was carried out under three differentconditions: (1) in 6 M urea, where all forms ofC2S/C10N-RNase T1 are unfolded (Figure 3A), (2) inthe absence of urea and NaCl, where the reducedprotein is unfolded, but the oxidized protein is folded(Figure 3B); and (3) in the presence of 2.5 M NaCl,where all forms of the protein adopt a folded,native-like conformation (Figure 3C).

Three major differences became apparent from thecomparison of the kinetic curves in Figure 3A to C.(1) The initial PSH

SH:PSSGSH reaction was very slow in 6 M

urea, its rate increased about threefold when ureawas absent, but then decreased again by a factorof 3 in 2.5 M NaCl. (2) The mixed disulfide PSSG

SH

accumulated transiently at a high concentration inthe presence of 6 M urea (Figure 3A) or of 0 M urea(Figure 3B), but not in 2.5 M NaCl (Figure 3C).(3) The formation of the double-mixed disulfide PSSG

SSG

was strongly favored over the intramolecular ex-change reaction to form PS

S in the presence of 6 M urea(Figure 3A) and in the absence of NaCl (Figure 3B).

Virtually no PSS molecules could be detected, when

the protein was oxidized in 6 M urea by 5 mM GSSG(data not shown). Therefore, the concentration ofGSSG in Figure 3A was reduced to 3 mM to allow forthe formation of a small amount of PS

S molecules.The formation of the protein disulfide bond in the

PSSGSH :PS

S reaction was very slow in 6 M urea andin 0 M urea/0 M NaCl, but accelerated strongly in2.5 M NaCl (Figure 3C). Therefore, it could competeefficiently with the formation of PSSG

SSG from PSSGSH and the

yield of PSS molecules was strongly increased. The

decrease in the rate of the initial step (PSHSH:PSSG

SH ) incombination with the accelerated formation of PS

S

in the PSSGSH :PS

S step led to the observed strongdecrease in the concentration of the intermediate PSSG

SH

in Figure 3C.The reoxidation in 2.5 M NaCl was also measured

in the presence of a mixture of 10 mM GSSG and1 mM GSH (Figure 3D). Under these conditions thedecrease of PSH

SH and the increase of PSS showed similar

time courses, which suggests that the intramolecularPSSG

SH :PSS reaction is very rapid, and thus the for-

mation of PSS is limited in rate by the initial formation

of the mixed disulfide (PSHSH:PSSG

SH ). As in Figure 3Conly a small amount of PSSG

SSG was formed, which couldbe converted to PS

S, because 1 mM GSH was presentin this experiment.

The rate constants of oxidation and reduction

Reduction and oxidation experiments, as shownin Figures 2 and 3, were carried out in 6.0 M urea,in 0 M urea/0 M NaCl, and in 2.5 M NaCl in thepresence of six to eight different concentrations ofGSH and GSSG or combinations thereof. The timecourses of the species PSH

SH, PSSGSH , PSSG

SSG, and PSS obtained

under a particular set of conditions were then fitted

Figure 2. Kinetics of: A, reduction of oxidized C2S/C10N-RNase T1; and B, oxidation of reduced C2S/C10N-RNaseT1 at pH 7.0, 25°C. A, Denatured, oxidized C2S/C10N-RNase T1 was reduced by 5 mM GSH in 6.0 M urea, 0.1 M Hepes,2 mM EDTA. The time courses of (Q) the oxidized protein (PS

S), (w) the mixed disulfide (PSSGSH ), and (W) the reduced protein

(PSHSH) are shown as a function of the time of reduction. B, Reduced C2S/C10N-RNase T1 was oxidized by 2 mM GSSG

and 2 mM GSH in 0.1 M Hepes, 2 mM EDTA. The time courses of (W) PSHSH, (w) PSSG

SH , (q) PSSGSSG, and (Q) PS

S are shown asa function of the time of oxidation. The concentrations of the individual species were measured by gel electrophoresis.The curves were obtained by fitting the experimental data to the kinetic mechanism in Scheme (I). The resulting valuesfor the rate constants are given in Tables 1 and 2.

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Figure 3. Kinetics of oxidation of reduced C2S/C10N-RNase T1 under various solvent conditions in 0.1 M Hepes, 2 mMEDTA (pH 7.0) at 25°C. The conditions for oxidation were: A, 3 mM GSSG in 6.0 M urea; B, 5 mM GSSG in 0 M urea/0 MNaCl; C, 5 mM GSSG in 2.5 M NaCl, after a preincubation of the reduced protein in 2.8 M NaCl for one hour; and D,10 mM GSSG and 1 mM GSH in 2.5 M NaCl after a preincubation of the reduced protein in 2.8 M NaCl for one hour.The time courses of (W), PSH

SH, (w) PSSGSH , (q) PSSG

SSG, and (Q) PSS are shown as a function of the time of oxidation. The

concentrations of the individual species were measured by gel electrophoresis. The curves were obtained by fittingthe experimental data to the kinetic mechanism in Scheme (I). The resulting values for the rate constants are given inTables 1 to 3.

jointly to the kinetic model in Scheme (I) to determinethe microscopic rate constants of the individual stepsin Scheme (I). For the analyses it was assumed thatthe concentrations of GSH and GSSG remained con-

stant during thiol/disulfide exchange, because theywere much higher than the protein concentration.

The rate constants that resulted from these analy-ses are given in Table 1 for the experiments in 6 M

Table 1. Rate constants for disulfide bond formation and reduction inC2S/C10N-RNase T1 in the presence of 6 M urea[GSSG] (mM) 1 3 3 — 5 —[GSH] (mM) — — — 5 2 DTT

k1MD (s−1 M−1) 0.081 0.11 0.12 — 0.12 —k−1MD (s−1 M−1) — — — 0.042 0.065 —k2MD (s−1 M−1) 0.013 0.01 0.02 — 0.033 —k−2MD (s−1 M−1) — — — — 0.066 —kintra (s−1) 6.8 × 10−6 7.1 × 10−6 7.9 × 10−6 7.0 × 10−6 7.0 × 10−6 —kr (s−1 M−1) — — — 0.05 0.077 0.05

Reduction and oxidation were carried out in the presence of the indicated concentrations of GSH andGSSG. Rate constants were determined by a joint non-linear least-squares fit of the kinetic data obtainedby electrophoretic analysis of trapped refolding and reduction reactions, using GSSG and GSH as reagent,except where indicated by DTT. The value of kr in the presence of DTT was obtained from the rate ofreduction of the disulfide bond by DTT. The kinetics of reduction by DTT were pseudo-first order atthree different DTT concentrations, and the product of reduction was the fully reduced protein.

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Table 2. Rate constants for disulfide bond formation and reduction inC2S/C10N-RNase T1 in 0 M urea/0 M NaCl[GSSG] (mM) 0.4 3 5 1 2 2[GSH] (mM) — — — 1 1 2

k1MD (s−1 M−1) 0.24 0.27 0.32 0.24 0.31 0.33k−1MD (s−1 M−1) — — — 0.1 0.17 0.12k2MD (s−1 s−1) 0.063 0.043 0.052 0.083 0.084 0.082k−2MD (s−1 M−1) — — — 0.16 0.14 0.11kintra (s−1) 6.3 × 10−5 6.1 × 10−5 6.5 × 10−5 5.7 × 10−5 5.7 × 10−5 6.7 × 10−5

kr (s−1 M−1) — — — 0 0 0

The rate constants were determined by using GSH and GSSG, as described in the legend to Table 1.

urea, in Table 2 for 0 M urea/0 M NaCl, and in Table 3for 2.5 M NaCl. The profiles calculated for theindividual species of Scheme (I) by using these rateconstants agreed well with the experimental dataat all concentrations of GSH and GSSG. For theexperiments that are displayed in Figures 2 and 3 thecalculated profiles are shown by continuous lines.The rate constants obtained at different glutathioneconcentrations were consistent within experimentalerror. It should be noted that not all rate constantscould be determined with equal accuracy underall conditions. In particular, the rate of reduction(PS

S:PSSGSH ) could only be measured in the presence of

urea, because the native oxidized protein is stable inthe absence of urea (cf. Figure 1).

NaCl and urea influence the rates of thiol/disulfide exchange not only because they changethe stability of folded conformations of a protein,but also because they affect the ionization equilibriaof thiol groups. Thiol/disulfide exchange is deceler-ated when the concentration of urea is increased(Creighton, 1977, 1978; Darby & Creighton, 1993) andaccelerated when urea is replaced by an ionic de-naturant such as GdmCl. To examine these influenceson the thiol/disulfide exchange between unfoldedC2S/C10N-RNase T1 and glutathione, we carried outcontrol experiments in denaturing solvents of highionic strength, i.e. in 4 M GdmCl and in 4 M urea plus1 M NaCl. The results of these experiments areshown in Table 4. The rate constants of all thiol/disulfide exchange reactions were increased in 4 MGdmCl, or when 1 M NaCl was added to a 4 M ureasolution. In further control experiments the reactionbetween reduced DTT and GSSG was measured atpH 7.0, 25°C as a function of NaCl and of ureaconcentration (data not shown). The addition of

1.0 M NaCl led to a 2.0-fold increase in the rate of thisthiol/disulfide reaction, the addition of 4.0 M ureadecreased it 1.6-fold.

Tables 1 to 4 provide a wealth of information on allsteps of the oxidative folding of C2S/C10N-RNaseT1. The experiments in the presence of varying con-centrations of GSH and GSSG yielded rate constantsthat were generally consistent with each other,suggesting that the model in Scheme (I) provides anadequate description of the thiol/disulfide exchangereactions between glutathione and the protein underall these conditions.

In the following we concentrate on the rateconstants k1MD and kintra (cf. Scheme (I)), which reportdirectly on the interdependence between the exist-ence of folded structure and the formation of thedisulfide bond. The rate constant k1MD was measuredunder various unfolding conditions (Table 1), andvalues of 0.11 s−1 M−1 in 6 M urea, 0.26 s−1 M−1 in 4 MGdmCl and 0.39 s−1 M−1 in 4 M urea plus 1 M NaClwere found. When the reduced protein is in a foldedconformation (in the presence of 2.5 M NaCl), thethiol groups are still able to form mixed disulfideswith GSSG, its rate constant is, however, slightlydecreased to 0.14 s−1 M−1.

The intramolecular exchange between the freeprotein thiol and the mixed disulfide with gluta-thione (PSSG

SH :PSS, cf. Scheme (I)) is strongly depend-

ent on the conformation of PSSGSH . When the protein is

unfolded, this reaction is very slow and its rateconstant kintra shows values of 7 × 10−6 s−1 in 6 M urea,8 × 10−5 s−1 in 4 M GdmCl and 6 × 10−5 s−1 in 4 Murea/1 M NaCl. When PSSG

SH is in a folded confor-mation (in the presence of 2.5 M NaCl) the PSSG

SH :PSS

reaction is strongly accelerated, and kintra increases to3 × 10−3 s−1 (Table 3).

Table 3. Rate constants for disulfide bond formation and reduction in C2S/C10N-RNase T1 in the presenceof 2.5 M NaCl[GSSG] (mM) 0.4 5 5 10 1 10 10 20[GSH] (mM) — — — — 1 1 1 1

k1MD (s−1 M−1) 0.16 0.13 0.10 0.14 0.14 0.15 0.15 0.15k−1MD (s−1 M−1) — — — — 1 0.46 0.5 1.9k2MD (s−1 s−1) 0.11 0.15 0.1 0.12 0.13 0.17 0.14 0.12k−2MD (s−1 M−1) — — — — 0.24 0.63 0.41 0.49kintra (s−1) 1.9 × 10−3 2.6 × 10−3 2.4 × 10−3 3.3 × 10−3 2.6 × 10−3 3.0 × 10−3 3.4 × 10−3 2.7 × 10−3

kr (s−1 M−1) — — — — 0 0 0 0

The rate constants were determined by using GSH and GSSG as reagents, as described in the legend to Table 1.

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Table 4. Rate constants for disulfide bond formationand reduction in C2S/C10N-RNase T1 in thepresence of 4 M GdmCl and 4 M urea + 1 M NaCl

4 M urea4 M GdmCl 1 M NaCl

[GSSG] (mM) 5 — 5[GSH] (mM) — 5 —

k1MD (s−1 M−1) 0.26 — 0.39k−1MD (s−1 M−1) — 0.19 —k2MD (s−1 M−1) 0.17 — 0.11k−2MD (s−1 M−1) — — —kintra (s−1) 8.7 × 10−5 8.0 × 10−5 4.1 × 10−5

kr (s−1 M−1) — 0.3 —

The rate constants were determined by using GSH and GSSGas reagents, as described in the legend to Table 1.

2.0 M urea. A slight decrease is noted between 2 and3 M NaCl.

The profile for kintra in Figure 4A strongly resemblesa protein folding transition, and it could reflect thestability of the mixed disulfide PSSG

SH . In native-likefolded PSSG

SH intramolecular disulfide exchange isstrongly accelerated probably because the remainingfree protein thiol is in close proximity to themixed disulfide with glutathione. The data for kintra inFigure 4A can indeed be well analyzed by assuminga two-state equilibrium between folded (N) andunfolded (U) molecules of PSSG

SH (USSGSH _NSSG

SH ), whichdiffer strongly in their values for kintra. An excellentfit is obtained by using the formalism of Santoro &Bolen (1988) and by assuming that the kintra valuesof both the unfolded and the folded protein varylinearly with the concentrations of urea and NaCl.The midpoint of the transition of kintra in Figure 4A isat 1.2 M NaCl. At this concentration of NaCl kintra ofN is about 150-fold higher than kintra of U.

Conformational stability of the mixed disulfidespecies P SSG

SH

The good quality of the fit to a two-state unfoldingtransition does not constitute proof that the curve forkintra in Figure 4A reflects indeed the conformationalstability of the PSSG

SH species. In the transition regionthe reaction mechanism is complex because both thereduced protein PSH

SH and the mixed disulfide PSSGSH can

exist in the folded or the unfolded conformation, anda mechanism as in Scheme (II) is required to describethe kinetics of oxidation. Scheme (II) is basically aduplication of Scheme (I). It is composed of twohalves that represent the oxidation kinetics of theunfolded (U) and of the folded (N) forms of theprotein. These two halves are linked by the folding

Dependence on protein conformation ofthe intramolecular thiol/disulfideexchange reaction

To further investigate the dependence on proteinconformation of the intramolecular disulfide ex-change reaction we measured the kinetics of oxi-dation by 5 mM GSSG (as in Figure 3B and C) in thepresence of 6 to 0 M urea (Figure 4A, left panel) andof 0 to 3 M NaCl (Figure 4A, right panel). The valuesobtained for kintra from the analysis of the observedkinetics are shown in Figure 4A.

The protein is unfolded in the presence of urea,and the slight increase in kintra from 0.7 × 10−5 s−1 (in6.0 M urea) to 2.0 × 10−5 s−1 (in 2.0 M urea) reflects theintrinsic dependence of kintra on urea concentration.kintra increases strongly when NaCl is added (cf. theright panel of Figure 4A), and a maximal value of2.7 × 10−3 s−1 is reached near 2 M NaCl. This value ismore than 100-fold higher than the value of kintra in

Figure 4. Dependence on urea and NaCl concentrations of the measured values for kintra at pH 7.0, 25°C. A, The valuesof kintra (W) at the indicated concentrations of urea and NaCl were determined by oxidation experiments as in Figure 3A to C. (w) Values of kintra in the presence of 1000 PU/ml prolyl isomerase. The line represents an analysis of the dataaccording to the two-state model by using the method of Santoro & Bolen (1988). The midpoint of the transition is at 1.2 MNaCl. The base lines for the unfolded and the folded molecules are given by: kU

intra = 1.8 × 10−5 s−1 − 1.7 × 10−6 s−1 M−1 × [urea],and kN

intra = 3.5 × 10−3 s−1 − 3.7 × 10−4 s−1 M−1 × [NaCl]. B, Comparison of the normalized transition of kintra (W) with thenormalized equilibrium unfolding transitions of the PSH

SH form (w) and the PSSGSSG form (q), as taken from Figure 1.

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Scheme (II). Coupling between the kinetics of oxidationand the conformational transitions of C2S/C10N-RNaseT1. This Scheme is basically a duplication of Scheme (I). Theright and left halves represent the mechanisms for theoxidation of the unfolded form (U) and of the foldedform (N). They are linked by the folding equilibria of thereduced form, of the mixed disulfide with glutathione, andof the oxidized protein. Only oxidation reactions areshown, because the experiments were carried out in thepresence of GSSG only, GSH was absent.

constant for the stability of the mixed disulfide inequation (4):

KMD = [USSGSH ]

[NSSGSH ] (4)

The combination of equations (3) and (4) yieldsequation (5):

KMD = [USSGSH ]

[NSSGSH ] = kN

intra − kappintra

kappintra − kU

intra(5)

These considerations show that indeed the de-pendence on NaCl concentration of the measuredvalue kapp

intra (Figure 4A) follows the stability curve ofthe mixed disulfide PSSG

SH provided that the equili-bration reaction between the folded and unfoldedforms of the mixed disulfide (NSSG

SH _USSGSH ) is fast

compared to the redox reactions (cf. Scheme (II)).Actually, it is difficult to evaluate whether thiscondition is met, because the rates of unfolding andrefolding of both the reduced protein and themixed disulfide depend strongly on the NaClconcentration. Whether equation (5) is appropriatefor analyzing the kinetic data in Figure 4A can betested experimentally, because the unfolding andrefolding reactions can be accelerated by prolylisomerase.

The equilibration reactions between folded andunfolded forms are slowest in the transition region,and at 1.25 M NaCl they show time constants of570 seconds for the reduced protein (Figure 5A)and of 470 seconds for the double mixed disulfide(Figure 5B). These reactions are very well catalyzedby prolyl isomerase and both are accelerated about20-fold, when 1000 PU/ml prolyl isomerase areadded (Figure 5). At 0.5 M NaCl the time con-stants of uncatalyzed folding were 233 seconds and88 seconds for the reduced protein and the doublemixed disulfide, respectively, and 1000 PU/ml prolylisomerase accelerated folding about tenfold (data notshown).

Accordingly, the reoxidation experiments at 0.5 Mand 1.25 M NaCl were repeated in the presence of1000 PU/ml prolyl isomerase. The resulting valuesfor kapp

intra are shown as open circles in Figure 4A. Theycoincide well with the values that were measured inthe absence of prolyl isomerase. This coincidenceprovides strong evidence that under the conditionsdescribed for Figure 4A the folding reactions ofthe reduced protein PSH

SH and of the mixed disulfidePSSG

SH are sufficiently rapid relative to the disulfideexchange reactions. This indicates that equation (5)holds and that the profile for kapp

intra in Figure 4Aindeed follows the stability of the mixed disulfidePSSG

SH .The normalized unfolding transition of the mixed

disulfide (NSSGSH ), as derived from the kapp

intra values, isshown in Figure 4B in comparison with the foldingtransitions of the reduced protein (PSH

SH) and of thedouble-mixed disulfide (PSSG

SSG), as taken from Figure 1.The mixed disulfide with glutathione is less stablethan the reduced protein, and at 1.0 M NaCl there is a

equilibria of the reduced protein, the mixeddisulfide, and the oxidized protein.

Under the conditions of the electrophoretic analy-sis (pH 9.5 and low salt) the conformational stateof the individual species is not conserved, and wecannot discriminate between the folded and theunfolded forms of the various species in Scheme (II).Their sums are monitored as a function of time andexperimentally an apparent rate constant kapp

intra isderived from the kinetic analysis of the formation of(US

S + NSS) as shown in equation (1):

d([USS] + [NS

S])dt = kapp

intra·([USSGSH ] + [NSSG

SH ]) (1)

According to Scheme (II) USS and NS

S are formedfrom USSG

SH and NSSGSH , respectively, and the change

of their concentrations with time is described byequation (2):

d([USS] + [NS

S])dt = kU

intra·[USSGSH ] + kN

intra·[NSSGSH ] (2)

The combination of equations (1) and (2) yieldsequation (3). It relates the measured value kapp

intra withthe microscopic rate constants kN

intra and kUintra and with

the relative concentrations of USSGSH and NSSG

SH :

kappintra = kU

intra· 11 + [NSSG

SH ]/[USSGSH ] + kN

intra· 11 + [USSG

SH ]/[NSSGSH ]

(3)

The concentrations of both USSGSH and NSSG

SH are afunction of time (cf. Scheme (II)) and equation (2) isnot easily integrated. When, however, the equili-bration between USSG

SH and NSSGSH is much faster than

all the thiol/disulfide exchange reactions, then theratio between USSG

SH and NSSGSH in equation (3) remains

constant and can be replaced by the equilibrium

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Figure 5. Kinetics of refolding of: A, the PSHSH form: and B, the PSSG

SSG form of C2S/C10N-RNase T1 in the absence (filledsymbols) and in the presence (open symbols) of 1000 PU/ml prolyl isomerase at pH 7.0, 25°C. Refolding was monitoredby the increase in fluorescence at 320 nm. The refolding conditions were 1 mM protein in 1.25 M NaCl, 0.1 M Hepes, 2 mMEDTA (pH 7.0); the refolding solution for the reduced protein contained in addition 4 mM DTT. All refolding reactionswere monoexponential processes. The refolding of PSH

SH in A showed time constants of 567 seconds for the uncatalyzedreaction and 26 seconds for the catalyzed reaction. The refolding of PSSG

SSG in B showed time constants of 474 seconds forthe uncatalyzed reaction and of 24 seconds for the catalyzed reaction.

difference of about 5 kJ mol−1 in the Gibbs free energyof stabilization between PSSG

SH and PSHSH.

The unfolding equilibria of the reduced protein PSHSH

and of the mixed disulfide PSSGSH are linked with the

thiol/disulfide exchange reactions of the folded andthe unfolded forms as shown by the thermo-dynamic cycle in Scheme (III). As a consequence thedifference in the conformational stability betweenPSH

SH and PSSGSH can also be determined from the ratio of

the equilibrium constants KNSSG and KU

SSG for the for-mation of the mixed disulfide between the reducedprotein and GSSG in the folded and unfolded forms,respectively. KN

SSG and KUSSG were calculated from the

kinetic data in 2.5 M NaCl (Table 3) and in 0 Murea/0 M NaCl (Table 2). Their values were 0.3 and2.3, respectively, indicating that PSH

SH and PSSGSH differ by

5.0 kJ mol−1 in conformational stability, which is inexcellent agreement with the value derived from

the two-state analysis of the folding transitions inFigure 4.

This destabilization results from the decrease instability of the folded conformation of PSSG

SH and notfrom an increased stability of the mixed disulfidein the unfolded protein. The equilibrium constantKU

SSG = 2.3 is as expected for an unfolded protein(Darby et al., 1994) when the decreased reactivity ofCys103 is taken into account. Similar conclusionswere reached for variants of T4-lysozyme and DsbAwith single cysteine residues that could form revers-ibly mixed disulfides with thiol reagents (Lu et al.,1992; Zapun et al., 1994).

Together, this confirms that the dependence onNaCl concentration of kapp

intra indeed reflects the stab-ility of the mixed disulfide PSSG

SH . The attachment ofa single glutathione moiety reduces the confor-mational stability of the protein by about 5 kJ mol−1.When a second glutathione is bound no furtherdecrease is noted, and the stabilities of PSSG

SSG and PSSGSH

are very similar (cf. Figure 4B).

Dependence on protein conformation ofmixed-disulfide formation with glutathione

The rate constant of formation of PSSGSH (k1MD, cf.

Scheme (I)) is shown in Figure 6 as a function of theurea and NaCl concentrations. As noted before, k1MD

increases with decreasing urea concentration andreaches a maximal value of 0.48 s−1 M−1 in the pres-ence of 0.25 M NaCl. Between 0.25 M and 1.25 MNaCl we note a sharp decrease in k1MD, followed bya continuing small decrease with NaCl concen-tration. A quantitative analysis of the profile for k1MD

is difficult. A comparison with the value for k1MD

in 4.0 M urea plus 1.0 M NaCl (the open circle inFigure 6) suggests that the increase in k1MD between6.0 M urea and 0.25 M NaCl is largely caused by the

Scheme (III). Thermodynamic linkage between thereaction of reduced C2S/C10N-RNase T1 with glutathioneand reversible protein unfolding.

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Figure 6. Dependence on urea and NaCl concentrationof the measured values for k1MD at pH 7.0, 25°C. The valuesof k1MD (W) at the indicated concentrations of urea and NaClwere determined by oxidation experiments as shown inFigure 3A to C. The value for k1MD in the presence of 4 Murea/1 M NaCl (w) is also shown.

k−1MD and k−2MD equal to 0.5kex and 1kex, respectively(cf. Darby & Creighton, 1993).

We find a value of k1MD = 0.3 to 0.4 s−1 M−1 for theformation of the mixed disulfide in the unfoldedprotein chain (in 4 M GdmCl or in 4 M urea/1 MNaCl). This value decreases to 0.11 s−1 M−1 in 6 Murea. Disulfide exchange reactions are decelerated inthe presence of urea (Creighton, 1977; Darby &Creighton, 1993), presumably because the pK valuesof cysteine thiols are increased when urea is present(Donovan et al., 1959). In 6 M urea the rate constantk1MD for forming the first mixed disulfide is abouteightfold higher than the rate constant k2MD forforming the second mixed disulfide. This is signifi-cantly higher than the factor of two which is expectedon statistical grounds (Darby & Creighton, 1993). Itsuggests that Cys6 and Cys103 of C2S/C10N-RNaseT1 differ in nucleophilicity when reacting with GSSG.Cys6 is flanked by uncharged residues, but Cys103is flanked by the side-chain of Glu102 and thecarboxy terminus at Ala104. These two negativecharges decrease the reactivity of Cys103 in twoways. They increase its pK value, and they lead toelectrostatic repulsion with glutathione, which isalso negatively charged. These unfavorable inter-actions are partially screened in the presence of saltand therefore the ratio k1MD/k2MD is reduced to lessthan 4 in the presence of 4 M GdmCl or 4 M urea/1 M NaCl. The influence of the local charge distri-bution around cysteine residues on the kinetics ofdisulfide exchange was studied in detail by Snyderet al. (1981).

Because the reactivity of Cys103 is reduced wesuggest that in most PSSG

SH molecules the glutathionemoiety is attached to Cys6. This conclusionmust remain tentative, however, because the twoalternative PSSG

SH species could not be separatedexperimentally.

Intramolecular disulfide formation in theunfolded protein

The disulfide bond between Cys6 and Cys103closes a very large loop with n = 96 interveningresidues, which is far above the average value ofn = 15 (Betz, 1993). As a consequence the rateconstant for the intramolecular reaction (kintra) is verysmall in the unfolded protein. The tendency to forma disulfide bond depends on the effective concen-tration (Ceff) of the free thiol relative to the mixeddisulfide with glutathione. Ceff can be derived bycomparing the rate of this intramolecular exchangereaction (kintra) with the rate of the intermolecularexchange reaction with GSH (k−1MD) of the samemixed disulfide, and the ratio kintra/k−1MD gives akinetic measure for Ceff (Creighton, 1983; Darby &Creighton, 1993). The Ceff values of various unfoldedpeptides and proteins with disulfides in the presenceof urea vary approximately with n−5/2 as expected forunfolded polymers when excluded volume effectsare taken into account (Chan & Dill, 1991; Darby &Creighton, 1993). With its value of about 1.5 × 10−4 MCeff of Cys6 and Cys103 follows the n−5/2 relationship

general effects of urea and NaCl on the intrinsic ratesof thiol/disulfide exchange as discussed earlier. Thethreefold decrease in k1MD between 0.25 M NaCl and1.25 M NaCl correlates well with the NaCl-inducedfolding transition of the reduced protein, whichoccurs between 0 M and 1.25 M NaCl (cf. Figure 4B).This suggests that the measured value for k1MD

follows the conformational stability of the reducedprotein, and that the PSH

SH:PSSGSH reaction is decelerated

three- to fourfold when the reduced protein is in thefolded state.

Discussion

Formation of mixed disulfides with glutathionein unfolded C2S/C10N-RNase T 1

The reactions between protein thiols and lineardisulfide reagents are very well studied (Szajewski &Whitesides, 1980; Shaked et al., 1980), and the kineticmechanism in Scheme (I) provides an adequatedescription for the thiol/disulfide exchange betweena protein chain with two cysteine residues and gluta-thione (Creighton, 1986, 1992; Snyder, 1987; Darby &Creighton, 1993). Thiolate anions are the reactivespecies in all these reactions, and the rate constant ofthiol/disulfide exchange kex depends on the pH of thesolvent and on the pK values of the nucleophilic, thecentral, and the leaving thiol groups. Szajewski &Whitesides (1980) suggested an empirical relation-ship to calculate kex from these data. For exchangereactions between the cysteine resi-dues of an unfolded protein chain and of glutathionekex is approximately 0.1 s−1 M−1 at pH 7.0 and 25°Cwhen all three thiol groups show identical pK valuesof 8.8 (Snyder, 1987). Under this assumption therate constants in Scheme (I) can be derived from kex

when the number of free thiols and the number ofalternative exchange reactions are taken into account.k1MD and k2MD should be equal to 2kex and 1kex, and

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and is as expected from a compilation of Ceff valuesfor unfolded proteins and peptides with muchsmaller disulfide-bonded loops (cf. Figure 6 of Darby& Creighton, 1993).

In unfolded oxidized C2S/C10N-RNase T1 the rateof reduction by GSH of the Cys6–Cys103 disulfide(kr = 0.05 s−1 M−1) chain is about twofold smaller thanthe reverse reaction of the reduced protein withGSSG (K1MD = 0.1 s−1 M−1). This is as expected frommodel studies and indicates that in the unfoldedprotein the disulfide bond is accessible to solvent andas reactive as GSSG.

In summary, these comparisons show that in 6 Murea all species of C2S/C10N-RNase T1 are maxi-mally unfolded. The Cys residues are accessible tosolvent and their reactions with glutathione followthe rules that were established for the thiol/disulfideexchange in small molecules.

Interdependence between the formation ofordered structure and the formation of thedisulfide bond

The kinetics of formation of the Cys6–Cys103disulfide bond are strongly affected when NaCl isadded to induce ordered structure in the reducedprotein and in mixed disulfide intermediates. Wefind that the initial formation of the mixed disulfidebetween the reduced protein and GSSG followsthe NaCl-induced folding transition of the reducedprotein. The rate of this reaction is decreased aboutfourfold when the reduced protein adopts a native-like conformation.

In contrast, the subsequent intramolecular disul-fide exchange reaction is strongly accelerated bystructure formation. Its rate follows the confor-mational stability of the mixed disulfide PSSG

SH and isincreased about 100-fold when PSSG

SH is in a native-likefolded state. These opposing effects of a folded pro-tein conformation lead to a change in the mechanismof oxidative folding.

Under conditions where the reduced protein isunfolded, the formation of mixed disulfides withglutathione is much faster than the intramolecularstep when 1 to 5 mM GSSG is present (as in thisstudy). The intermediate PSSG

SH accumulates and thereaction with a second molecule of GSSG (in thePSSG

SH :PSSGSSG step) competes efficiently with the slow

formation of the intramolecular disulfide bond (inthe PSSG

SH :PSS step, cf. Scheme (I)). As a consequence

PSSGSSG accumulates, and only a small amount of native,

oxidized protein is formed at a very low rate underthese conditions.

Under conditions, where native-like structurecan form in the reduced protein and in the mixeddisulfide, the initial reaction with GSSG is slightlydecelerated, but the intramolecular exchange reac-tion is extremely accelerated. Now kintra is much largerthan k2MD × [GSSG] and the formation of PS

S is stronglyfavored over the competing formation of PSSG

SSG

(cf. Scheme (I)). Because the intramolecular step is sorapid, the nature of the rate-limiting step changes,and the initial formation of the mixed disulfide with

glutathione becomes the slowest step of oxidativefolding.

In summary, the presence of an ordered proteinconformation strongly increases the rate constant kintra

for the formation of the correct intraprotein disulfidebond over the formation of non-native disulfidebonds, and consequently the yield of native proteinis strongly increased. In our model protein with asingle disulfide bond the non-native disulfide bondsare formed intermolecularly with glutathione. Inproteins with several disulfide bonds an increase inthe value of kintra for forming the native disulfides, butnot for forming non-native disulfide pairings couldsimilarly increase the yield of molecules with correctdisulfide bonds (Peng & Kim, 1994; Peng et al., 1995).

A high value for kintra might also be importantfor the de-novo folding of proteins in an oxidizingenvironment such as in the periplasm of Escherichiacoli, to avoid the accumulation of double-mixeddisulfides. The formation of ordered structure is notthe only way to increase the intramolecular disulfideexchange. Zapun et al. (1993) noted that in modelreactions kintra increased more than 1000-fold whenDsbA is used as an oxidant rather than GSSG.

Importance of the mixed disulfide species foroxidative folding

The mixed disulfide PSSGSH is chemically unstable

because it undergoes intramolecular disulfide ex-change and thus its conformational stability couldnot be determined at equilibrium. It could bederived, however, from the dependence of kintra

on NaCl concentration and from measuring thereactions with glutathione of the folded and unfoldedforms of the reduced protein according to thethermodynamic cycle in Scheme (II). The twoapproaches gave consistent results and indicated thatthe covalent attachment of a glutathione residuedestabilizes the reduced protein by about 5 kJ mol−1.

The conformation of PSSGSH is of central importance

for the oxidative folding because it controls the rateof the intramolecular disulfide exchange reactionkintra. The presence of ordered structure in the mixeddisulfide can have two opposing effects on thisreaction. It can increase the effective concentrationsof the reacting thiols and thus enhance the rate ofprotein disulfide formation. It can, however, alsointerfere with the formation of the transition statefor disulfide exchange by steric hindrance and byproviding a hydrophobic environment, which in turnwould increase the pK values of the thiol groups.Both effects would be unfavorable and decrease therate of forming the intramolecular disulfide bond.

In the oxidative folding of RNase T1 these problemsseem to be avoided. The conformational stability ofreduced C2S/C10N-RNase T1 decreases when themixed disulfide with glutathione is formed, and theregions around Cys6 and Cys103 become locally un-folded. Such a local unfolding reaction is supportedby two pieces of evidence. The formation of thesecond mixed disulfide with glutathione PSSG

SSG is(1) virtually independent of protein conformation in

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its rate (cf. the values for k2MD in Tables 1 to 3), and(2) it did not lead to a further decrease in stability(cf. Figure 4B). Thus, the disruption of the orderedstructure around Cys6 and Cys103 by the firstmodification reaction seems to expose both themixed disulfide with glutathione and the secondthiol. This local unfolding reaction enables C2S/C10N-RNase T1 to take advantage of the higheffective concentration of the thiols provided by anoverall folded conformation and, at the same time,avoid an increase in the energy of the transition statefor the disulfide exchange reaction.

Such favorable conditions are not always metin oxidative folding. The variable domain of theimmunoglobulin light chain cannot form a mixeddisulfide with glutathione when the reduced proteinis in a native-like folded state, and, once the mixeddisulfide with glutathione is formed, the proteinunfolds (Goto & Hamaguchi, 1981). As a conse-quence, the formation of the disulfide bond ofthis protein should best occur when it is stillunfolded. This illustrates the advantage of a veryrapid formation of the disulfide bond prior to folding,which can be accomplished by thiol/disulfideoxidoreductases, such as DsbA (Frech & Schmid,1995). Problems are also encountered in the for-mation of the Cys5–Cys55 disulfide bond of thepancreatic trypsin inhibitor (Creighton, 1992; Darby& Creighton, 1993; Weissman & Kim, 1991), which isburied in the native protein and forms very slowly.Darby et al. (1995) proposed that the formation ofsuch disulfide bonds is generally slow and a majorrate-determining step of oxidative folding.

The folded conformation of the mixed disulfidePSSG

SH is important for intramolecular disulfide bondformation in C2S/C10N-RNase T1, not only at highconcentrations of NaCl, where this conformationactually predominates (cf. Figure 4), but also at lowsalt concentrations. Since kN

intra is about 100-fold higherthan kU

intra, even in the presence of only 0.25 M NaClabout 80% of the protein disulfide bonds are intro-duced in the folded form of PSSG

SH and the apparentvalue kapp

intra is already increased fivefold, although thefolded form is populated only to about 5% at equili-brium. This demonstrates that even under conditionswhere the stability of this folded intermediate is verysmall, it is used in the oxidative folding of mostmolecules. Its presence at low concentration acceler-ates protein disulfide formation and suppresses thecompeting non-productive reaction with a secondmolecule of GSSG.

These results illustrate the role of intermediates inprotein folding in general. Intermediates are neces-sary, because they contribute specificity to thefolding process, and because they can direct thefolding process to an efficient and fast pathway. Theirstability should remain low, however, to avoidkinetic traps, to maintain a high flexibility and towarrant good accessibility for the oxidant whendisulfide bonds have to be formed. They illustratealso that productive intermediates need not bepresent at high concentration to exert their role forfolding. Rather, highly stable or excessively folded

intermediates may interfere unfavorably with pro-ductive folding. Several examples for such an inter-ference are now known (Kiefhaber et al., 1992;Sosnick et al., 1994; Elove et al., 1994; Weissman &Kim, 1991; Creighton, 1994; Zhang & Goldenberg,1993).

The complex interrelation between confor-mational reactions and thiol/disulfide exchange asshown in Scheme (II) indicates that the pathwaytaken from the reduced and unfolded state to theoxidized and native state can vary significantly. Thechoice of a pathway through Scheme (II) is deter-mined not only by the stability of the individualspecies, but also by the kinetics, i.e. by the relativerates of competing reactions. These rates depend onthe solvent conditions and on the nature and concen-tration of the oxidant. The pathway of folding canthus easily change when folding catalysts (such asprolyl isomerase) and efficient biological oxidants(such as DsbA) are present.

Materials and Methods

Materials

The C2S/C10N variant of RNase T1 was purified (Mayr& Schmid, 1993; Mayr et al., 1994) from E. coli cells trans-formed with a plasmid carrying a chemically synthesizedgene (Quaas et al., 1988). Periplasmic peptidyl-prolylcis/trans isomerase from E. coli was a gift from N.Takahashi (TONEN Corp. Nishi-Tsurugaoka, Japan). A1.4 nM solution of this enzyme had an activity of 1 PU/mlwhen measured as described by Fischer et al. (1984). Theactivity unit of prolyl isomerase is defined by Mucke &Schmid (1992). GdmCl and urea (ultrapure) were fromSchwarz/Mann (Orangeburg, USA), Hepes (sodium salt),iodoacetic acid (sodium salt), iodoacetamide, oxidized andreduced glutathione were from Sigma (St Louis, USA).Dithiothreitol (DTT) was from Fluka (Buchs, Switzerland).All other chemicals were from Merck (Darmstadt,Germany). The concentrations of GdmCl, urea and NaClwere determined by the refraction of the solutions. Theequations correlating the refractive index with the concen-trations of GdmCl and urea are given by Pace (1986).The respective equation for the concentrations of NaClsolutions is given by Mucke & Schmid (1994).

The concentrations of GSSG stock solutions at pH 7were determined by the absorbance at 248 nm using anabsorption coefficient of 382 M−1 cm−1 (Chau & Nelson,1991). The concentrations of reduced DTT and GSH insolutions were determined by Ellman’s assay in theabsence of GdmCl, using an absorption coefficient of14,150 M−1 cm−1 at 412 nm for the 4-nitrothiophenolateanion (Riddles et al., 1983).

Spectroscopic methods

For optical measurements a Hitachi F4010 fluorescencespectrometer, a Kontron Uvikon 860 spectrophotometer,and a Hewlett Packard 8452A diode array spectropho-tometer were used. The concentrations of the various formsof C2S/C10N-RNase T1 were determined spectrophoto-metrically by using the absorption coefficient of thewild-type protein (e278 = 21,060 M−1 cm−1; Takahashi et al.,1970).

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Reduction of C2S/C10N-RNase T 1

Typically 5 mg of C2S/C10N-RNase T1 were dissolvedin 500 ml of 0.3 M Tris-HCl (pH 8.7), containing 6.0 MGdmCl, 2 mM EDTA, 50 mM DTT and incubated at 25°Cfor at least three hours. Reduction was stopped by adding30 ml of 3.0 M HCl. The reduced protein was immediatelyseparated from the reagents by gel filtration over a FastDesalting Column HR 10/10 (Pharmacia, Sweden), equili-brated with 1 mM HCl. Fractions containing reducedC2S/C10N-RNase T1 were lyophilized and stored at−20°C. The content of free thiol groups of the reducedprotein was determined by Ellman’s assay in the absenceof GdmCl. 2.1 to 2.3 free thiol groups per C2S/C10N-RNase T1 molecule were found.

Preparation of the double-mixed disulfide P SSGSSG

About 2 mg of reduced C2S/C10N-RNase T1 weredissolved in 500 ml of 0.3 M Hepes (pH 7.0), containing6.0 M GdmCl, 2 mM EDTA, 0.2 M GSSG and incubated at25°C for at least three hours. The reaction was stopped byadding 10 ml of 3.0 M HCl. The protein was immediatelyseparated from the reagents by gel filtration over a FastDesalting Column HR 10/10 (Pharmacia, Sweden),equilibrated with 5 mM NH4 acetate (pH 5.0). Fractionscontaining the PSSG

SSG species were lyophilized and stored at−20°C. The protein showed no free thiol groups in Ellman’sassay, gave a single band in native polyacrylamide gelelectrophoresis and a single peak in RP-HPLC analysis. Itsmolecular weight as measured by mass spectrometry, was11,694, which is close to the theoretical value of 11,693.

Preparation of carboxymethylated andcarbamidomethylated C2S/C10N-RNase T 1

About 2 mg of reduced C2S/C10N-RNase T1 weredissolved in 500 ml of 0.1 M Hepes, 2 mM EDTA, 0.2 Miodoacetate or 0.2 M iodoacetamide (pH 7.0) and incubatedat 25°C for 5 minutes in the dark. The protein was immedi-ately separated from the reagents by gel filtration over aFast Desalting Column HR 10/10 (Pharmacia, Sweden),equilibrated with 5 mM NH4 acetate (pH 5.0). Fractionscontaining the carboxymethylated C2S/C10N-RNase T1

(RCM-C2S/C10N-RNase T1) or carbamidomethylatedC2S/C10N-RNase T1 (RCAM-C2S/C10N-RNase T1) werelyophilized and stored at −20°C. The proteins showed nofree thiol groups in Ellman’s assay, gave a single band innative polyacrylamide gel electrophoresis and a singlepeak in RP-HPLC analysis.

Equilibrium folding transition

The various forms of C2S/C10N-RNase T1 (all at 0.5 mM)were incubated at 25°C in the presence of 0.1 M Hepes,2 mM EDTA (pH 7.0) and varying concentrations of NaCland of urea for at least three hours. The fluorescenceemission of the samples was measured at 320 nm (10 nmbandwidth) after excitation at 268 nm (1.5 nm bandwidth)in 1 cm × 1 cm cells. The samples with reduced proteincontained additionally 4 mM DTT. The transitions wereanalyzed by assuming a two-state transition between thefolded (N) and the unfolded (U) conformations (Santoro &Bolen, 1988).

Refolding kinetics

Refolding kinetics were typically initiated by a 100-folddilution of the reduced protein (in 10 mM HCl) or of the

PSSGSSG species (in 0.1 M Hepes, 2 mM EDTA (pH 7.0)) to the

final conditions. The kinetics were followed by the increasein fluorescence at 320 nm (10 nm bandwidth) after excit-ation at 268 nm (1.5 nm bandwidth) in 1 cm × 1 cm cells.All kinetic experiments were carried out in 0.1 M Hepes,2 mM EDTA (pH 7.0) at 25°C. Samples with reduced pro-tein contained additionally 4 mM DTT. The final proteinconcentration was 1 mM. In some experiments 1000 PU/mlprolyl isomerase was present. The observed kinetic curveswere analyzed as a sum of exponential functions by usingthe program GraFit 3.0 (Erithacus Software, Staines, UK).

Thiol disulfide exchange reactions

The reduced protein (in 5 mM HCl) was diluted to a finalconcentration of about 50 mM in 0.1 M Hepes, 2 mM EDTA(pH 7.0) at 25°C. The exchange reaction was started byadding the reduced protein to an appropriate volume of astock solution of GSSG and/or GSH. After variable timeintervals aliquots were withdrawn and disulfide exchangewas quenched by adding 1/3 volume of 1 M iodoacet-amide in 0.1 M Hepes, 2 mM EDTA (pH 7.0). After reactionfor five minutes at room temperature in the dark, thealiquots were frozen at −70°C. The reaction in the presenceof urea and/or NaCl was carried out in an analogousmanner, the protein was incubated, however, in 0.1 MHepes, 2 mM EDTA (pH 7.0) for one hour at 25°C in thepresence of the final concentration of NaCl before startingthe reaction.

The aliquots were subjected to native electrophoresis on15% (w/v) polyacrylamide gels at 5°C using the dis-continuous buffer system of Ornstein (1964). After stainingwith Coomassie brilliant blue R250 the gels were scannedwith a LKB UltroScan XL laser densitometer. Relative areasof individual species were determined by normalization ofthe peak areas by the total integrated area of all peaks.

The reduction of oxidized glutathione by reduced DTTwas followed by the increase in absorbance at 286 nm(Iyer & Klee, 1973; Creighton, 1975). The kinetics weremeasured in 0.1 M Hepes, 2 mM EDTA and varying con-centrations of NaCl and urea at pH 7.0, 25°C. The kineticsof thiol/disulfide exchange were analyzed as a second-order reaction.

Analysis of the oxidation and reduction kinetics

The microscopic rate constants of disulfide exchangewere determined from a joint non-linear least-squares fitof the time courses of the species PSH

SH, PSSGSH , PSSG

SSG, and PSS

(as obtained from the electrophoresis experiments) to thekinetic mechanism in Scheme (I). For the fit the programEASY-FIT (Schittkowski, 1993) was used. In this procedurethe differential equations are used that describe thedependence on time of the concentrations of the variousspecies in Scheme (I).

AcknowledgementsWe thank K. Schittkowski (Universitat Bayreuth) for

making his program EASY-FIT available to us, A.Schierhorn (Universitat Halle) for performing the analysesby mass spectrometry, and M. Mucke, T. Schindler, andStefan Walter for discussions of this work. It was supportedby grants from the Deutsche Forschungsgemeinschaft, theFonds der Chemischen Industrie and the European Union.

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Edited by R. Huber

(Received 28 March 1995; accepted 12 May 1995)

influence of protein conformation on disulfide bond formation in the oxidative folding of...

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