Intermediates in the refolding of reduced ribonuclease A

.J. Xol.

Bid.

129, 411-431

(1970)

Intermediates in the Refolding of Reduced Ribonuclease A THOMAS E. CREIGHTOS Medical Research Cbuncil Laboratory of Molecular Biology Hills Road Cambridge CB2 2&H: England (Receiced 19 October 1.978) The intermediates with one, two, three or four disulphide bonds which accumulate during unfolding of native ribonuclease and refolding of the reduced protein have been trapped by rapid alkylation with iodoacetate and separated by ionexchange chromatography. They have been characterized to varying extents by their enzymic activity, electrophoretic mobility through polyacrylamide gels, disulphide bonds between cysteine residues, the environments of the six tyrosine residues as indicated by ultraviolet absorption and fluorescence spectra, interactioI1 with antibodies directed against) eitller t’he trapped unfolded reduced protein or the native folded protein, and for the disruption by urea of any stable conformation producing a change in molecular shape. Correctly refolded ribonuclease was indistinguishable from the original native protein, but. virtually all the intermediates with up to four disulphide bonds formed directly from the reduced protein were cnqmically inactive and \u-

folded by these criteria. transition

to the

intermediates.

fully

Unfolding reduced

The intermediates

of native

protein,

with

ribonucloase

was an all-or-none

no accumulation

of disulphide

in refolding are separated from the fldly

folded

state by the highest energy barrier in the folding transition; t,hey may be considered rapidly interconvertible, relat’ively unstable microstates of the unfolded protein. The measured elements of tllo final conformation are not acquired during formation of the first three disulphide bonds, but appear simultaneously wit)h formation of the fourth native disulphide bond. These observations with ribonucleaso are qualitatively similar to those madcx previously in greater detail with pancreatic t)rypsin inhibitor and suggest a possible general pattern for the kinetic process of proteirl unfolding and refolding.

1. Introduction Bovine pancreatic ribonuclease A is the protein most thoroughly studied with respect to unfolding and refolding; the very extensive early studies have been summarized by Anfinsen (1967,1972,1973), Anfinsen & Scheraga (1975), Baldwin (1975), Nemethy & Scheraga (1977), Pace (1975), Richards & Wyckoff (1971), Tanford (1968,1970), and Wetlaufer & Ristow (1973). More recently, bhe reversible unfolding of the protein with the four disulphide bonds intact has been confirmed to be a two-state transition with an absence of stable partially-folded intermediates (Ginsburg $ Carroll, 1965: Westmoreland & Matthews, 1973: Privalov & Khechinashvili, 1974; Nall & Baldwin, 1977). Tn spite of this, the kinetics of refolding are complex, primarily due to two slowly-int,erconv~rt,~d forms of t,hc fully unfolded protein which refold at different, 4I1

412

T.

E.

CREIGHTOS

rates (Baldwin, 1975,1978; Brandts e.t al., 1975; Schmid & Baldwin. 1978). Refolding with the four disulphide bonds intact is relatively rapid, the slowest) step having a half-time of 30 seconds under normal conditions. Refolding of reduced RNAasef which accompanies formation of the four disulphidti bonds has been studied extensively since the initial work of Anfinsen and colleagues (Anfinsen, 1967:1972,1973) ; the formation of enzymatically active protein under a variety of condit’ions and after various modifications of the protein has been rcportctl from a number of laboratories (e.g. Ahmed et al.. 1975: Andria & Taniuchi, 1978: Chavez & Scheraga, 1977 : Hantgan et al., 1974: Kato & Xnfinsen. 1969: Schath et al.? 1975; Taniuchi, 1970; Takahashi et al.. 1977). However. very little is known about the pathway of refolding, even though t#he four disulphicle bonds may he us~l to t’rap the intermediate states (Creighton, 1977~1978). Disulphidc bond formatioi~ has generally been observed to proceed more rapidly t,hen appearance’ of’ tAnz>mcb activity, suggesting intermediate states with incorrect, disulphide bonds (e.g. Antinsct~ bonds present i 11 tjlrtl et al., 1961). Hantgan et al. (1974) examined t,he disulphidc entire renaturation mixture, but could detect only those present, in t,he corr.ec:tl>. renat,ured protein. Procedures have been developed for directly trapping and charact~rrizing intrrmediates which accumulate during disulphide bond formabion and breakage in the> somewhat smaller bovine pancreatic trypsin inhibitor and have been used to elucidat (’ in outline the pathway and energetics of the protein conformational transitions which occur in the unfolding and refolding of this protein (Creighton. 1978). ‘l’htt same procedures were used to demonstrate directly the presence of intermt&att* disulphide-bonded states in the refolding of reduced RNAase and to examim their. kinetic roles in the refolding process (Creighton. 1977~). This approach has nolv been extended by isolating and characterizing those intermediates which accumulatt~ in unfolding and refolding. making possible a preliminary comparison of the refolding of this protein with that of BPTZ. Qualitatively similar results have been fount1 with both proteins, suggesting that they may reflect a general mechanism for l)rotGn unfolding and refolding.

2. Materials and Methods (a) Naterials Ribonuclease was the phosphate-free protein of \Vorthington Biochemical Corp. (lot, RAF 8BB). For some experiments, the ribonuclease was further purified by CM52 cllroacid (82 Ci imol), matography as below. Iodo-[2-14C]acetic acid (50 Ci/mol). iodo-[“HIacetic and [35S]mercaptoethanol

were obtained from the Radioctlernical Centrrs Ltd. iodoacetic a.cid \v6’r(A mixed wit.11 trot,-ratlioReti\-r, iodoacetic acid that had been recrystallized from WI,. T11o “5S~1aballed mercaptoethaclol (11 mCi) was converted to [35S](HOEtS)2 by mixing it with IWO ml of 0.1 bl-(HOEtS), (non-radioactive) in 0.1 x-Tris.HCl (pH 8.0) to permit thiol-disulphide interchange. t311erl leaving it in the presence of air for at least 2 days for further air oxidation.

Amersham.

(20 Ci/mol)

The 3H- and 14C-labelled

(b)

!Prap&ng

and isolation

of intermediates

i,~ cmfoldiny

or refoldiny

Fully reduced RNAase was prepared as before (Creighton, 1977~). Solutions containing thiols were made up fresh and all solutions were doaerated by rrduced pressure. For t Abbreviations used: RNAase, bovine pancreatic ribonuclease A; BI’TI, disulphitlc: GSRG. glntathionr trypsin inhibitor; (HOEtS),, hyd roxyethyl RNAase, fully rcducrtl, carboxymethylated RNAasc.

bovine pancreatir. ~litiulphitk: R(‘M-

INTERMEDIATES

IN

RIBONUCLEASE

REFOLDIXG

4 13

longer than about 20 min, the solutions were kept under an atmosphere of Nz. The fully reduced protein isolated by gel filtration or native RNAase were diluted into,

incubations

or dissolved in, respectively, the standard buffer mixture of 0.10 M-Tris.HCl (pH 8.7), of a 0.20 nr-KCl, 1.0 m&r-EDTA at a final protein concentration of 30 pM. After addition concentrated solution of the appropriate disulphide or thiol reagent, incubation was continued at 25°C for the desired period of time, then 0.25 vol. 0.5 &I-iodoacetic acid (neutralized with an equivalent amount of KOH and in 0.25 &r-Tris.HC!I (pH 6.8)) was added to block rapidly all free thiol groups. Within 2 to 3 min, the protein was isolated by rapid gel filtration on an 8 cm x 37 cm column of Sephadex G25 equilibrat’ed wil-h 0.1 b%-acetic acid. The prot’ein solution was lyophilized and then dissolved completely in 39 ml of 0.01 nl-HCl; the pH was raised to 6-2 by addition of 1 nr-imidazole, and 0.4 ml of 0.1 &r-EDT-A added. The clear solution was then applied to a 1.5 cm x 80 cm column of CM-ctllulose (CM52, Whatman Biochemicals) equilibrated with the chromatography buffer, 0.02 fir-imidazole.HCl and 1 rnM-EDTA (pH 6.2). Elution was at room temperature \vith a linear gradient of 0 111to 0.2 ;M-NaCl in the chromatography buffer (2.0 1 tot,al vol.). 7’110 absorbance at 275 nm was monitored continuously. The desired protein species wer(’ dtx-salted by gel filtration on Sephadex G25 in 0.1 &i-acetic acid, lyophilized, dissolve<1 iii wat’er to a protein concentration of 0.2 m&r, and stored frozen. (c) Immunochemical

procedures

Antibodies to native RNAase and to fully reduced carboxymethylated RNAase were protein, emulsified in complete Freund’s prepared by injection of 1 mg of the appropriate adjuvant (Difco), into each rabbit periodically over a period of 4 months. Two-dimensional double-diffusion analysis of the precipitation of the various protein species by the antisera was carried out in Ouchterlony plates (Meloy, Springfield, Virginia, U.S.A.), using 20 ~1 of antiserum or 2 pg of protein for each well. (d) Oth,er naethods concentrations of all forms of RNAase were determined from the absorbance at 275 nm, using the molar absorbance value of 9200 (Anfinsen et al., 1961). Absorbance spectra were measured with a Cary 17 spectrophotometer, fluorescence spectra wit11 a Perkin Elmer MPF 3 fluorescence spectrophotometer. Polyacrylamido gel electrophoresis \vas carried out using the discontinuous buffer system of Reisfield et al. (1962). RNAasc enzyme activity was measured using 2’,3’-CMP by the method of Crook et al. (1960). Radioactivity was measured in a Beckman LS-250 scintillation counter, using Aquasol (New England Nuclear) as scintillant. Diagonal maps (Brown & Hartley, 1966) and separation of other peptides after digestion by trypsin, chymotrypsin or thermolysin wer’c performed by electrophoresis at pH 2.1, 3.5 or 6.5, essentially as described previousl) (Creightoii, 1975). WII:

3. Results (a) Trapping

and isolation

of intermediates

in refolding

Intermediate species with one, two, three, and four disulphide bonds accumulate during refolding of reduced RNAase when the disulphide bonds are formed via thioldisulphide exchange with a linear disulphide reagent like GSSG, since the rate of formation of each disulphide bond is determined primarily by the essentially constant rate of reaction of the reagent with the protein thiol groups (Creighton, 1977c). When the intermediates are trapped by rapid reaction of all thiol groups with iodoacetate. the intermediates are stable, and those with different numbers of protein intramolecular disulphide bonds have different net charges, due to the different number of acidic carboxymet,hyl groups (or possibly mixed disulphides with acidic glutathione). They may be separated readily by ion-exchange chromatography on CM-cellulose, as

414

I’.

E.

(‘REIGHTOS

the fully reduced carboxymethylated protein. wit,h no disulphide bands. has essentially, no net charge at neutral pH and consequently does not bind t*o CM-cellulose, whiles the native protein is basic and binds quite strongly. Species w&h one, two. or thret protein disulphide bonds should have corresponding intermediat,c elution positions : t,hey should also be distinguishable by their kinetics of accumulation during refolding. one-disulphide species accumulating before two-disulphide species. etc. Chromatography profiles of t’he RNdasr species t,rapped at various times during refolding of initially reduced protein with 0.2 mM-GSSG are shown in li’igurr I. ‘I%(~ order of elution from the column and the kinetics of accumulation identity unambiguously t,he major peaks of one-, t.wo-. t.hree-. and four-disulphidt: sprciw. which aw designated by the corresponding Roman numbers: this was confmwtl by their content of 14C when 14C-labelled iodoacctate was used to trap them. In addition to the readily-identified major peaks, there were minor peaks t’hat eluted between t,hrm. often with intermediat,e 14C cont,cnt. These minor peaks wer‘r not) studied in detail. as they probably arise in a number of ways. e.g. (a) som(a covalent heterogeneit,y of t,hc original reduced protein (see below). (1)) anomalous ohromat,ographit? baha\%nr ot and (c) carbox?-methyla’tion of protein g~~n~l)s some of the normal intermediates, other than t,hiols. Virtually quantitative recovery of the protein was obtained ikm these columns, so it is unlikely that any major species \vas orerlookecl. The reduced protein, with no disulphides and eight carboxymethyl groups. invariably emerged in a complex peak for reasons that, art’ not clear. ‘l’hwtb was a sir& symmetrical major peak of one-disulphide species (I) and a single major peak of twodisulphide species (II): but t,he three-disulphide species u.erc cluted in two partiallyresolved peaks (IllA and I1 IB). The four-disulphidc species wtw t,lut)ed in t\\.o willresolved peaks, but only t~he second (X) was enzym icall!, act,ivct and corrt:ctJy r~~foltlrcl, After ext’ensivc periods of refolding. about 80’!,, of the prot~ein \\.as t,luted it) t Iit. RNAase. hut about 20’$, elut.ed ctarlier iu peak N. which corresponds t,o natiw peak (N’). Protein eluting at’ t)his position was also prwent in t8hc initial protein. bt4orcl reduction, but significant amounts wrt: also found in refolded prot*ein from RNAaw which had been chromat~ographically purified. so cht least, some of it \cas generated during the unfolding and refolding process : it was not caused hy carboxymethylat ion of the protein by iodoacetatr. The various species may also be scyarated hq’ pol~~crglarnide gel &~ctrophormis. where mobility is a function of bot8h the numbrr of ttisulphitlc~ bonds (i.e. net charge) and the compact,ness of the protein (Creighton. 197X). Both the initial mixtures and the major species isolated by ion-exchange chromatography wre so analysed (Fig. 2). The electrophoretic patterns of the t,rapped mixt8urt:s applied t*o tlw CM-cellulosc~ columns in Figure 1 were essentially the same as those reported preriowly (Creighton. 1977~) and were entirel) consistent with those expected of mixtures with the compositions indicated by the chromatography profiles. The elertrophoretic mobilitirs of the isolated species confirmed the number of protein disulphidc bonds in each major species inferred above. The electrophoretic profiles also indicated varying degrerh of heterogeneit)y in the species IT, IIIA, TITB. and IV. giving some’ measure of the numbers of different species. presumably with the same number, but different pairings, of disulphide bonds. and of their different shapes. Species N’ and N wert’ homogeneous and rirt8ually indistinguishable from nativt, RKAaw : t)hew wah also some protein witlh na,tive-like mobility in peak IT’, although most of the protein ill h’ and ilu’ also ?hIted frOIl1 a this peak migrated considerably more slowly. ~~Jeclt’S

ISTERMEDTATES

TN

RJRONU:(‘I~EASE

REl~OLDISG

415

Fro. 1. Chromat’ographic separation of disulphide intermediates trapped in t,he refolding of reduced RNAase. Fully reduced RNAase was refolded after addition of GSSG to 0.2 rn~ for the indicated t,imes, when all disulphide bond formation, breakage or rearrangement was quenched by addition of iodoacetate (sometimes labelled with l*C , 6.7 mCi/mol) to 0.1 M. Aft,er desalting amI lyophilization, the protein was chromat.ographed on CM-cellulose to give the illust.rat.ed chromatopraphy profiles. The solid curve is the protein concentration, as measured by t,he absorbance at 275 nm. Whew the iodoacet,ate was 14C-labelled, the content of carboxvmethyl groups per protein molecule is indicated by the open circles, using the scale indicated by the broken lines. R is the fully reducrtl RNAase, while the reman numerals indicate the number of intramolecular disulphide bonds in thv protein molecules of each major peak. For the 25 h mixture, t,he refolding was stopped by addition of 14C!-Iabelled iodoacetate (67 mCi/mol) to 0.012 M and incubated for 40 min; in this case, the concentration of carboxymet,hyl groups indicated by t,he radioactivity is plotted directly. The solid circles give the concentration of active enzyme. measured by the activity towarc ?‘!:I’-CMP relative to that of native RNAase. The small peak at the beginning of each trace is dur to a low molecular weight contaminanl in thP imidazale used to adjust the pH of the sample applied to t,hr column.

416

T.

E.

CREIGHTOK

FIG. 2. Polyacrylamide gel electrophoresis of species trapped during refolding of reduced RNAase. About 10 pg of each protein, isolated as in Fig. 1. was applied to the gel with the diswas at continuous buffer system of Reisfield et nl. (196”)1 , which runs at pH 3.8. Electrophoresis 60 V for 4 h at room temperature. The gels were stained with Ctromasnie brilliant blue and scanned by a densitometer.

column of Sephadex G50 in the same position as native RNAase, indicating that both are compact and monomeric. The various species of RNAase were compared with the original RNAase fol enzyme activity using 2’,3’-CMP as substrate (Crook et al., 1960). Reduced carboxymethylated RNAase had no detectable activity (
bonds present at various stages of refolding

The disulphide bonds of the intermediates are probes of the conformational properties of the polypeptide chain at each stage of refolding (Creighton, 1978), so the pairings of the eight cysteine residues in disulphides were examined by diagonal electrophoresis (Brown 82 Hartley, 1966) of proteolytic digest’s of each major species,

INTERMEDIATES

IN

RIRONUCLEASE

REFOLDING

417

techniques which had been verified in the similar studies with BPTI (Creighton, 1978). The non-native species were generally digested with trypsin followed by chymotrypsin, as this produced specific cleavages of RCM-RNAase with each of the eight cysteine residues on separate peptides (see below). Diagonal maps were generally made by electrophoresis at pH 2.1, pH 3.5, and pH 6.5. The fully reduced protein, R, gave no indication of any disulphide bonds, but the species I. II, IIIA, IIIB, and IV indicated a very great multiplicity of disulphide bonds, and none of them appeared to predominate. It was apparent that each peak of protein contained a number of different species with different pairings of cynteinc residues in disulphide bonds. The quantit,ative frequencies with which each of the eight cysteine residues were involved in disulphide bonds, irrespective of which cysteine residue they were paired wit)h, were then determined in each of the major species by reducing all disulphitle bonds of the trapped species and blocking these cysteine residues with 3H-labelled iodoacetate. All the species then yielded fully reduced carboxymethylated RNAase which cliflered only in the location of the 3H label. The intermediate species gave the correct incorporation of 3H expected from the number of intramolecular disulphicle bond$: protein species T, II: IITA, and IIIB incorporated 2.4, 4.0, 6.4 and 6.2 carboxymethyl groups per protein molecule, respectively. These values and the direct measurements given below indicate that only a minor fraction of cysteine residues was involved in mixed-disulphides with glutathione, which would also be measured by this procedure. The relat,ive distribution of 3H was determined by (1) mixing each protein with uniformly [14C]carboxymethylated reduced RNAase as an internal standard, (2) digestion by trypsin followed by chymotrypsin, (3) separation and identification of c,ach of t’he cysteine-containing peptides, and (4) measurement of the 3H:14C ratio. The results illustrated in Figure 3 indicate that all eight) cysteine residues were involved significantly in disulphitle bonds in each of the one-, two-, and three-disulphi& species, although cysteines 65 and 72 were uniformly involved more than t,he other cysteine residues. The spectrum observed with the one-disulphide species (1) was close to that expected for a statistical pairing of cyst’eine residues (Kauzmanu. 1959), in which case the frequency of pairing should be proportional to nm3i2, whertl 11is the number of residues between the two cysteine residues. Thermdysin digests of species N’, N, and native RNAase gave peptide maps which were qualitatively indistinguishable, indicating t,hat both X’ a,nd N hat1 rc~foltlrd lo form the four correct disulphide bonds. (c) Con,formmtional

restrictions

on refolding

Int.ramolecular protein disulphide bond formation using thiol-disulphide exchange with a disulphide reagent, like GSSG or (HOEtS)L 2 is a two-step process, in which initial reaction of reagent with a protein thiol. to form a mixed-disulphide, is followed by int8ramolecular thiol-disulphide exchange with the t’hiol of a second cysteine rrsidue, which reflects the conformational properties of the polypepticle chain. The mixed-disulphide accumulates only if there are conformational restrictions on the ability of a cysteine residue to form rapid1.y a disulphide with any of the other free was examined cysteine residues (Creighton, 1978). A ccumulation of mixed-clisulphides using 0.4 mM-35S-labelled (HOEtS), as disulphide reagent, which gave the same kinetics of refolding and intermediates w&h BPTI as 0.2 mM-GSSG (Creighton, 1977a). The major intermediat’es in refolding with this reagent gave chromatographic

418

T.

L C

I 26

E.

I 40

CREIGHTOIV

I

I

58 65

I

72

I 84

95

110

J

Residue number of pr~mory structure

FIG. 3. Participation of the 8 cyst,eino residues of RNAase in disulphide bonds of t,he trapped intermediates. Intermediate species I, II, IIIA, and IIIB, trapped with non-radioactiveiodoacetatt, and isolated as in Fig. 1, at a concn of 0.1 rniw were reduced with 1 mlvr-dithiot,hrrit,ol in 0.1 31. groups were carboxyTris.HCl (pH 8.0) and 6 M-guanidinium chloride for 1.5 h at 25°C. Thiol methylated by addition of iodo-[3H]acetate (3.1 Ci/mol) to a concn of 3 mx. Aft)er incubat,ion fat 2 h, the proteins were desalted by gel filtrat,ion in 0.1 nr-acetic acid and lyophilized. ApproximateiS 160 nmoles of each protein were mixed with 15 nmoles of fully reduced RN&se which had been uniformly carboxymethylated in the same way v&h iotlo-[2-‘4C]acetat,c (4.4 Ci/mol), and then digested with 0.4 mg t,rypsin in 1.3 ml of 0.2 M ammonium acet,ate (pH 7.5) for 2 h at 35”C, folIowed 2 h. The mixtures wcw by addition of 0.2 mg of chgmotrypsin and incubation for a furt,her lyophilized and the 14C-labelled peptides separated by rlectrophorcsis at, pH 2.1, pH 3.5 ant1 pH 6.5 u&l pure. The digests of the various prot,eins gave identical peptide patterns. Carp was taken to minimize oxidat,ion of the carboxymet,hyl cysteinn pept,iden, as oxidation was observetl to cause 3H exchange of the peptides with the solvent,. pnpt~itlc WCPP tktwminctl by sointillat~ion aortril The relat,ivr levels of 3H and l4C of t,ho purified ing and normalized by t.he number of “H-labelled cysteinr residues of each species. The idontitieti of the cysteinc residuc,r were detrrmined unambiguously by quantitative amino acid analysis of each peptide: the 8 cyst,eine residues were found in the following peptides: Cys26, residues 26 to 31; Cys40, residues 40 t,o 46; Cys58, residues 47 to 61 ; Cys65, residues 62 to 66; Cys72, residues 65 to 73; Cys84, residues 80 to 85; CysSS, residues 91 to 98; <‘ysllO, residws 105 to 11.5.

profiles (Fig. 4(a)) very similar to those obtained with GSSG (Fig. 1). suggesting that the same intermediates are formed and that mixed-disulphidrs are relatively minor. A mixed-disulphide with the neutral HOES prevents react’ion with acidic iocloacetate, so each mixed-disulphide makes a trapped species one unit charge more basic, and the mixed-disulphide species in t,his case elut’c aft’er t)he normal species. The distribution of 35S was consistent with significant, accumulation of mono-mixeddisulphide forms of the one-, tcvo-, and three-disulphide species. as the radioactivitv was found only between the normal major intermediates and the accumulation was related t’o that, of the preceding normal intermediate (Fig. 4(a)). There may also haw been a very small amount of the mixed-disulphide form of the reduced protein: It. The original protein used for unfolding and refolding had been purified chromatographically, but the minor species eluting between the major int)ermediat,es were still detectable over t#he mixed-disulphides in the chromatography profiles.

INTERMEDI~%TES

IX

RISOXUCLEASE

REFOLDING

FIG. 4. Accumulation of mixed-disulphidos of RNAaae and the effects of 6 nl-guanidinium chloride during refolding. The chromatography profiles in (a) were of species kapped at the intlicated times of refolding with 0.4 mM-(HOEtS),; all conditions were t.he same as in Fig. 1, except that the disulphide reagent was 0.4 m@%](HOEtS), (11 Ci/mol). Refolding in (b) was for 4 min under the same conditions, except that 6 ivr-guanidinium chloride was added to t,he refolding mixturfs. (--------) I’rot,ein concentrat,ion, as measured continuously by the absorbance at 27.5 nm; (0) the concentration of HOEtS-, as measured by the radioactivity of the collected fractions.

The locations of the mixed-disulphides on the eight cysteine residues of each of the pooled species were examined by separating the t’ryptic plus chymotryptic peptides The results indicated by electrophoresis, then locating the 35S bvy autoradiography. that t.he mixed-disulphides were present significarnly on all eight cysteine residues of the major one-, two-, and three-disulphide intermediates: consequently, there must be a multiplicit’y of such mixed-disulphide species. The same techniques repeated w&h BPTI yielded only accumulation of specific mixed-disulphides with certain irnermediates as reported previously (Creighton. 1977a). Conformational specificity in the intermediates and conformational restrict)ions on tlisulphide bond formation may also be detected by the effects of denaturing agents, such as G ivr-guanidinium chloride, on the spectra of intermediates and on the kinetics of disolphide bond formation. The rate of the thiol-disulphide exchange reaction of model thiols wit’h (HOEtS)2 has been shown t’o be virtually unaltered by 6 pirguanidinium chloride (Creighton, 19773). The resulm of refolding of reduced RNAase in

420

T.

E.

CREIGHTON

the presence of 6 M-guanidinium chloride are illustrated in Figure 4(b). The peak of one-disulphide species (I) was indistinguishable from t’hat, normally observed, but Dhe t’wo-disulphide peak (II) was distinct,ly different. : sorne\vhat surprisingly: mixrddisulphide forms of both accumulated. Very small amount,* of t,hrce-disulphide species were eluted from the column, but significant amount)s of protein \v~~r~’ irreversibly stuck to the top of the column. This was even more apparent, lvith mixtures in which disulphide bond formation was permit,tcd to proc~d for doubly t,he period of time. thereby generating primarily t,hrt\r- and four-disulphide specirr : in t,his case, most, of the protein appeared to be precipitat’ed at the top of t)he column 1 and only minor amounts of three- and four-disulphide species ww t~lutJetl. ‘l’h(L precipitation of the majority of the t’hree- and four-disulphidc species formed W&Y denaturing conditions is indicative of their relative insolubility under non-denaturing conditions. The random int’ermediatos of BPTI have brcn noted to be significantzly less soluble than t,hc normal intermediates, and t,ht x same appears to b(i trutx of th(i RNAase three- and four-disulphide species. The spectrum of species generated in 6 M-guanidinium chloriclc should approximatcl t,hat in which disulphide bonds are incorporated in a random, statistical manner. so it may be concluded t’hat, the normal one-disulphide species may be such a’ random mixture, the normal two-disulphide species less so: t,hc normal three- and fourdisulphide species are probably very non-random.

(d) Conformational (i) Spectral

properties

of trapped

intermediate.s

properties

In order t,o determine whether the six tyrosinc residues of each of t#he trapped species were buried in a hydrophobic environment, as in nativo RNAase, or accessible to the polar solvent, as in RCM-RNAase, the absorption spectra of the trapped species were measured. Similar to t’he observations of others (e.g. Anfinsen et al., 1961), the wavelength of maximum absorbance was shifted from 275.4 nm in RCM-RNAase t’o 277.7 nm in native RNAase. which resulted in a large difference spectrum with a maximum difference at 286.8 nm (Fig. 5). The spectra of intermediates I. II, IIIA. IIIB, and IV were very similar to that~ of RCM-RNAase, while those of species X’ and N were virtually indistinguishable from native RKAase (Table 1 and Fig. 5). The difference spectra were measured with solutions which gave the same absorbance at 295 nm in 6 M-guanidinium chloride with Wl M-Nash, denaturing conditions which should remove any conformat’ional effects on the spectrum (Edelhoch: 1967), but the resulting difference spectra gave indications of somewhat low protein concentrations with species I, II, IIIA, IIIB and TV. N evertheless. it is clear from the absence of a positive difference peak at 286.6 nm relative to RCM-Rh’Aase (Fig. 5(a), (b) and (c)), that intermediates I, II, IIIA, and lIIB had no significant fraction of t)yrosine residues in an environment like that, in nat’ive RNAaxe. Intermediate IV gave indications of both native-like and accessible tJyrosine residues (Fig. 5(c) and (d)). which probably arise because of the presence of bot’h native-like protein and unfolded protein, as is apparent from other results (Figs 2(b) and (6)). Fluorescence of the six tyrosines of RNAase is quenched in the native conformation, in part due to the proximity of the disulphide bonds (Cowgill, 1967); native RNAase was found to have only 35(1/i, of tOhe fluorescence of RCM-RNAase (Table 1). The fluorescence of the inbermediates decreased in the order R :,> 1 > T I > IIIA > IIIH

INTERMEDIATES

IN

RIBONUCLEASE

421

REFOLDISG

1000 : :: b :: 0 b = F

-1000

0

0

-1000

--IO00 II

I

I

,” Tp \\ ;:

;:

- -1000

L’ i /; !,‘: :,iReduced 1 Wavelength

I 260

I

I 280

I

I 300

--2000 ,

I,, 320

340

(run)

FIG. 5. Difference spectra of the trapped species of RNAase WKWS RCM-RNAase ((a), (b) and (c)) or native RNAase (d). The buffer was 0.1 M-Tris.HCl (pH 8.7) and t’he protein concentration in each case was 50 q, as determined by the absorbance at 295 nm in 6 iw-guanidinium chloride plus WI >INaOH. The proteins labelled R, I, II, IIIA, IIIB, IV, N’, and N were isolated as in Fig. 1. ln (r) (. . . .) native RNAaso; “reduced” refers to RCM-RNAase. TAYLE

Spectral properties

of trapped

Absorption

Intermediate

Reduced carboxymethylated R I I1 IIIA IllB IV iv’ N Native RNAase

I

RhTAase

maximum (nm)

RNAase

275.4 275.4 275.5 275.7 275.8 275.6 276.0 257.4 277.6 “77.7

(ii)

Urea-gradient

fluorescence intensity 100 101 84 76 66 60 54 35 36 35

The prot,eins designated R, N’, 1\’ or by the Roman numerals spectral measurements were made at 25°C in 0.1 wTris.HCl (pH 50 p~ and 12 PM for absorption and fluorescence, respectively. measured at 304 nm, the wavelength of maximum emission for all length of 275 nm.

,B IV > N, so the primary factor may be quenching conformation must also be important, as incorrectly fluorescent than native RNAase.

Relative

were isolated as in Fig. 1. Thfl 8.7) at protein concentrations of The fluorescence intcnsit,y was samples, with excitat,ion wave-

by the disulphides. However, folded species IV was more

electrophoresis

One test for the presence of a stable ordered conformation is to look for its disappearance upon addition of a denaturant, such as urea. This may be carried out most

422

T.

E.

CREIGHTON

readily by electrophoresis of a band of protein through a polyacrylamide slab pt.1 in which there is a gradient of urea perpendicular to the direction of electrophorwis: unfolding of the protein by urea is indicat,ed by a decrease in mobility of the protrill. due to sieving by the gel (Creighton, 1979). Native RNAase has been shown to undergo a co-operative unfolding transition in about 5 br-urea at pH 4, while RCM-RNAasc and reduced RNAase did not,, so this method was used to examine the trapped spcciw. As shown in Figure 6. species I, II. I1 IA. and Il.IB undcrwcnt no apparent unfolding transitions. their mobilities only decreased very slightly with increasing urea (WIcentration. just as did RCM-RNAase. The same was true of the major portion ot species I\‘, but t’hat fraction with a normal native-like t~leot~rophoret~ic mobilit> (Fig. 2(b)) underwent a co-operative folding transition like bhat, of native RNAaw. but. at, a substantially lower urea concentration. When unfolded by urea, this fiact iota had about. the same mobilit,!: as the remainder. Refolded species N was indistinguish able from nat,ive RNAase, but specicx N’ appeared t.o consist) of at lrast~ two fractions. one which was unfolded by urea like nat’ive RNAase. the ot,her \vhich untlt~rwnt ir TOP

OH Urea

OM

Urea

FIC~. 6. Urea-gradient electrophoresis of native RNAaw and of tlw trapp+btl species. ‘l’h(t slab gel of acrylamide contained a linear concentration gradient of 0 M to 8 ~-urea, superimposed on an inverse linear gradient of 15% to 11 o/0 acrylamide to compensate for efkcts of urea on the elcctrophor&c mobility; t.he buffer was 0.06 M-Trisacetate (pH 4.0). About 60 pg of native RN.%aacb were applied to the gel, and allowed t.o migrate electrophorotically H. short way into the gel; thcln 8 similar amount of the appropriate protein species, isola.trd as in Fig. 1, wa3 appliotl. Elwtro phoresis was continued for 4 h at, 16°C.

IS'I'1~:IZ1\11~:I)I.\'I'ES

IS

IllIiOS~T~‘I,1E.\S15

similar unfolding at a slightly lo\~w~ urea conwnt,rat,ion. mobilitjies were indistinguishable. (iii) Immunfochemical

li1~F’OI,l~T;1~:

12::

Their folded and unfolded

analysis

Antibodies are now recognized as valuable probes of protein conformat’ion (&la. 1969 : Sachs et al.. 1972 ; Crumpton, 1974: Reichlin. 1975) and antibodies against’ t*hr native and the RCM forms of HP7’1 haw: been used to analyze the conformational properties of t’he intermrdiat8es in refoldin g of reduced BPTL (Creighton et al.. 1978). Two-dimensional double-diffusion was found to provide. with one except’ion. a general division of the intermediates into t,hose with essentially unfolded and t,how with refolded conformations. Accordingly, antibodies against1 native RNAase and against RCM-RNAase were prepared which gave no risible cross-reaction, consi&nt with their being specific for the folded and unfolded conformations, respectively, as had been demonstrated previously by Brown (1962). As is illustrated in Figure 7; the trapped species R: f, I I, I I IA, and I I fB formed precipitation bands only with ant,ibodies against RCM-RN;\asr : t,hey \zere indistinguishable from RCM-Rn’Aasr Anti-N

424

T.

E.

CREIGHTOS

in that they gave complete fusion of bands in each instance. Species 1V gave precipitation bands with both antibodies, which seemed likely to be due to the presence of both native-like and non-native-like species, as detected by electrophoresis (Fig. 2(b)) : this was confirmed by obtaining intersection of the two precipitate bands upou diffusion of species IV versus the two antibody preparations in adjacent, ~11s. The refolded species h’ and N’ interacted only with antibodies against) natives RNAase and were indistinguishable by this criterion from native RNAasr.

(e) Kinetics

of disulphide

reductiorr of native RNAase

Elucidation of the kinetics and energetics of protein folding pathways also rcquirrs examination of the pathway of unfolding, which in this case entails reduction of the four disulphide bonds of RNAase. Accordingly, purified native RNAase was unfolded under conditions identical to those used in refolding, with t,hc sole exception that dithiothreitol was used rather than a disulphide reagent, ;io as Do shift, the redox potential of t,he cysteine interaction to greatly favour the reduced form. The species

xi “5 FIG. 8. Reduction of the disulphide bonds of native RSAase. Chrornat”graphy-pu’itind 1tSAasr (N) at a concn of 30 p~was incubated under N,at 25°C in 0.10 >f-Tris.HCl (pH 8.7),0.20 51.K(‘1. 1 mu-EDTA, also containing 10 mrmr-dithiothreitol, for either 3 h (top) or 20 h (bottom), when disulphide reduction was quenched by addition of iodo-[2-14C]aoetate (14 mCi/mol) to a final concn of 0.1 M. After 2 min, the protein was desalted, lyophilized, and chromatographed as in Fig. 1. Protein concentration, as measured continuously by the absorbance at 275 nm; (0) (---) the concentration of oarboxymethyl groups as measured by the radioactivity of the collected fractions, divided by 8, the number of cysteine residues per molecule of fully reduced RNAase (R). There was no significant amount of radioactivity in any of the other fractions.

generated under these conditions were trapped and separated by the same procedures used in refolding (Fig. 1). The rate of disulphide reduction of native RNAase is quite slow, presumably due to the many non-covalent interactions stabilizing the folded state, and native RNAasc disappeared with a half-time of approximately 10 hours (Fig. 8). The primary product was fully reduced RNAase, with no significant accumulation of one-, t’wo-, or three-

INTERMEDIATES

IN

RIBOKUCLEASE

REFOLDING

425

disulphide species. The rate-limiting step under these conditions must be reduction of the first disulphide, bond, the remaining three disulphides then being reduced relatively rapidly, suggesting that significant unfolding of t,he molecules occurred upon reduction of the first disulphide. Very minor amounts of folded species with altered chromatographic properties were generated during the reduction process, but they did not incorporate carboxymethyl groups from the iodoacetate and their possible significance for the reduction process is not clear. The reduced RNAase generated was observed to be separable into t’wo peaks chromatographically, and the relative proportion of the peak eluting earlier appeared to increase with time (Fig. 8). Both peaks of reduced RNAase had incorporated eight carboxymethyl groups and were virtually indistinguishable bJ electrophoresis, as in Figure 2, at low pH, but were separable at pH 8.6. A second redu&ion treatment of the separated peaks did not alter the chromatographic properties of the first peak, but converted some of the second peak into protein eluting in t’he first. It thus appears that some covalent modification of reduced RKAase occurred during the reduction process which makes the RCM-protein elute earlier from the column, a process which would be consistent with the kinetics apparent in Figure 8. This unknown phenomenon may be related to the complex chromatographic behaviour observed with the residual reduced protein trapped during refolding (Fig. 1).

4. Discussion (a) Trapping

of intermediates

The experimental procedures developed for elucidating the folding pathway of reduced BPTI (Creighton, 1978) should be appropriate for any protein in which the xt’ability of the folded state is dependent upon one or more disulphide bonds, and this has generally been confirmed here with RNAase. The first requirement is that intermediates accumulate to significant concentrations; this need not be true for other intermediates in protein folding, but it is ensured with disulphide intermediates by using conditions in which the rates of formation of all disulphide bonds are comparable in magnitude. With a disulphide reagent like GSSG or (HOEtS),, the ratelimit.ing step in formation of a protein disulphide bond is often the initial reaction of t’he reagent with any of t’he free thiol groups of the protein: so that rate generally slows slightly as disulphide bond formation proceeds, t’hereby ensuring significant accumulation of intermediat)es with one, two, three, etc., disulphide bonds. It, is important that the conformational properties of the protein, not the disulphide reagent, determine the intermediate st’ates; this was confirmed with both BPTI and RNAase by finding that the same spectrum of species was generated with either GSSG or (HOEtS)2, suggesting no specific effect of the disulphide reagent. Having accumulated such intermediates, they must be trapped in a stable form by rapidly reacting all thiol groups of the solution, thereby quenching disulphide bond format’ion, breakage or interchange under appropriate conditions. The quenching procedure used here, i.e. addition of iodoacetate to 0.1 M, has been shown to t’rap faithfully the intermediates of BPTT, and it is assumed that this is also the case with RNAase. TTse of the acidic iodoacetate introduces neb charge differences between molecules with different numbers of disulphide bonds, thereby permitting t’heir

426

T.

E.

CREIGEITOK

separation by electrophoresis or ion-exchange chromatography. The conformations of the trapped, isolated intermediates then may be analyzed in depth : in particular. the cysteine residues involved in the disulphide bonds are direct links bet,wern t,ht primary and tertiary structures. The roles of the int,ermediat’es in the folding transition must, be elucidated by kinetic studies, t,he initial results of which \vith wduwtl RNAaw have already been report’etl (Creighton. 1977~). hut not with JWI’I, nigh One phenomenon detected in t’hr,‘s stud?; with RSAaw, the heterogeneity of the protein produced during the (~xperirnrnt~al rilaniplllat~iolls. for example, the native-like species N’ and the two or mow forms of RCM-REAaw (Figs 1 and 8). It is very probable that this hcterogencity of bot’h t hc native-like an(l RCM forms n-as due t,o differences in covalent, st ructuw. rather t’han conformat’i(jll, ;IB RCM-RNAase appears to be fully unfolded and spcciw K’ containctl at least, t u 0 species which remained dist,inct, throughout t,hr urea-induwd nnfolding transition (Fig. 6). Both instances of heterogeneity were det,ectablr by methods wparat,ing ott the basis of cherge (e.g. rlect,rophort:sis or ion-cLxchangt> chrolnat,ographv) at’ nwtral or alkaline, but not acidic, pH values. so the diflerenw could be dw to deamidatioti of glutamine or asparagine residues. Alternatiwl>-. the hct~erogeneity could Iw dr~c~ to the treatment wit,h thiols. as Watkins & Benz (1978) detected unkno\\.n structural modifications of nat.ive RP;Aase upon treatment with In~,rcaptoet,llatlol in thtb prcwnw of air. Several ot,her invest,igators have recent,ly wportcd t lw generation of supt2oxides, free radicals, and peroxides during air-oxidation of t hiols. u%ich wnlrl rt:ac*t with various groups of the protein (Misra, 197-l: Costa f4 nb.. 1977). L\‘hatcs\-w it5 cause, t#his phenomenon must be controlled beforr a dt%ail(bd charactt~rizatio~~ of’ t 11~3 folding pathway will he possible.

The intermediate species which accumulate during refolding havr been charactwizctl thr nature of the cyst,eine rwiduw to varying extents by their enzyme activity. gels. involved in disulphide bonds (Fig. 3). their mobilit?; through polpacrylarnidt~ which depends upon their compact~ness (Fig. 2): thr canvironment’ of t,heir tIyrosine residues as reflected in their spectral proper&s (Table I ). their interaction with antibodies specific for either the unfolded or fully foldod states (Fig. 7), and t,he effect of urea on any stable conformation (Fig. 6). and they have been compa~retl to the inttbrmediate species generated under denaturing conditions (Fig. 4(b)). Reduced RNAase appears t,o be grossly unfoldrtl, WPI~ under non-denat wing xtudirs of it:: physical properties (c.g. conditZions. as has been demonstrated by many Tanford, 1968; Pflumm 8: Beychok, 1969: Tarnburro et al.. 1970: Garel, 1978). This and &ctrohas generally been confirmed here hy it,s spectral. irlimunoc~lcniical. phoretic properties, and by the absence of any unfolding transition producrd by IIW~. This is undoubtedly due to the stabilizing role of the covalent interaction brtwtwn cysteine residues (Bchellman, 1955 : .I ohnson (~1ab.. 1978) and of the ma,n\r IN’IIcovalent, interactions with neighbouring atoms of each of the four tlisulphidc: bonds, which are integral parts of the folded conform:tt,ion (Let &, Richards, 1971): thaw close-packed interactions would br expected to Lw far lws stable witOh cyhtcGnc> t hi01 groups in the absence of disulphide bonds. The mixture of one-disulphide int,ermediat,w appearwl t.o contain all 28 possihlc~ disulphitle-bollc~ed q&es in proport,ions close t,o t.hosr cxpwt ad on a st,atist ical basis

INTERMEDI.4TES

IN

RTBOSUCLEARE

REFOI,l)ISC

427

(Fig. 3), and to be fully unfolded as judged by spectral, immunochemical, and elect’rophoretic criteria. They also were similar t’o the presumably random one-disulphide species generated in 6 M-guanidinium chloride and exhibited no unfolding transition produced by urea. Similarly, the two-disulphide intermediates also appeared to be unfolded and to consist of a large number of disulphide cross-linked species, although probably not all the 210 isomers theoretically possible and presumed to be generatetl The three-disulphide intermediates, which were in 6 fir-guanidinium chloride. separated bg chromatography into two populations, contained at least’ six different disulphide-linked species resolved by electrophoresis, but probably only a small fmction of the 420 possible isomers. In spite of this non-random spectrum of disulphide bonds. t’he normal trapped three-disulphide intermediates appeared to have no st,ablc folded, compact conformation. The majority of the four-disulphide species (IV) were fully resolved by ion-exchange chromatography from correctly refolded RNAase and appeared 60 be essentiall) unfolded with incorrect disulphide bonds, although there was also present a unique electrophoretic and immunochemical properties, some species with native-like enzyme activity, and a stable conformation more susceptible to unfolding by urea,. The fully refolded protein was indistinguishable from natjive RNAase by all these criteria, although it’ also contained a second species separable by ion-exchange chromatograph,y, which was probably due to covalent modification of the protein (see (a) above). (c) The pathmy

and energetics

of unfolding

ad

refolding

The kinetics of unfolding (Fig. 8) show that’ reduction of the first disulphide bond of nat,ive RNAase is the rate-limiting step. while the kinetics of refolding reported previously (Crcighton, 1977c), which are entirely consistent’ with the further observations made here, demonstrated that the slowest step in the formation of RNAase with native-like electrophoretic mobility is the format’ion of t’he fourth correct disulphide bond. The pat’hwag of unfolding and refolding may then be represent,ed as I\’ R,

I- -

11 -

111 slow

// slow

\

pi

where R is reduced RNAase, I, IT, ITT, and IV are mixtures of one-, two-, three-, and non-native four-disulphide species trapped here. and N is folded RNAase with the four correct disulphide bonds linking cysteines 26-84, 40-95, 58-110. and 65-72, which would include the apparently covalently modified species K’ observed here: however, the significance of the unidentified species in peak IV with native-like electrophoretic mobility (Figs 1 and 2) is not yet known. Each of the steps in the above pathway involves disulphide bond formation or breakage, involving disulphide or thiol reagents, respectively, which have been omitted for clarit,y. It cannot be ruled out that. conformational transitions not involving alterations of disulphide bonds are rate-limiting at some stage; in particular, c&tram isomerization of peptide bonds adjacent to proline residues may be slow on the time-scale used here (Brandts et al., 1975; Schmid & Baldwin, 1978).

428

T.

E.

CREIGHTOX

This is clearly the simplest possible representation of the pathway, as 1: II, II 1. and IV have each been found to consist of multiple species. There must then bc a corresponding multiplicity of microsteps of disulphide bond formation and breaka.pe at each stage. There probably are also numerous unimolecular steps of varying rates by which the various disulphides of I. II, or II1 are rearranged by intramolecular thiol-disulphide exchange. The designations R, I. II, and III also include any specirs in which cysteine thiol groups are involved in mixed-disulphides with the reagent. :o as t,o include t#hiol reagent-catalyzed protein diaulphide interchange, which must involve. at least transiently, protein disulphide breakage. There must, also be prt’~lt in III. although not, necessarily in large amounts. at least one species with tBhrc~c~ t,o S upon forming t,he fourth native-like disulphide bonds which i s converted disulphide bond, in contrast to the other three-diaulphidr species which produccl IV. The many details of this pathway will onlybc determined byidentifyingthr individual intermediates and elucidating their kinetic bchaviour. The kinetics of reduction of native RNAasr observed herfx (Pig. 8); whew t t-1(process was all-or-none. contrast, with other rcbports that on(’ or two disulphidch may be reduced selectively (Sela et al.. 1957 : S~rnn~m e/ ~1.. 1967 : Sperling of ~1.. 1969): this should have been detected here. The reasons for this di~crrpancy are not clear, as no at8temptj was made to reproduce exactly t81reprwious cxperimrnts. The previous st#udy of the kinetics of refolding (Creighton. 1977c) observed that, t’hc\ first and second disulphide bonds \verc: formed rt:atlil,v in retlucctl RNAascx, \\.ith apparent half-times for the intramolecular steps of a fe\\, rnilliscconds. whilt> I hv fastest steps in forming the third and fourth disulphides Id hdf’-tinw of I at~tl 20 seconds. respect’ively. In spite of t’he rapid rat)tx of forming thtx sc~utl tlisulphidv bond, significant proportions of the one-disulphidt intcrmetliatjc~s \vcrc observctl to accumulate as mixed-disulphides with the reagent (Fig. 4(a)). indicating a half-tirnc, of several seconds for the intramolecular step in forming t,hc second tli~ulphidc~ bontl. The t’wo observations are not incompatible, but indicate ~5wide spectrum of irka,molecular rates for this step in the various one-disulphitle intermcdiatcxs. The pr(+~uh study would have measured t’he most rapid steps. \vhik t’he mixctl-disulI)hitles tldwt the slowest. There must be conformat,iona,l restrictions on forming thth s~ontl. t bird. and fourth dixulphide bonds. favouring formation of some and inhibiting format ion of others, even though the trapped int’ermediatas appear t’o 1~ u~~f’oltl~~d by t hca criteria used tinrc>. The wide variety of one-: two-. and three-disulphitle species formrtl frotll rr~lucctl RNAase. especially the apparently random rnixturr of one-tfisulphidc intermrtliatcs, reflects the unfolded nature of reduced RNAase and thr weakness of’ any conformational interactions present, at the early stages of disulphidc formation. t~\~ttrl t.hough of all the interact,ions present in native RNAase, only those involving cyst,cGnc residues are not possible. Conformational interactions bt:t\r.een groups closc~ along the linear polypeptide chain (“short-range interactions”) might be impurtantS for folding, but Obey are not det,ectable at t,he early stages of refolding. Virtually all the t,rapped intermediat’es were found t,o he unfolded by a varictty ot criteria, which is fully compatible with the knobvn co-oprrativit’y of protein folding : a folded conformation is not stable until virtually all the stabilizing int,eractiotls arc present (e.g. Taniuchi 8: Anfinsen, 1969). However, disulphide formation at the t,hrt.cLand four-disulphide stages clearly was not random. so the nature of the conformational forces which must be operat’ive remains to be detected. Otjhcr spect,ral studies of the

ISTERMEDTATES

TN

RIRONUCLEASE

REFOLDISG

429

kinetics of refolding of reduced RNAase using circular dichroism and rotatory dispersion (e.g. Anfinsen et al., 1961; Schaffer, 1975; Takahashi et al., 1977) have generally concluded that polypeptide secondary structure tends to appear before enzyme activity, so it n-ill be of interest to examine the trapped intermediates in this w-a-. If they do t’end to have secondary structure, any such conformation must not be recognized by antibodies toward native RNAase, and the tyrosine residues must) still be exposed to the solvent. It seems clear Ohat reduced RNAase does not, refold by t’he simple sequential formation of the four native disulphide bonds. nor does it acquire parbs of the nativelike conformation during the intermediate steps of disulphide bond formation; instead, all aspects of the native-like conformation appear simultaneously when t)he fourth correct disulphide bond is formed. This appears to be fully compatible with most previous observations of disulphide bond formation in RNAase (e.g. Anfinsen et al., 1961: Kato & Anfinsen, 1969; Taniuchi, 1970: Hantgan et al., 1974), but, contrasts with other conclusions that, refolding of reduced RNAase and of t’hermallyunfolded RN$ase, with the four correct disulphides intact’, occur by simple sequential acquisition of elements of the final native conformation (Burgess & Scheraga, 1975; Chavez & Scheraga, 1977). The experimental evidence used to support the sequential pathway of Burgess & Scheraga (1975) would require the unfolding of RNAase to occur via stable partially-folded intermediate states, but there is overwhelming rvidence that’ t’his is a two-state folding transition, uith no stable intermediat,es (Ginsburg & Cerroll, 1965 ; Westmorela#nd $ Matthews: 1973 : Privalov & Khechinashviii, 1973; Nail & Baldwin, 1977). The experimental observations which suggestctl step-wise unfolding of the native conformation probably reflect the effects of thcb probes used to monit’or unfolding. The immunochemical observat’ions presented hew arc in direct, contrast to those of Chavez & Scheraga (1977), who concluded that, antibodies direct’ed against different portions of the native conformation det’ected t,heir appearance at different rates during disulphide formation in reduced RNAasr. However, Chavez & Scheraga (1977) did not, quench disulphide formation or r’c. arrangement before the prolonged immunochemical measurements, and it seems clear f -om their data that varying extents of formation of refolded RNAase during t’hcse measurements, rather than varying rat’es of folding, account for their results. Disulphide intcrmediat,es must be t’rapped in a stable form by blocking all thiol groups of the protein, although t’his does leave open the qucshion of the effects of such blocking groups on the conformation of the protein. The kimtics of refolding of reduced RNAase were entirely consistent, \vith those observed in detail in refolding of reduced BPTI, where it was also shown that! the three disulphide bonds are not formed in a simple sequential manner. In this case. tlw slowcst~ step is just prior t’o forming a nat.ivr-like species with two disulphide bontls. as the third and final disulphide is not an integral part of the folded conformation of BPTl and may be formed and broken v&h very little perturbation of the f&led conformatJion. The greatest difference between refolding of the two proteins is the number of disulphide intermediates which accumulated. Formation of the initial disulphidr bond in reduced BPTI is nearly random, yet only two specific one-disulphide species accumulate substantially, because the first disulphide bond is rapidly rearranged intramolecularly t,o that, of eit’her of these two most stable intermediates. In cont#rast, the one-disulphide intermediates of RNAase were an apparently random mixture, indicating that all had comparable energies. The restrictions on conforma-

430

T.

E.

CREIGH’I’OX

tional flexibility which are necessary for refolding occur witah RiXAaw at a late1 stage of disulphide formation. This could be envisaged as resulting from the much smaller size and greater disulphide bond density of BPTI (3 disulphides in 58 residues) relative t.o RNAase (4 disulphides in 124 residues). which could permit in BPTI a greater fraction of stabilizing interactions with one or a few disulphide bonds. The non-sequential pathways of refolding of reduced RX=\ase and BP’J’I may btl rationalized as being due to the general co-operativity of protein folding, whereb? u%h the rat’+limiting tjransition partially folded states are unstable, combined occurring at, the very final stages of refolding. The rate-limiting step in refolding of both proteins is the formation of the last correct. disulphide bond required for a st)ablr: native-like conformation, while it’ is tile breakage of t#his bond which is ra.tt:-d&wmining in unfolding. The slow rate of the latter I,‘s: undoubtedly tiuc> to the unfavourable energetics of perturbing the native conformation to reduce onta of t,he required disulphides, each of which appear to be integral parts of the nat,ivc: conformation. under the The energetics of protein unfolding appear to control t,he refolding pathway same conditions. A high-energy barrier to any significant. perturbation of t,he natiw conformation would seem a reasonable means of maintaining a protein in its active form, so ib might bc expected that, pat#lrways of refolding of ot,lrrr prottains will also tw determined by this hightbst energy barrier. It’ swnls trnlikcly that such considcrstiow will apply only to protein folding iuvolving disulphide bond formation. as uudcr the conditions used here the formation. breakage, and rearrangcnrent of disulphide bonds would be expected to be similar to t,ha.t of hydrogen bonds (Creighton. 1978).

REFEKEYCES A Ahrned, A. K., Schafkr, 8. \V. & IVetlaufer, D. B. (JQ7.5). .J. Hid. Andria, Cr. & Taniuchi, H. (lQ78). ./. Biol. Chem. 253, 2262 2270. Anfinsen, C. B. (1967). Harvey Lectures, 61, D&l Ifi. Anfinsen. C. B. (1972). Rio&em. .J. 128, 737-749. anfinsen, C. B. (1973). Science, 181, 223-230.

Anfinsen, C. S. & Seherapa, H. A. (1375).

Advan..

(‘hew,. 250, 8177 8-kX2.

I’roteiw Chew. 29, 205 300.

Anfinsen, C. B., Haber, E., &la, M. & White, F. H .1 Jr ( 106 I ). I’roc. .Val. .2cncl. Sci., U.S.A. 47. 1309.-1314. Baldwin, R. L. (1975). dnnu. Kev. Biochern. 44, 4% -IT:i. Baldwin, R. L. (1978). Trends Biochem. Sci. 3, 66 -68. 14. -LR.5% 49(X1. Brandts,
TNTEKMEUIATES

IN

RIBOSUCLEASE

HEFOLUISG

43 I

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