Electrophoretic analysis of the unfolding of proteins by urea

J. Mob. Biol. (1959) 129. 235-264

Electrophoretic

Analysis

of the Unfolding

of Proteins by Urea

T. E. CREIGHTON

Mrrlical

Research Coumil Laboratory of Molecular Hills Road. Cambridge, Enjgland

Biology

(Received 5 S’eptember 1X8) The lmfoldin,< of several proteins by llrea has been followed by eloctroplloresis of a band of protein throng21 a slab gel of polyncrylamido in wllich there was a gradicxrlt, of urea concentratiott porpendioular to the direction of electrophoresis. Unfoldirlg \vas invariably manifested by a marked reduction of mobility, preslunably due to lnolecular sieving of the clxpanded polypeptide chain by the polyacrylarnide gel. The procedtwe pro\-ides a continuous two-dimensional pattern oftlie t+f&ct~ of urea on tile sliape oftlrr protein and is especially sensit,ive t,o microIlcterogeneity of the protein. inhibitor, ribonl&rase, lysozyrne. trypsirl Experiments with pancreatic stapllylococcal nucleasc, snd cytocllrome c c:ll>-tnotrypsill, cllymotrgpsinoper1. RY~~Cconsistcrlt with t’hr results of otllws ltsinK orthodox rrwt.hods and confirm t,llr \ralidity of t’lle metllod. TVtwre tmfoldirlp occurred, it \~as generally rapidI> rc~vwsibl(~ ant1 tile curves WYI-c entiwl>~ cotrsistent \vitll tllr prcsencc of only th(l nativcl anti t IIO fXly unfolded states. &~~un albumin gave mcjre complex curl’es at~d a rrtnarkable illust’rat,iorl of lnicro-lIr,tero~c,neit?-. P-l,act,oploblllirls A and B and ovalblnnln refold very slo\vl~- arid t’lle Iilrfoldrd molecules appear4 to ccluilihrate prrferent,ially \vitlr compact. but rlo~~-nat,i\-P, forms at 101~ III’~N c:oncelltlatiolrs.

1. Introduction The many experimental studies of protein unfolding and refolding (reviewed by Tanford, 1968,197O; Baldwin, 1975: Pace, 1975; Creighton, 1978) have generally required the use of a denaturant’ to destabilize t,he native conformation sufficiently to produce the unfolded state. The t)wo most widely used denaturants have been urea and guanidinium chloride; urea was initially favoured but has since been supplant,ed by the somewhat more potent’ GuHClt. The precise physical explanation of thp denaturing ability of t’hesc t’wo reagents is not’ clear, but studies with model caompounds suggest that both function by increasing the aqueous solubility of t’he hydrophobic portions of proteins (Nozaki & Tanford. 1963; Wetlaufer et al., 1964: Pranks & l&gland, 1975) while maintaining the hydrogen-bonding capability of t,he aqueous solvent, (Roseman & Jencks, 1975). The unfolding of proteins by these reagents has generally been studied experirnent,ally by measuring one or more ph,vsical parameters of the protein as a function of the concentration of the reagent. The properties of small proteins usually change little, if at all, at low concent.rations of bhe reagent until that concentration range is

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‘t’.

E.

(‘J
HTOS

reached where a relatively abrupt unfolding of t,he protein vccurs; beyond the unfolding t,ransition, the physical properties of the protein change little. if at all. 1Jdcr appropriate conditions. t,he unfolding of small prot,cins is usually wvcrsible UJX~ dilution of the denaturant’, and refolding vcwrs \vit,hin pwcisely the ~mc range vf denat,urant concentrations as did unfolding. ‘I’hr equilibrium folding transit,ion generally follows a smooth curve with a, singlit infltJct,ion point, : mw~~vw. the samt’ transition is generally measured by all appropriate l)robt:s of t,h(, prvteiil corlforrnat,iou Such observations are cnt’irely consistent \+.itll a t\+ o-statcl t,ransition i tl \vhich vnl! the fully folded (N) and fully unfolded (U) stattxs are poputatr~tl signiticalltty : sIJIIz=e

I’

(1)

Although an abrupt’ transition is obsrrvrd, urea and GuHCl have> gc.nwally brcn observed to produce unfolding by gradualI>- and continuously increasing t.lw apparent. rate of unfolding while simultaneously decreasing the rate of refolding, resulting in a continuous change in the value of thr rquilibrium constant for the twwstat,c transition (1) ; the abrupt t’ransition occurs when its value is in the narrow range around unity. Such observat’ions have been extrapvlat~ed to t,hc absence of denaturant (Tanford, 1970; Pace. 1975) to provide a dynamic description of a marginally stabhx folded pro&in under non-denaturing contlit~ionh. has been The apparent absence of stablu part(ially folded stat(es i tl such transitions best confirmed by the agrccment~ bctwwn thrb appawnt enthalpies of the folding transit,ion measured calorimetrically and that measured by t h[h trmprrat8urt~dependence of the apparent equilibrium corlstant~ for the transition (Privalov K: Khcchinashvili, 1974; Pfeil & Priralov. 19i6rx.O.~). This co-operat,ivity of protein folding is wt,ll-r:stablishecl for tlwst? small. stabIt> proteins which have been well studied, but. it is not known whether it is rcpresentativc of’ globular proteins in general. Indeed, taxceptional brhaviour hah been reported for c (Myer. some other proteins : for example. thcl folding t)ransitivns of cytochrvmo 1968), bovine E-la&albumin (Kuwajima rt (11.. 1976). bovine ca’rbonic anhydraw 13 (\Vong & Tanford, 1973) and RtcxLphyboc,oc,c,u.u ~1!1re~.spcnicillinass (Robsou KS Pain, 1976 ; Carrep & Pain. 1978) as measurrtl \vitlr differwt prvbw apparcntl!do not coincide: and t,he folding t,ransitions of cytochromt~ /a (Mg’rr. 1968), carbonic anhydraw (Wang & Tanford, 1973 : \ITong & H amlin. 1974). and immunoglobulins (Azuma ef ~1.. 1972) have not, been smooth. but haw hat1 t\\,o or more inflection points. Thew vbservat,ions suggest the presenw of thrw or mow different states of tlw protein, but, t,hey need not invalidate the generality of’ co-operat,ivity of protein folding. as other explanations for such behaviuur are possible. (I) The protein population st,udietl might 1~ heterogeneous in covalent, strurt8urca. individual sub-pvpulatic,ns unfolding in t,wo-stat,e transitions at differcwt denaturant concentrations ; this ~\.oultl not bc apparent, \vit,h most methods in general IIS~L.(2) ‘l’h(l third state could be aggregaks of insvlublt> under nvnthe unfolded protein, as unfolded prot~eins art’ grnwallv is of%tw obvious (e.g. London d nl.. 1974: denaturing conditions : precipitation Orsini & &ldberg: 1978), but, small aggregatrs uced not be risible. Complex folding transitions have been observed most oft,en \rit,h proteins which unfold at low tlcnaturant concentrations, where the denaturant is less cffcct,iva in solvating thrx lmfolded protein. (3) The stability of t.he folded state may be dependent upon binding of a small molecule, which is present in insufficient concentrations for refolding aftfar release upon unfolding. (4) The folded protein might. collsist of‘ mrIltiplt% independet1t~

UNVOLDING

domains trations, (Rowe $ Kenjamin. Privalov. domains. Kinetic

OF PROTEINS

RY

UREA4

2.35

which unfold and refold independently at different denaturant concenfor which there is considerable indirect evidence wit,h several proteins Tanford, 1973; Celada et aZ., 1974 ; Raibaud & Goldberg, 1977; Teale & & 1976,I 977; Taylor & Silver, 1976; Yonath et al., 1977; Tiktopulo 1978). The co-operativity of folding could then apply to individual struct,ural

observed in t,he unfolding and refolding of proteins. even two&ate behaviour at equilibrium (Baldwin, 1975), could bc tiut> t,o t h(b awumulat~ion of t8ransient,. u&able partially folded states. but much oft hc Icinet ic complcxit,v has been found upon thorough invest’igat.ion (Gart.1 & Baldwin. 1973.1975 : Garrl ei! al., 1976: Hagerrnan &. Baldwin. 1976 : Nail 85 Baldwin. 1977 : Sal1 et ~1.. 1978) to be due to slowly interconverbed sub-populations of t,he unfolded prott!in. This phenomenon may be general (Brandtx et al., 1975 ; Pohl, 1976; Hagerman 1977) alld due to slow c:is-trans isomerization of peptidc bonds, particularly thaw it(ljact~Ilt to proline residues (Brandt,s PI al.; 1975,1977; Sehmid & Baldwin, 1978u.h). ‘I’hr~ precise nature of t,he unfolded protein is generally the least certain aspect, of prot,tlin unfolding and refolding. Under denaturing conditions. where the solvent is probably fax-ourablr for all parts of a protein, an essentially random polypeptidt vhairl is believed t)o be approached (Tanford. 1968.1970) 111 which rotations about a,11 hontls occur independently and transiently, just as in the appropriate small molecules. ‘l’hv f’ul1!, UJlfOkkd state of a protein thrrl consists of a very broad SpeCt'rum of t~ransirnt t.onfoJ.rrlatioJis~ so that, each IJJOkc!Uk of a population probably has a unique coolfiwnation at an>- instant of t.ime. The nature of t.he unfolded state under nondrnat~uring conditions, where it is only transient, is much less certain. Indications of ilIly ~io1i-ra,n(lo~i~icss in an unfolded protein are often t,akcn to indicate the persistence of’ stabh~ conformation, even though peptide fragments are not known to maintain st,ablt c:orlti,rmat,ions. A more plausible cause of’ non-randomness of an unfoldrad fwot’c*in wo111d bc the predominance, dw t,o relatively favourablc energies, of a subset of thft rna11~~ conformations comprising thr fully unfolded state, but t>hcrc would \till bc ver?’ large, numbers of readily interconvert~ed conformations. One means of tlist’inguishing bet n-een t’hese t’wo limiting possibilities is to look for a distinct unwhich should occur if any stable confolding t#ra,nsition upon adding a denaturant. forrnstiou were present init’ially, whewas only a gradual changr of conformational propt’rt,ies wt~uld be expect,ed if initially there were only a sub-set. of unfolded COJlf’orrnations ((1.g. Aune et al., 1967 : HolladaJ- $ Puet,t,, 1976). ‘l’l~tw rnally uncert’ainties about protein folding t’ransit,ions arise primarily from twhnical limitIatJions in studying protein conformation in solution, so there is a need f’or ntw. c~speriment.al approaches to the problem. One physical propert,y in which folded and unfolded proteins differ markedly is t,he shape and size of the molecular \~ol11111eoccupied by the polypeptide. folded proteins being remarkably compact relativts to a random polypeptidc. yet this parameter is not8 oft,en measured for lack of iI convctnit%nt mt%hod. The shape of a protein affects the rat<> at which it migratw through polyaorylamide gels (Crambach 8: Rodbard, 19’71). a,nd the rate of electroI)howtic migration through such gels has been shown to be sensit,ive to the compact11ws of trap@ inbermediates in protein folding (Creighton, 1978). Electrophoresis rrla>’ Ite c*arrit,d out. in the presence of non-ionic denaturants, such as urea, so it seemed ~)ossibl(~ t,hnt) the folding transition of proteins could be visualized by electrophoresis of Iwot,ttins in a polyacrylamide slab gel in \f.hich thrrc was a concentration gra(lient

t how

\vhich

complexities exhibits

238

T. E. CREIGHTON

of urea perpendicular to the direction of migration, so that, molecules at varying positions along the gel were migrating in varying concentrations of urea. Such twodimensional urea-gradient gels have been used in purifying oligonucleotidea b) Gross

et al.

qualitative

(1976). It information

is demonstrated about

urea-induced

here

t,hat

they

folding

not possible with the other t~echniqurs in yrxwral

may

transkions

also

bo used

of proteins

to

gain

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2. Materials and Methods All proteins were obtairled co~nmercially atltl 11sc~1withollt tiwtt~er pnrificatioll. Bovine pancreatic trypsin inhibitor was a gift from Bayw A(:. Bovillc pancreatic rihonucleaso A was either the chromatoprapllically pln%icd fornl from Koclr-Light (batch 7~0. 74158) OJ tile phosphate-free form from Wortllin#orr (lot RAF’ 8RB) : ttrc, t,\vo prcxparatiolrs bellavcd identically. Bovine a-lactalbumin xas ttlr gra(Iv 11 of Sigma (lot, S6C-8020). Bovine j3lactoglobulin was a salt-free, freeze-dried prcparatioll from Britisll Drug Houses cont,aining both A and B variants. Horse lrcart f~rric~-toClIrofnc~ c was t,llr type VI of Sigma (lot 57C-7070). Bovine u-cliymotrgpsiri \vas tlicb 3 c~rystallizctl salt-free protein from Worthingt,on (batch CDT 37B601) : box,irw ct I?-1nc,tl,~psirloUf~ll A bvas t,lle 6 :: carystallizcd was from P.-L. Biochemicals type II from Sigma (lot 11 IC-8170). Hen (xgg-\vtrit P lyxozyrrw (lot 267.9). Staphylococcal (micrococwd) tr~~~l(~aso was frown Worthingt,on (b&cl1 KFCF’ u as from Britisll Drug Hol~srs. Hc11 egg 57B556). Crystallino bovine SCI‘I~I alhtllnitl ovalbumirr was the cryst,allized gracl
Acrylamide gel electrophoresis XVAScarrirtl or11 iI, a GE-4 ~(11 rloctrophoresix apparatns (Pharmacia) because (1) the prl slabs arc> squar,’ (82 mill > 82 ~nm) and tllrls may bc gels rrray tw prepared simrrltanoolwly in ttrc> readily rotated by 90”, (2) up to 8 idcnticat it) bufYc1 MC-8 gel slab cast,@ apparatus (Pllarrnacia). atid (3) tlie gel is immerse(l throughout the elrctrophoresis so that ttlc, tcmpc~ratllr<~ ~nay tw cont,rolled rradily ; Ilo\\,el’er, any slab gel apparatus probably may b(x uwd witll only minor alterations. acrylamide solut,ions in t I I( The urea-gradient gels were made by E’trotol”~t?-lnt~rizin~ gel slab apparatus in whicll a gradient of ,wea tract bwn formed by mixing two acrylamidc solutions with the two limiting conrcnt,rations of 11r~a (~~sl~wlly 0 x and 8 -“I). The acrylamid,, solution with tile lower concentratiorl of 111-w was fed into thcl bott,om of the gel casting of ltwa. follo\z-rd b?; more of t)lle solution apparatns first, followed by the linear gradietlt to with the higher urea conwlltrat iml, rising 1tI(s ~,i\trlral tlensit,y of ttie llrea solution stabilize the gradient. Ttte \olrunes UWY~ :rtljustc,tl so tllat, ttlcl top and bottom I cm of the slab gel were at, t,lle two limit.ing ln~~a corlc.ctrtri-rtiotls, cl it,tI ttlc linear IZI’W gradient irl the middle. The linear pradient was formtcl by atIding, wittl rapid mixitlg tlrlder a stwam of nitrogen gas, the high urea solutiolr to a Prc’letcrrnilrct~ \.olrime of the low urea solution with one channel of a peristaltic pump. \vtrilc wrnovirlg to the prl slab the solution from the mixing chamber with twn channels of tllcx sa1nc pump. The two urea solutions were identiral itr all wspccts but, ttrc concentration of urea arid \vas gcneralll acrylamide. as a slight gradintlt, (from 15’!,, to I I (y;,, \v/\-) of acrylamidr present to compensate for the cffecats of ur(sa OII t I)(%clwtroptioretic properties of proteins xvhich did not involve unfolding (SW Results. swt iolr (a)). Tte concentration of t,he crosslinking X, N’-metllylene bisacrylamide was kept constant at, 0.0067 that of acrylamidc. atltl any 111-w. t.he mixtnrc: contained the In addition to the acrylamide. bisacrylarnide. ~1’.S.~~‘,~~‘-tetr~~~tt~rylrnctl~~l~r~e~liarnir~~~ (adjusted to the desired buffer, 0.12 9/b (v/v) appropriate pH with acetic acid). and 5 pp ribc,flavin/ml. The solutions were deaerated under rednced pressure wit11 an aspirator bcfow castJirlg tile p-1. the urea gradimit, slabs ~vew insertrd into the PlectroAfter photopolymerization, phoresis apparatus so that tltt, uwa gradient was pc~rpcwdicnlar to the direct,ion of ekct,rogel, hvith no urea. Tlx phoresis. The buffer was the sarnc as tllat, it1 t11v l”)l~ac:r)-liLrlridc

UNFOLDING

OF

PROTEISS

HY

UREA

“39

c~lt~ct,l,oplrorr~sisbluffers used were: 0.05 %I-Tris-acetate at pH 4.0 (0.05 M-acetic acid titrated to pH 4.0 with Tris), 0.05 nz-Tris-acetate or Tris.HCl at’ pH 8.0 (0.05 wTris titrated to pH 8.0 with acetic acid or HCI. respecti\.-ely), and Tris-horate/EDTA at pH 8.6 (5.45 g Tris, I .55 g boric acid, 0.29 g EDTA per litre). Approximatol\50 to 80 ,LK of protein in 50 ~1 of buffer \vere layered onto the top of tlrts ~1bwrol was added to increase tile densit),. ccl ; of tllf, protein solution contained no rwea, r) Ii:ithclr lnc:t 11>,1grwn or brornophenol l)lrlc~ \var usually- added t,o the sample as a marker. ‘I’lw c,lcctropltorc~sjs was pewrally startctl by applying about IO r&/gel; aft,er the marker tlycl Ilad tstlttlr’ed tllcs gel several mm, ttle crlrrent was increased and kept constant at clearly 20 nA/gel. During the course of thus electroplloresis (2 to St)), the telnperat,lwt- of t Ilr I)uff~r was peuerally kept constallt, at’ brtwvccrl 15°C and 20°C. After complet,iorl of t,II(l c,lcc:troph~,r~sis. tllc gels ww-~ stained ovrrnipht~ in 0.1 ($A(w/v) Coomassie brilliant blr~cacid. The gels wew I,, loo,, (XV,/\.) trichloroacetic acid plus 10% (W/V) sulpllosalicylic . Io mct~llanol or IO”:, t,richloro~~ct~tic~ ctrntainc3l I)y diffilsiorl against, cit,tlcr 5’y0 acetic, acid, i.gO’ ;witl.

3. Results (a) Kationale of two-dimensio~~al urea-grdien,t

elrctrophoresis

‘I’he moIecu]a.r dimensions of prot,eins change markedly upon unfolding frurn the, very compact close-packed globule of a normal folded protein to tile unfolded polypeptide chain (Tanford. 1968). Such changes in shape affect, the rate of migration through concentrated gels of polyacrylamide and may be brought about by the inclusion of varying concentrations of a non-ionic denaturant, such as urea. To this PM]. two-dimensional polpacrylamide gel slabs were prepared in which there was a horizontal. ront,inuous, linear concentration gradient of urea perpendicular to thr v&ic*al tlirwtion of electrophorrtic migrat,ion. An homogeneous sample of prot,ein applietl uniforml!~ across the top surfaw of tho gc>l. perpendicular to the direction ot’ migration, then migrates in continuously varying cwnccntrations of urea across th(, slab. ‘I’hc uwa concentration varies only \+Niin the gel slab, not within t,hfb WI 0 applic~d sample. .‘;I) all unfolding and refolding transitions of the protrirt take pIact> within t,llc gel (luring t,he elect,rophoret,ic migration. ‘l’hc interpretation of the gel patterns is facilitat,cd by considering first’ the effect of folding transitions on the electrophoretic migration at a single concentration of urea. i.e. within a t,hin vertical slice of such gels. (.Por references and details, see the *Ypprndix.) Multiple conformational states of a protein will only be separable by c~lcctrophorcsis if t’heir interconversion is slow relative to t,he time required for elechrophorrsis. which in the case of the experiments reported here was about two hours. For repitl interconversions, the protein molecules will migrate as a single band with a rnobi1it.y tlr~termined b,y t.he weighted average of the mobilities of the individual conformational forms present. The widt,h of’thc: band of a pure protein will be determined by diffusion whcstt interconversions are very rapid. but the band will be broadened if the half-timw for conformational t’ransitions are grrater than about> 0.01 the duration of t*hc cx~xGnent. For t’ransitions with half-times within an order of magnitude of the duration of the experiment. t’he original form of the protein will persist but wi]] h
“40

‘1’.

I’:.

CItE:lGH’I’oS

the various urea concentrations; the same pat,tern should be obtained irrespective of t,he original conformat,ional state of the protein applied to the gel. The effects of slol+t,ransitions on the mobilit’y, which may be important, at only certain urea Concentrat’ions, should be apparent by obtaining different, patterns when starting with the folded or the unfolded forms of the protein. Some examples of gel patterns predict& for two-stat’e folding transitions of varyjng ratcxs arca presented in the Appendix. The practical aspect’s of the urea-gradient gels were invest,igatetl using as test protein bovine pancreaCc trgpsin inhibitor. a protein \vhosc folded conformation. and therefore its mobility through polyacrylamitlc gels. should not be affected by donaturants such as 8 .yI-urea (Vincent ~2 nl.. I!171 : Karplus et cxb., 1973: Masson & Wiit,hrich, 1973). Howaver, it’s c,lectr~opllorct,ic: tnobility in 15”;;, acrylamide gels was ill 8 wI-\lrf’a observed to vary oont~inoously \vith t hr IIIW cwnwntrat ion. migrating \vit.lI onl>- O.(i t’ho normal mobility. ‘I’lrk ~~11f~1101t1f~~~011 u.8~ a,lso cvide1it \\4tll otlrf~r proteins aricl huggests an cftixct’ of lit’(‘a on tlrc, hiwing I)ropfy$if~s of’t IIf’ polybc’rylarllifl,~ gscl. Thf~ gel patjtcrns could be wrrwtc~d visually or by ttwasuring the Illobilitic~;i 01 ot.her proteins relat,iw t,o that, of BP’I’I. which was oftw included wit/l t.hc protein under investigation as an internal st,andard. Ho\vwer, it, was fourd pwfwable to compensate for the rffwt of t)hc> urea 1)) superimposing on t,hc urea gradient an inverse gradient of acrylamide concentration (generally from 15”/;;, to 11 “/,,) so as to produce comparable int’rinsic mobilities of’ most prot,citw in 0 >I and 8 Jr-urea. The compensation of t#hc t \\.o effects \ras not thxact and somrwhat variable. as small prot,eins like BPTI oftcsn migrat.ed xomt~what t~ow rapidly in the middle* of’ t,lre urea gradient than at t,hrl t\\ o r~xt~rcmw (SW Figs 1.2.3.5 arid K for examples). hut, it, produced acceptable patterns \\.ith most of th(b prott~ina stutlicd.

The rwersible unfolding by II vmict~~ OF f~f~rlat~uratlts of’ ribonuclease (Richards & Wyckoff. 1971). wit.h it,s f’our tlisulphide bonds kept, intact.. has generally been observed t,o behave as cxpcactrd for a tIwo-statc> t,rarrsition at equilibrium (Wahuddin & Tanford, 1970; Baldwin: 1975; lUal1 & Bald\* in, 1977), excluding minor perturbations of the folded Aate (Benz & Roberts, 1975: \Vest~moreland & Matthews, 1973). The unfolded st’att is heterogeneous kinetically. prol)ably due t,o slowly interconvert,ed unfolded forms with eit,her corrwt or incorrecl~ isomers of’ peptide bonds adjacent t,o proline rrsiducs (Garel & Baldwin. 1973.1975: Krw1dt.s rl al., 1975; Baldwin, 1978: Schmid & Baldwin, 1958a.h). Unfolding of ttlcl protein by urea (e.g. Harrington hz Schellman, 1956: Sela et nl., 195i: Sclson B Humnwl. 1962; Barnard. 1964: Greene & Pace, 1974) has been observed to occur at about 7 hl-urea at neutral pH, but at at lo\\w pH values. The half-t-imes for the correspondingly lowrxr concentrations appthar to be no greater than a folding transit,ion at. the various urea concentrations few minutes (Nelson & Hummel. 1962; Barnard. 1964). The urea-gradient gel patterns wc:rc f~nt~irc~l~consist)ent with the observat)ions of others, as is best illust,rated at pH 4 in Figurcl 1. LOIV concentratIions of urea produced little or no change in t,hc elwtrophoretic mobility of ribonuclease until the concent,ration reached about 4 nr. where a relat.iwlp sharp change in mobilit,y occurred, corresponding to the expected unfoldinp of t.hr> protein. At concentrations of urea above this transit,ion, lit,tle or no changtx in tnobilit,y occurred, consistent with the prot,ein being fully unfolded. The band of protein \z’as continuous and sharp throughrclativcbl?; mpid c,cpClibrat(ion of t!hs folded and out t,he transition rtgion, indicating

Reduced corboxmefhyloted

.-z % : .5

-

‘ ,/

* -

I( Native

V

l

Bu

Urea

FIG. 2. l:wa-gratlient clectrophoresis of hen egg-white lysozyme. The minor band was BF’Tl added as an intwnal standard. The buffer was 0.05 Jr-Tris-acetate (pH 4.0). The linear gradient of 0 JI t,o 8 $1 was supwimposed on an inverw lirrew gradimt, of 15 “() to 11’:6 arrylamidc. Elcrtruphorwis towards thv cat,hotle was at I6YJ.

242

T . E . CREI(‘HTOJX r

unfolded stat’es. Also, identical patterns were obtained when t,he ribonuclcase applied to the gel was either native or unfolded in 8 &r-urea. At! pH 8.0, only small changes in mobility were observed at the highest concentration of urea, corresponding to only partial shifting of the conformational equilibrium toward the unfolded state (Barnard: 1964). These results are entirely consistent with the t,uo-state behaviour of this protein observed by ot’hers, and illustrate t)hat unfolding may be detected elrct,rophoretically even when the unfolded st,ate i,c: highly constra’ined by four tlisulphidc bonds. Ribonuclease in lvhich the four disulphide bonds have been reduced and the eight, c,yst,eine residues carboxymethylated is generally accepted as being a fully unfoldrtl polppeptide chain even in t’he absence of denaturants (Tanford, 1968). Accordingly, no unfolding transition was observed in urea gradient gels at pH 4.0 (Fig. I) or pH 8.6 (Fig. 3). Reduced ribonuclease with free t)hiol groups also produced no signs of unfolding in the urea gels at pH 4.0, whrrcl oxidat’ion during electrophoresis is negligible.

The folded state of lysozyme with its four disulphide bonds intact) (lmoto et ab.. up to 8 i+I at 1972) is well-characterised as being resistant to urea concent,rations normal temperatures and pH values (e.g. Hamaguchi. 1958 : Tanford, 1968 : Warren & Gordon: 1970 ; Snape et al., 1974) alt,hough it) is unfolded by GuHCl (Tanford et ~1.. great,er than 1966); pH values of less than 3 (Greene & I’ace. 1974) or temperatures 30°C (Warren & Gordon, 1970) are required for fill1 unfolding by 8 bI-ur3a. Accordingly. no unfolding transitions were observed in urea-gradient, gels at, pH 4.0 (Fig. 2) or pH 8.0. The mobility of lpsozyme actually incrcbased somewhat, relative to that of BPTI (Fig. 2), with increasing urea concentrations: this may be due to inhibition of reversible aggregation (Sophianopoulos & Van Hold?. 1964). a relaxation of t’he folded conformation to a more compact, st#at,e (Snapcb Pf al.. 1954). or simply an artifact of the gels, but the viscosity of lysozyme has been reportrtl to bc decreased by 8 I\z-urea at low ionic strength (Warren & Gordon: 1970). Reduced lpsozgme with the eight free thiol groups migrated more slowly than folded lysozyme at pH 4.0 but gave no indication of any further unfolding induced by urea, exactly comparable to the results obtained with reduced ribonuclease. and consistSent, with it being unfolded in the absence of urea. (cl) Bovine a-hctalbumiu The amino acid sequence of a-lactalbumin (Brew et al., 1967) has been found to be homologous with hen lysozyme and compatible with its having a similar folded conformation (Browne et al., 1969), although by many criteria the folded st’ate of alactalbumin appears to be less compact or stable than that of lysozyme (e.g. Iyer & Klee, 1973; Kuwajima et al., 1976). X variety of spectral st,udies of the denaturation of a-la&albumin (e.g. Kronman et al., 1965: Bugai et al., 1973; Sharma & Bigelon-. 1974; Kita et al., 1976; Maruyama et al., 1977: Kuwajima et al.. 1976; Kuwajima. not, consisttent wit,h a simple two1977) have observed rat,her complex behaviour state model. Denaturation as a funct’ion of GuHCl concent’ration, pH. and tempera1977) in terms of ture have been interpreted (Kuwajima et al., 1976; Kuwajima, three states, the native, the fully unfolded: and a t#hird state of int,ermediate conformation which is stable at, low pH values and at, int,ermcdiattr GuHCl concentrations,

OM

pH 4~0

Bovme

Q - Loctolbumin pH 8.6

RCM-

RNose

244

T. E. ~JAEIGMTOX

although the third Aate was only detectabk by far-ult,raviolet circular dichroism, and the interprt:tat)ion of such data is complicated by solvr~nt effects on the spectral parameters and a tendency of thr protAn t 0 aggregat’c untlcr conditions wflere t,hr presumed third st,at’e is det&t~tl (Kronrna~ I $ ;\ntlrt)ott i. 1964 ; Kronman ut nl.. 1965 : Sharma & Bigelow, 1974). The unfolding 1)~ urcba has not bt:c~ invcstigatc~d in detail, but has been concluded to br similar to that t)y ($nHCl anal to lead to complete unfolding (Sharma & Big&w, 1974). The electrophor&ic mobility of cc-lactalbumin was observed Tao decrease with increasing urea concent’ration at, pH 4.0 (Fig. 3), but, it’ was a gradual change. without an abrupt, transition like that observed wit,h other proteins. Surprisingly, there was no significant change in mobility in urea-gradient gels run at, pH 8.0 with either 0.05 &I-Tris-acetat’e buffer or \i:ith 0.05 RI-‘Fris . HCl buff’er cont’aining 1 rn~-EDTA. At8 pH 8.6 t,herct \vas a gradual transition bct,\vern 3 M and 7 &r-urea (Fig. 3). \vhich could be consisbentf with a complex unfolding taansition. These observations arc not inconsistent, \vith t,htx obsc:rvat,ions of’ Kuwajima et ml. (1976) and suggest that unfolding of t,his protrlin is compltts. Reducrtf K-lactafbumin with tight frtltb t hiol ,qoups showtd no variation in mobilit,) at pH 4.0. except8 that, t,ho protein failctl to t,nt,tar*t I)(x go1 at urea concc~ntrations lower than about 2 M. appart:nt,ly due to t~xtcnsivc aggregation. ‘rhtt rcduccd protein is very insofublc undrr nc,ll-tlcrlaturitl~ conditions. but s;(‘cms t’o have no folded conformation disrupted by urea, ~vt~l t bough it s circular tlichroism spect,rum is rcport’etl to be like that. of tht% t,lrirtl, interm&att~ state propostxd by Kuwajima (1977).

a-Chymotrypsin is derived from chprnot,r?-psillo~etl X Ir)v proteolytic cleavage to cleave the single polypeptide chain into thretl segments, yet, the t\vo molecules have very similar conformat.ions (Kraut,. 1971 : Klo~. 1971 ) amI have similar susceptibilities to denaturants. ‘l%t: urea tlt~nat~urat ion of’ t8hcsc t,\\.o proteins, keeping intact the five disufphides which fink covalrntly t~ht~ three pofypeptidt: segments of GCchymotrypsin, has been studied extensively and generally observed to behave as 1964; Martin & expected for two-state behaviour (Neurath ef al.. 1956: Brandts, Bhatnagar, 1966; Herskovit,s et al., 1970; (ireenc bt Pacp, 1974). Unfolding is generally observed in an abrupt transit.ion at a urea concentration which decreases with decrease in pH; at pH 4.0 this concentration was usually observed to be 5 to 7 >I. ‘l?he half-times for unfolding and refolding were usually no more than a few minutes. The denaturation of a-chymot,rypsin observed with urea-gradient electrophoresis was entirely consist,ent n.ith the observations of &hers. wit,h an abrupt transition t,hr ot7ymot.rypsinoger1 split, into starting at about 6 &f-urea (Fig. 1). Surprisingly. two similar bands, differing only slightly in t)h(Lurt:a concentration at which unfolding started. However, t,hc chymotrypxinogen used I\ as found by sodium dodecyl sulphatr/ pofyacrylamide gel electrophoresis of t,he reduced protein to cont’ain about) half intact, chyrnotrypsinogen and about half apparently rlc?ochpmotrypi;inc,gen. which has been cleaved proteolytically at the peptide bonds of residue 146 or 148, or both (see Kraut. 1971). The neochymotr~~psinoge~l probably was tbo species which unfolded at the lower urea concentrat,ioll. as it hat1 a slipht,ly fo~vcr mobility when unfolded, as WOUND be expected for a c~lt~a\-c~cl cross-lirrltetl pol~~WI)fitl(~. It stlollltl I,(~ Ilot~td that all t,tlt:sc~ proteins in Figurcb 4 gavci tlic, 8;bnlt (*hara(+(~rist ic, (‘11rv(‘>.

cl-

Chymotryprin TOP

V

Chymotrypsinogen

A

.

TOP

V

(f) rS’tuphylococca1

nucleas~

The folded conformation of staphylococcal nuclease (Cotton S: Hazen, 1971) is known to be disrupted by high concentrat’ions of urea (dnfinsen et al., 1971), but only the conformational transit’ion t,hat occurs at low pH (below about pH 4.3) has been studied in detail (Epst,ein et al.. 1971u.h). It appears to be a very co-operative two-state transition, alt.hough some perturbations of the native conformation were observed t,o precede the major transition. Urea-induced unfolding was found to occur at, very low concentrations at’ pH 4.0. where the folded state is only marginally stable in the absence of urea, but at higher concentrat’ions at pH 8.0 (Fig. 5). The mobility of t’he unfolded protein was only 0.3 that of t’he folded protein at pH 8.0, presumably due to t’he absence of disulphide cross-links in the protein; the difference in mobilities was somewhat less at pH 4.0. probably due to the protein being folded only part’ of the time in the absence of urea at this pH. Tn both cases, t’ht: prot’cin bands were continuous, indicating rapid equilibration of the folded and unfolded forms of the prot,ein, but there were “spurs”

246

pH

4.0

BPTI

FIG. 5. Crea-gradient 0.05 mTris-acetate (pH t)o both protein samples imposed on inverse linear phorrsis was towards the

c,lcctropIlorcsls of ~taphylococcal nucloaac. The buffer at the t,op WAS 8.0). that at. the bottjom 0.06 mTris-acetatr (pH 4.0). BPTI was added as an int,ernal standard. The linear gradients of 0 M to 8 BZ were supergradicnt,s of 15 0; to 11 I;, (top) or t,o lo”/, (bottom) acrylamide. Electrocathorie at, 157’.

of unfolded protein, suggesting that, a fraction of t’he molecules could not refold rapidly at the urea concentrations in the t.ransition region. It is likely that this reflects either structural or conformational heterogeneity of the protein used. Except for this phenomenon, the protein mobility of the major band changed smoothly with urea with a t.wo-state concentration throughout, the transition region. fully compatible transition for the majority of the protein molecules.

(g) Ho/w

cytochrome

c

The folded structure of cytochrome c (Dickerson & Timkovitch, 1975) is critically dependent upon the heme group (Stellwagen et al., 1972; Fisher et al., 1973), but’ it is covalent,ly at,tached to the protein through thioether links to cysteines 14 and 17. so t,hat unfolding and refolding are unimolrcular processes. Two other heme-protein interactions important for stabilisat,ion of the folded conformation (Fisher et al.. 1973: Stellwagen $ Babul, 1975) are the His18 and Met’80 ligands of the heme iron. The heme iron is also important for the unfolded state, as its affinity for ligands appears to be so great that histidine residues of the unfolded protein tend to bind to it (Babul & Stellwagen, 1971), even in 9 M-urea, where the protein is otherwise f’ull~ unfolded (Myer. 1968; Stellwagen. 1968). Many complexities in the unfolding ot cytochromr c have been observed (e.g. Myer, 1968: Tsong, 1977). but. the available e\;idencr appears to be consistent with t.hem being perturbations of otherwise fully folded and fully unfolded states by the affinity for ligands of the hcme iron (e.g. Stellwagen & Babul, 1975), rather than additional conformational states determined by the prot,ein. The kinetics of unfolding and refolding of the oxidised form of tjlr(l protein are complex and not understood (e.g. Tsong, 1977), but this may also reflect t’he presence of the heme group, a s these complexities are not’ present \vith the reduced protein (McLendon & Smith. 1978). The unfolding of horse ferricytochrome c has generally been observed to occur under normal conditions with about, 7 M-urea (Myer, 1968; Stellwagen, 1968: Hcrskovits et ~1.. 1970: Tsong, 1975). Electrophoresis at pH 8.0 (Tris-acetat,e buffer) in urea-gradient’ gels produced no change of mobility until about. 7 or 8 -v-urea. but the unfolding transition was discontinuous, as expect.ed for slow unfolding (si(hfx Appendix). which was surprising as unfolding and refolding of t,his protein have generally been observed to occur rapidly (Tkai et nb., 1973; Tsong, 1976): the unfolded protein seemed to be aggregated, as it barely migrated in the gel. At pH 4.0, where the protein is only marginally stable in the absence of urea, unfolding was produced by lovver concentrations of urea and the transition was smooth and continuous (Fig. 6). consistent’ wit’h rapid int’erconversion of fully folded and fully unfolded st)at,es. However, t’he unfolded protein was oRen observed to split into a second. somewhat diffuse, band which migrated somewhat more rapidly a,nd appeared to refold onlv at slight’ly lower urea concentrations : only verp minor quantities of this species are visible in Figure 6. This phenornenon \vas not reproducible. and its moltscular origin and significance for folding are not clear. Very similar Curves were ob tamed Fvhen the protein was initially in 8 hr-urea, although there was generally a more alo\\-l~~ migrating band of presumably aggregated protein in t,he transitiotl region.

(h) Bovil,e .serum alhumirr The folded conformation of serum albumin is not known, but it appears to be a long ellipsoidal structure approximately 140 A long by 40 A4 wide (Bloomfield, 1966: Squire et nl., 1968), probably consisting of three globular domains, as indicated by fragmentation studies (e.g. McMenamy et al.: 1971: Peters & Feldhoff, 1975) and b> the covalent structures of the homologous buman and bovine prot.eins (Brown, 1976 : Saber ~1:n.1.. 1977) ; the 17 disulphide bonds link cysteine residues o&y within each of

248

Horse ferricytochrome

t

Bovine

OM

serum

albumin

> 8~

after the major transition, there was a significant, but, more gradual: decrrasc in tht, mobilit,irs of both monomer and dimer. The t,wo stages of unfolding may be a result of independent folding of the multiple domains of the folded protein, for which therf is some evidence (Teale & Benjamin, 1976.1977: Taylor L%Silver, 1976). or of stepwise disruption of any higher order struct,urc: produced by int,eracbions betwetn doma,ins. Identical pat.tcrns were obtained when t,he free thiol group had been react,& wit It iodoacet,amide. which indicates that rearrangements of the disulphide bonds hi, thiol-disulphide clxchange had not, accompanied t,hr urea-induced unfolding. Thih would suggest that unfolding in 8 M-urea is not complete, as intramolecular disulphide rearrangement should then be rapid under t,hese conditions (e.g. Creighton, 1977). Other studiw of urea denaturation of serum albumin (e.g. Kauzmann & Simpson. 1953 : Kauzmann & Douglas, 1956: McKenzie et al.. 1963; Taylor et al., 1975) haw also observed the primary conformational change ,s t)o be rapid. but’ disulphide intwchange to b(b slo\~ The remarkable complexity of t.hese patt,erns must, be due to intrinsic hctrrowneitv of t.he protein, which has previously been suspected (e.g. Kaplan & Fost,er. ‘9 ., 1971 : Hagtnmaier & Fost.er, 1971), hutf not, dctectetl tlirect.ly. The multiple forms of t h<> monomeric prot.ein are detected here only by t,heir slight,Ip different. suscrptihilities t,o unfolding bp urea. The microhet,erogencitis not limited to the state of thcl t hiol group of the free cysteine residue (cf. Fuller Noel & Huntw, 1972). nor is it. due to proteolytic cleavage in the cent#ral portion of the polypeptide (cf. Zurawski et al.. 1975). If it is dw t’o binding of diverse ligands (cf. Rosseneu-Motreff et al.. 1973). their Ibinding cannot bts disrupted by high conacntrations of urea. as most’ of the curv(‘s were continuous, indicating rapid equilibration of distinct foldetl and unfolded st,atcs. Hetcrogmcit’y in the primary structuw or diwulphidc bond pairings (Sogami et (cl., 1969) has not, bwn reporkd, but raw alterations at, a number of sites would not. be f,asily drt,cct.rd. In cont,rast. to the abrupt. unfolding transitions of individual species ohserved hcrc~. spectroscopic and viscosity measurements of t,hc unfolding of serum albumin prepara-

250

T. E. C’ltEl(:H’I’OS

t,ions by urea (Kauzmann & Simpson, 1953: Frendsdorff et al.; 19530; callaghan & Martin, 1982: McKenzie et al., 1963) and by GuHCl (Wallevik, 1973) have shown relatively gradual changes as a function of denaturant concentration. This is undoubtedly due, at least in part, to the microhet,erogeneity of the protein detected here, but there may also be other conformational transitions occurring which do not alter the electrophoretic mobility. (i) Bovine fi-lactoylohulin Bovine /3-lactoglobulin is generally isolat,ed as a mixture of two genetic variants, A and B, which differ in primary structure at two positions: variant A has Asp and Val at positions 64 and 118, respectively, while B has Gly and Ala (Braunitzer et al., 1973). The single polypeptide chains of 162 residues of both variants are linked b.y two disulphide bonds, but one of the pairings has been proposed to vary between either CyslO6 with Cysll9 or CpslO6 with Cys121. with Cysl21 or Cysll9, respectively, having a free thiol group (McKenzie> $ Khan. 1972). Further potent’ial complexities in behaviour of this protein are its trntlency t)o dimerize at neutral pH, to bind various ligands; and to undergo minor conformational transitions with varying pH (see McKenzie, 1967). The two genetic variants A and B have gmerallp been observed to have similar properties; their denat,uration by urea has been studied extensively (e.g. Kauzmann & Simpson, 1953; Pace & Tanford, 1968: Alexander & Pace, 1971; McKenzie 8: Ralston, 1973; Greene & Pace, 1974). At pH values between 2.6 and 3.5, both /3lactoglobulins completely unfold rapidly and reversibly in an apparently two-state transition at urea concent.rations of between 4 >I and 6 M. depending somewhat upon 1968: Alexander & Pace, 1971 : the pH and t,he t,emperature (Pace & Tanfi,rtl. McKenzie & Ralst,on, 1973). This behaviour has been confirmed by urea-gradient, electrophoresis at pH 4.0 (Fig. 8). where a’ major, smooth and continuous transition was observed at, about, 5 M to A hI-urea. Thtx tww variantIs A and B were only distinguishable hy a slight splitt,iny of t,llt, bawl at urea concentrat8ions \vhere unfolding is just, st,arting. has been obscrveci previously at neutral pH values More complex behaviour (Kauzmann & Simpson, 1953: McKenzie & Ralston, 1973) in that unfolding (and presumably refolding) was quite slo\v at urea concent)rations within t’he transition region (about 4 M-urea at pH 7; Kauzmann & Simpson, 1953)> but much faster at, both lower and higher urea concentrations. Complexity is also indicated by the ureagradient electrophoresis pat,terns obtained at pH 8% (Fig. 8). The presumed A and B variants are well-resolved at this pH and show. little variat,ion in mobility until about 3 &z-urea. The bands arr not cont,inuous throughout the transition regions; the discernible, although diffuse. bands in t,he transition region are continuous only with those of the fully unfolded protcain. Virtually identical results were obtained when the protein initially applied to t’he gel was in 8 br-urea. Discontinuous bands across the transition region are expected for slow folding t,ransitions, but the non-coincidence of the extrapolated bands of folded and unfolded (see Appendix). It does prot,eins is not, consistent wit,h simple two-stat,e behaviour seem clear that in t,he transit,ion region both unfolded proteins are in relativel-y rapid equilibrium with non-native. but compact,. forms of the prot,ein. which predominate at, the lower concentrations of urea. The nativcb folded prot,ein must. be only slowly unfolded and refolded tsithin the t,ransition region. h more drastic example of such behaviour is given by ovalbumin.

Urea

BF‘TI

R-loctoclobulin 6.6

Urea

pH

FIG. 8 Urea-gradient clcctrophoresis of fl-lactoglobulins I and B. The buffer on the left was 0.05 >I-Tris-acetat,e (pH 4.0), on the right (pH 8.6). In both instancw, the linear gradients of 0 nz to 8 ~-urea were superitnposcd on inverse gradients of 15% to 11% acrylamide. a-; internal standard in the pH 4.0 gradient; a faint band of contaminatmg a-lxtalbumin (c~xnparc with Fig. 3) is apparent in the pH

OM

Bovine

Tris-borate/EUTd BPTI was added 8.6 gel.

iklatuw ovalbumitt is a polypeptidt: chain of 385 residues (MeReynolds et al., 1978) which has been covalcntl.v modified aft,w syttthwis by proteolyt,ic removal of thr amino-t,erttlillal met~hionine and acctglatiott of t tt(b nc’u’ x-amino group (Palmitc~r it nl.. 1978), phosphorylation of t\\ o writw twiduw (Milst#ein. 1968), and attachment of carbohydrat,e to an asparaginc residue (Let: & Montgomery, 1962). Only two of the six cysDeint+ residues are believed t,o bc linked in a disulphide bond (Fot,hergill $ E‘othergill, 1970). Most, preparations of ovalbumin are heterogeneous dw t,o only partial phosphorylation of the two serines. the possible presence of two genetic variants (Wiseman e!t nl., 1972), and some further unknown alterations that occur with ageing (Ta,borsky, 1974). The folded conformat,ion of ovalbumin is not, known. but) it exhibit,s the normal properties of a monomeric globular protein (Taborsky. 1974). ‘l’hc many Audies of thrh unfolding and refolding of this protein have general]), observed wry complex behaviour, undoubtedly due in part, to t.he hrterogencit,y of’ t’he protein and also to a tendency of t.h(, unfolded protein to aggregate, ofiett accompanied by formation of intermolecular disulphide bonds. However. a recent st,udy of the unfolding tjransition produced by GuHCl of purified ovalbumin, wit,h two phosphate groups per molecule, observed wry simple behaviour e&rely consistent with a t,u’o-state model (Ahmad R- Salahutltlitt. 1976). It’ is clear from marty studies (e.g. Simpson & Kauztttantt, 1953: Scht~lltnatt it CL/.. 1953: F’rcnsdorff et (~1.. 1953a: McKenzie et ab., 1963) that, t.he unfolding of’ ovalbumitt is exceedingly slo\\ at room temperature. This was confirmed hy urea-gradient rlect.rophorcsis, as there \vas no rffwt of urea coneent,rat,ions of up t,o 8 M on t.hc elrctrophoretic mobility of any of thr prot,ein species present in t,he commtwial preparation (Fig. 9). However. if t,iw in 8 >I-urea at, FiU’C for 6 min. followed b> ovalbumin was denatured by incubation ~‘as drasticall? t LVOhours at) room tcmperaturr. hefore el~~~trophoresis. thtl Inobility alt~rred. This is illustrated on a single gel in Figure 9, where nativcl ovalbumin uas first migrated into the gel? followwl shortly by unfolded ovalbumin. The heterogeneit,y of both the folded and unfolded protjeins is apparent. The significant observat’ion is t,hat although t,hc mobility of the native protein was not altered by urea. due to its nrgligiblc cattl of unfolding. the mobiliticn of all the merging int,o a single unfolded species increased c&h decrrasing urea concent,ratiotw, diffuse band at. low ttwa concentrat,ions u.itlt a tttobilit,y approaching. hut not. equal to. t.ttat, of the tlat,i\-c protein. Each of the unfolded species gave continuous bands of compact. but non-natiw. forms of across thta gel, suggestSing rapid cqui1ibration the protein. Refolding to the init,ia,l stat.{, was not dt%rct~able under thwe conditions at, any urea concentrations.

4. Discussion (a) Electrophoretic

a~tu1ysi.s of’ ptnte.itl, zoa~oldiny

The alterations in clectrophoretA mobility t,hrough polyacrylamide gels Avhich accompany prot.ein unfolding must, he clw primarily to changes in t,he shape, or molecular volume, of the polypeptidr chain. but alterations in net charge of the

Ovolbumin

Unfolded

Folded

OM Urea

protein. e.g. by changes in exposure of ionizable groups or in bound ions, would also affert the electrophoretic mobility. The change of shape upon protein unfolding hah usually becrr measured by the viscosity (e.g. Tanford, 1968); t,hert: was a reasonabl) linear. c*orrtxlat,ion between the relat,ive viscosities of the folded and unfolded forms of the rm)trirl?reported by others and the rt~lativc mobilitics of the two forms of thtb and cr.-lacta~lbumin undergo only protein obswr~~l here. For example. ribonucleaw small incwascs in viscosity upon unfolding (Tanfortl, 191%: Sharrna & Bigelow. 19il). due to t>hcl wnstraint,s of four disulphide bonds in each cast, and also exhibited onl? .zp the relative shapes of intermediatw in ref(Ming of reduced pancreatic trypsin inhibitor and reduced ribonuclcase tjrappt:d by t#hrir disulphide hnds (Crcighton. 1978). Other hydrodynamic studies of prot,ein unfolding have usetl wdirnrnta~tion velocity (Holcornb 85 Van Holdr. 1962) and gel filtration (Halvorson bz Ackrrs. 19’73; Xrharya & Taniuchi, 1976.1977). Thaw rnrthods ha\,e t,he advantage‘ over riswsity rrrcasurement~s in giving some rnt~asuw of the dist,ribution of t hr. hytlrotlylarnic paramekr arnonpst t)he moleculrs. thus perrnitt,ing t,hc tletwtion of’

254

‘I’ . E . C’REIGH’1’09

slowly interconverted isomers, or of heterogeneit#J-, it1 the protein population. Heterogeneity of protein samples will affect all studies of protein folding, but the methods generally used do not detect it’. The primary advantages of polyacrylamide gel electrophoresis are its high resolut,ion and its ready compatibility with a continuous variation of denaturing conditions in the second dimension. The resulting t,wo-dimensional patt’erns yieltl important information about, the number of species present, may detect, het’erogeneit,!, of the> protein. even if the multiple species only differ slightly in the concentration of urea at which unfolding occurs (as exemplified by serum albumin (Fig. 7)), and give sonw insight into t,he rates of the folding transition, while using very small amounts of protein with a minimum of effort. The method is ideally suited for comparing t.htk conformational stabilities of closely related proteins, as they may btx t~xaminctl simultaneously on the same gel. The validity of the method is apparent from thtx consist’ency of the results obtained with ribonuclease, lysozymc, chymotryp~in. cytochrome c and staphylococcal nuclease \+ith those obtained previously by others using the normal methods. The apparent disadvantages of the method are that it gives no information on what type of conformation might, be present. t,he data obtained are not of a quanGtative nature. and the environment in \vhich the folding t,ransitions t,ake place must br compatible with polymerization of acrylamidc (e.g. no reducing agents) and with electrophoresis (e.g. low ionic strength and pH values dist,ant from t’he isoelect,ric point of the protein). The method is not compatible \vith folding transitions involving more than one component (e.g. more t)han otw polypcptith~ chain, cofactors. ctjc.). into t,hc- gel. I’nfolding b? unless the additional components may hf. incorporated GuHCl may not be studied in t,his way. as such electrolyte 9I interfere \vitti t)hc electrophoresis. but5 other uncharged denaturant’s. such a.< alcohols, etc. could conceivably be used. IJnfolding studies with urea run t,he well-known risk of rcact’ion of cyanatc with the protein (Stark rt al.. lS(i0). but, no widt~ncr of‘ this was observr~d. (b) Creu-iraduced

ut~foldit~q

of

proteins

obtained here with ribonucleaw. The urea-gradient elect,rophorcsis patterns st~apliylococcal nuclcasr and cytochrome c wew chymot’rypxin. chymotrypsinogen, entirely compatible with two-stat’e models of protein folding transitions : at, low uwa concentrations the protein migrated with the tnobilit)y of the folded Aate, at high urea concentrations as the unfolded state. At intermediate concentrations there were intermediate mobilities due t,o a presumed rapid equilibrium between the tI3.o states ; th(x resultant continuous curve of the protein band then reflects t)hc equilibrium proportions of t#he two species and was smooth wit,h only a single apparent infection point. The t’wo-state nature of the folding transitions of t,hrsc: small proteins has been wellestablished (see Privalov, 1974). Two-state folding transit,ions produce little information about, the pat,hways b) which proteins fold and unfold. so much effort has kx~~ expended to find folding transitions in which intermediate states are detectable. An example here might be serum albumin, in which for each of tho micro-species of this protein there was a major transition at) lower urea concent,rations and a more gradual change in mobility at, higher concentrations, and unfolding appeared t,o be incomplete. In this case, the nature of apparent, step-wise transitions may be due to the probable multi-domain the folded stat,e of this protein. X- Lactalbutnin has bwn cwncludetl t’o exhibit, three

USFC)Ll)TSG

OF

t’ROTElN8

BY

UltEB

“$5

stable defined states in the unfolding induced by acid and by GuHCl (Kuwajima et al.. 1976: Kuwajima, I977), as the circular dichroism at 220 nm is reported to change upon unfolding under somewhat different conditions than all other parameters of An intermediate state of intermediate conformation is the protein conformat’ion. claimed t,o be present at acid pH, at intermediate concentrations of GuHCl, and in 1977). Urea-gradient electrophoresis of Ethe fully rcduccd protein (Kuwajima, lactalbumin at acid pH and of the reduced protein gave no indication of urea-induced unfolding transit,ions, but at, high pH the unfolding transition was not abrupt as \\-ith thr other proteins (Fig. 3). which could conceivably be due to a complex tranhit!ion. l’ofortunately, analysis of this protein is difficult as it undergoes t,he smallest change in mobility upon unfolding, presumablv due t,o its four disulphide bonds. l’hta ~mplrx behaviour exhibited by the P-lact’oglobulins and by ovalbumin is probably tlw to slo\\. rates of refolding and will bc discussed below. li:\-cw if a t,hircl conformation st,ate, ot,hrr than nat’ive and fully unfolded. were to hi tlctwtc~tl in equilibrium populations. its significance for the folding transition woultl not tw obvious. as this can only br tleducetl from kinetic studies. In spit.t of t,his, tllcrc is an unfortunate tendency for a presumptive third conformational state tletc~ctrtl at intermediate denaturant’ concentrat’ions to be considered to have int’errnrtliate conformational propert#ies and to be an intermediate on the folding pathway. It is very natural to describe any st,tlp-wisp action of a denaturant in a temporal fashion ((1.~. “first’ partial unfolding to produce a partially-folded intermediat,tl, thllowtl 1)~ complete unfolding of the int~crmediatr“). implying a kinetic sequence of ckventq for nhich there is no r\:idence. ‘I’ht\ wcontl problem with any “intermediat’e” stat’es detected at equilibrium is t’hat the>, can only be detected at intermediate concentrations of denaturant’. so that their apparent stablht~les under such conditions might reflect the mechanism of action of the tltnaturant more than the conformational properties of the protein per se. The fol(lrd st.at.cls of proteins are finely balanced energetically relative to the unfolded states. and any specific interactions between the environment and the protein map t)ip the conformational balance. The detailed mechanisms of action of denaturants sue11 as urea and GuHCl are still the subject of debate, but it appears from studies on model compountls (Eozaki & Tanford. 1963,197O; Wetlaufer et al.: 1964; Roseman & .Joncl+. l!)i5) and on the refolding of reduced BPTI (Creighton, 1977) that both act similarly by solvat,ing more equally all portions of the unfolded polypeptide. increasing the aqueous solubilit’y of the hydrophobic portions \vhile maintaining t,he Ir~drogcn-bonding capability of t’he aqueous solvent (Roseman & Jencks, 1975). The twwgrtic~ of hydrophobic interactions may be estimated by an empirical relationship (22 t,o 24 cat/AZ) between the surface area buried and the free energy of transfer from wat,cr t,o organic solvents, approximat.ing the interior of t,he protein (Chothia, 1!+74: Richards. 1977). Such a relationship also holds for the free energy of transfer of amino acid side-chains from wat’er t,o 8 &l-urea or to 6 r\l-GuHCl (Nozaki $ Tanforcl, 1!163.19’70: irrtlaufer et al.. 1964), illust,rat,ed in Figure 10. Most, of the amino acid &k-chains in X &f-urea define a linear curve with a slope of 7.1 Cal/as, suggest,ing ttrat th(t iwrmal hydrophobic interaction in lvater of 22 to 24 Cal/AZ is decreased to 15 to I7 ral!W in s-urea, althdugh this onl,v holds for buried surfaces greater than in thrcsholtl size of about 50 A2. Side-chains with polar atoms lie on the same curvy. sugg&ing t’hat 8 M-urea is just as proficient as \vater in hydrogen bonding. There aw clxw~)tions. as illustrated with histidinr. and with models for the pol,ypeptid(L

256

T . E ., C’REIGH’I’OS

backbone (Nozaki & Tanford, 1963,197O) which may be due to more specific interactions with the solvent. A similar relationship holds for 6 M-GuHCl (Fig. 10): and the greater slope of the line (8.3 cal/A2) and the slightly smaller threshold value are consistent with its greater effect,iveness as a denaturant Amino acid side-chains with polar atoms lie below the linear curve, suggestming that 6 M-GuHCl is slightly 1~s proficient at hydrogen bonding t’han is water‘. These empirical relationships confirm t’hra general explanations of how urea and GuHCl unfold proteins, but it should be not,4 that, the hydrophobic interaction. as defined above, is not abolished in 8 M-urea or 6 YI-GuHCl. but merely reduced energetically by about one-third. Unfolded prot,clins in these solvents might not have equal interactions with the solvent, in all conformations. and would then not’ 1)~)

z

H,O -+

Hz0 -+6u-urea

6~GuHCI

1

50

100

150

Accessible

0 surface

50 aieo

100

150

( %* )

FIG. 10. Relationship bet,ween free energy of transfcsr from wat,er to 8 JI-urea 01’ 6 &I-GuHCI and accessible surface area for soveral amino acids de-chains. The free energies of transfer WOI’P taken from Nozaki & Tanford (1963,1970), the accessible *urfaw ~TC&S from Chothia (1976).

entirely random coils. It should be noted that 8 M-urea and 7 M-GuHCl do not disrupt the a-helical conformation of synt’hetic polymers of hydrophobic amino acids (Suer $ Doty, 1966). There is also ample opportunity for relatively specific int,eractions between hhe denaturants and the protein (Schellman, 1978) and it is even possible to envisage such denaturants inducing helical conformations in unfolded polypept’ides (Tiffany & Krimm, 1973). It would not be surprising t,o find some helical conformation in unfolded proteins in the presence of such denaturants. but, this \vould not necessarily imply a role for the helices in refolding of the prot,ein. (c) Unfolded proteins

under note-denaturing

The general co-operativity of protein folding stability of partially folded intermediates and the has led to attempts to accumulate significant, 1975). However, mediates kinetically (Baldwin,

conditions

transit’ions, as manifest in t#he intwo-state nature of the t,ransitions, concentrations of unstable interonly when two or more disulphide

~;IZ’FOLI)TK-G

01’

I’ROTEISS

RY

UREA

257

bonds a,rcaformed or broken during the transition have intermediates been characterized unequivocally, since t,he disulphide interaction may be controlled kinetically t,o maximizr t,heir accumulation (Creighton, 1978). The kinetic importance for the folding path\+,ay of any intermediate can only be demonstrat)ed directly if there is a lag period in appearance of the fully folded state. corresponding to t’he measurable t,imo for appearance of the int,ermediat,a. No such lag period has been detected in the r.efolding of any protein not, involving disulphide bond formation, so it’ must IX ~~n~lutlrtl that the intermediates in refolding (which must exist’) are either formed very rapidly upon adjust,ment, of the conditions to favour refolding, but’ unfold even tnorc rapidly. or they are formed by the rate-limiting step in refolding and t’hen complete refolding much more rapidlp. The former intermediat’es would occur on the, pat,hway hqforvthe rate-limiting step. the latter after it. The latter intermediates would br transient, variations of the folded state. and are manifest, in the dynamic fluct,uations of tht, folded protein. ‘1’11~former iut)ermediates are probably the rnorr important for making prot’cin rrfi)ltliny occur on a finit)c time-scale, but, t,hey are essent)ially microscopic sub-stat’cn of t,llr unfolded state and may never be populat’ed substantially, even under refolding conditiotls. The question then arises as to the nature of the unfolded state of proteins rmtlcar non-denaturing condibions. but thew is wry litt,le experimental data, as it is normally a transient species. t’ossibl(~ candidates for study are t,hr reduced forms of those proteins which norrnall~~ wquiw disulphide bonds for stability of the native state, such as BPTl, x-lactalburnin. ribonuclease and lysozyme. They appear to remain unfolded under Iron-(lf~!latllrinp conditions in t,he absence of disulphide bond formation (e.g. Creighton cutal.. 19%: Gawl, 1978). but, there are many reports t#hat t,heir spectral properties are not t~how of random coils under non-denaturing conditions, and it is usuall? wnclutlrd t,hat they have elements of ordered struct,ure (e.g. Yutani et al.. 1968: \Vhit~c~ 1976: Takahashi et al., 1977). Such spectral measurements are not unequivocal. hut f’vcn if valid. an alternative explanation fvould be that a sub-set of the marl) conformations of the unfolded prot,rin arc favoured under thesr conditions, but the>n-oultl at ill bcb t,ransient* and rapidly interconvert,ed. In this case, the spect,rum of cwnformations present would be expect,ed to shift, gradually t,o the more random st,at’cJupon addition of denat’urant, while one or more co-operative transitions would bc r~xpwtrtl if thwc were st,able ordered structure present (Aunr et al., 1967; Hollada> & Pnrtt. 1976). Urea-gradient gels of the reduced forms of BPTl. a-lactalbumin. ribonuclr~aw and lysozpme (u-ith no blocking groups on the thiol groups) showed no c~vitlrnce of any unfolding t’ransitions. At most) there was a gradual. but slight. tlrcwasf~ of tnohiiity wit8ti increasing urea concentration. The natural of the unfolded state untler refolding condition;; may also be examined iF thfs protein refolds very slo~l~~. Tllis appears to bc the case wit,h the p-lacto, t’llc meanh by which proteins refold, but in t,his case it could also be argued that, they are t’hc reason \vhy these two proteins refold relat,ively slowly. The roles of such species can only 1~ determined bp kin&c methods, ()tllor rapid c,hnracterizations of unfolded proteins under refolding conditions havcb

“5X

‘1’. E . (‘RElGH’L’Oh-

used electrophoretic mobility of thermally unfolded chymotrypsinogen at) O’C1 (Hawley & MacLeod, 1976) and nuclear magnetic resonance and trapping of exchangeable hydrogens in the slowly refolding form of ribonuclease (Blum et al., 1978; Schmid & Baldwin, 19783). Two electrophoretically distinguishable forms of unfolded chymot’rypsinogen were detected. so one maS have been relatively compact, but the possibility that t,hey reflect, het’erogeneity of the protein, as observed her<> (Fig. 4), does not appear to have been ruled out. The nuclear magnet’ic rrsonancc’ spcct,rum of the slowly refolding form of unfolded rihonuclease did not d&ct an? conformat,ional int’era&ions of the four hist,idine residues with parts of the protjrin distant in the covalent structure (Blum et al., 1978) but slowly exchangeable hydrogens have been trapped (Schmid & Baldwin, 19780). Marc data about the unfolded stat,es of proteins under non-denaturing condit,ions arca required ; this mar be possible with more proteins using urea-gradient gel electrophorrsis hv decreasing that duration of thr electrophoretic separation relative to thr rate of’ refolding.

APPENDLX Predicted urea-gradient varying rates

electrophoretic

patterns

for

two-date

folding

Zone transport of an isomerizing molecule. such as elect8rophoresis molecule unfolding and refolding in a two-stat,c transition,

tmrmitions

qf

of a prot,ein

has been treated theoretically by Scholten (1961), Cann & Bailey (1961), Van Holde (1962), Cann & Oates (1973) and Halvorson & Ackers (1974), but t’he most useful expressions treatment, appears to be that of Mitchell (1976a.). who derived analytical for the concentration of protein as a fun&on of distance migrated after a period of mobilities time, given the values of k, and k, and of the intrinsic electrophoret’ic and diffusion coefficients of the two species Ir and N, and applied them to the folding transition of chymotrypsinogen (Hawley & Mitchell, 1975). The protein profile as a function of dist,ance for one set of conditions then corresponds to the electrophoretic pattern expected at a single urea concentratjion, i.c>. along a vertical line in a urea gradient gel. Simulation of a two-dimensional ureagradient gel requires calculation of a number of such profiles with different urea concentrations. Only the values of k, and k, were t,aken to change across the gel, both varying exponentially with the urea concentration (Fig. 11(a)), as is usually observed experimentally. Only t’he transition region was analyzed. with 100-fold variations of both k, and k,, to produce a 104-fold variation of t#he equilibrium constant for the folding transition, so that the fraction of both U and N expected at equilibrium varied from 1 o/o to 99yb (Fig. 11(b)). !rhe predicted protein profiles at 20 evenly spaced urea concentrations were t’hen calculated using equation 46a of Mitchell (1976a), to produce the two-dimensional contour maps of Figure 12, which should approximate the two-dimensional urea gels described in the text in the region of the folding transition, i.e. an elect,rophoresis time of 200 minut’es, electjrophoret’ic

c+2 Ia)

kN

I‘c E

,

-2 t’ m 4emIY\ c

/’

/’

/’

/’

/’

/’

/’ _<-----

/’

/’ ’ k”

_---------

/’

/

---

log(p/2)

/’

’

f”

3

arc:. 11. Illustration of assumed dependence of the kinetics of folding and unfolding as a function of the concentration of urea. In (a), the logarithms of the rat)? constants for folding (12~) and nnfolding (k,) are plotted as a function of t,he linear concent,ration of nrca, showing the presumed dependence of both rate constants. Only the (unspecified) urea concent,rat,ion range in which the values of both rate constant,s vary 10%fold around that concentration at which they are equal is shown. The absolute values of t,he rate constant,s were taken to vary, as indicated by the parameter (!. The minimum value of t,ho apparent rate constant for t’he folding transition (fig: L k, $- !i,. where k, -: k,) is indicated.

U’hatever the value of C, the relative values of k, and k, in (a) define the 104-fold depondenw of the equilibrium constant K shown in (b). This then defines t,hr indicated which arcs unfolded at equilibrium, fU.

fraction

of 0.1 and 0.2 mm per minute for U and N, respectively. and coefficient of 3.3 _. lo- 7 cm2 per second for both C and N. The rates at, which the unfolding and refolding transitions t’ake place when the mean respectively) determine t’he electrophoretic patterns either U or N is ueit’her very much greater nor smaller than the time of phoretic separation. The apparent rak constant (k,,,) of a unimolecular is given by k itpp = k, + k,. Inobilities

of molecules

a diffusion (k, and k,, lifet’ime of the electrotransition

folding transition, which is at a minimum (kF$‘) at th e midpoint of the equilibrium k, =mk,. The rates of unfolding and refolding were found to affect the electrophoretic patterns most conspicuously when kF&’ ranged from 0.1 to 10 times tht inverse of the elcctrophoresis time. Representative profiles are presented in Figure 12. when eit’her the fully folded or fully unfolded proteins were assumed to be applied to the urea gradient gels. With very rapid rates of unfolding and refolding t’hroughout the transition region. the widt’h of t,he protein band is de&mined by diffusion, the band should follow t,he equilibrium distribution (with the average mobility determined bp the fract,ion of the time spent in the two states), and the same profile should be obtained st,art)ing wit#h either fully folded or fully unfolded protein. AR the rate of the folding transibion clecreases. t,he width of the band should increase in the transition region, from which where

260

I’ . I3 . CREIGHTON Native

“-----

Unfolded

..__....,.. ... ... .. ....

-____

N

-----

- - - _

-------~_

.I..____..... ,..........

k Fi”, = 0.04 U------

min-1 ----

-,.,!

_,,_.._. I... .......,....

,,.......................

..I

..-.

. . . . . . ...*.....

k rk =O*Ol min-1 ” _ I I _ I _ _ _ -,,, ~ .,_,. =.--“‘= .................“““““‘~ ;,~,,‘,. ,,e

,,....‘:” N

,)...Y

,_../’

_.’ ,.: ,..’ ,..’..’ /

/

/

/

/ ,..’,/

........__..________.....~.................... ....

- -I-

,._..‘~ ..’ ,_.’ ...’ ,_.’ - - -

-

_

_

I . . .

~~ ~-.~.,5~..~,

,.-‘. /

/

/

,_.’ ,:’ ..I. .i.’

” , 1’ ’ _,,.,: .’ ---iiis2 ,,._.......__.,,,.......,,,...... ..“‘. ..-. <.: - - - - - - - -

* [Urea] Fro. 1%. Simulated urea-gradient gel clcctrophoresis patterns expected for two-state bchaviour for t,he range of rata constants of folding ant1 unfolding in which the kinetics affect t,he elcckophoretic pattern. The expected profiles at each concentration of urea were calculatotl using quat8icm 46a of Mitchell (1976a). Native, folded protein (K) was present initially for t,he p&terns on thr, left.. fully unfolded protein (U) on the right. The dependence of the rate constantjs for refolding (kN) and unfolding (k,) on the concentration of urea was that. illustrated in Fig. 11. The absoluk values of both rate constants were varied to give t)he indicated minimum values of the apparent rate constant. for the folding transition (kN + k,) at the midpoint of tQe transition. All of the elect,rophoret’ic patterns arc t.hose expcctecl after electrnphoresis for X09 min for a {U) form have eloctrophoretic mobilities of 0.2 protein of which t,he folded (X) and unfolded cnettioient. of mm/min and O-1. mm/mm, respectively, and both have% an c?ffe,ct.ivt: diffusion 3-3 x 10m7 cm2/s.

_

UNEOLDING

01’ PROTEISS

BY

UKE.4

261

the rate of folding may in theory be inferred (Mitchell, 1976%). With k$‘i: =-: 0.1 min-‘, the tram&ion region is quite diffuse, but the pattern is symmetrical and the same for both init’ial folded and unfolded protein (Fig. 12). With kzj: = 0.04 min-I. the transition zone is more diffuse, becomes somewhat unsymmetrical, and differs slightly when starting with unfolded or folded prot,ein. With k&$ = 0.02 min-l, a spur due to residual original protein appears in both cases, and the folded and unfolded protein give quite different, but nearly complementa.ry, patterns. When k:i,” : 041 min-I, prot’ein of intermediat’e mobility is virtually undetectable and t,hc original protein predominates. With even smaller values of kF$, this t,endency increases. until only the original protein is detect,able, there being negligible folding or unfolding during the electrophoresis. It should be not’ed that where a band of protein of intermediate mobility is detectable, it. follows very closely the expected equilibrium average mobility. Miss .Denisc Thomas mastered the art of preparing urea-gradient gels and performed lnost of tile experiments reported here. K. L. Baldwin provided constructive criticism of the manuscript., and S. A. Hawley kindly answered questions about the calculatio~lx r(tport,otl iI1 Figure 12. Bayer AC: kindly provided t,he BPTI (Tra,sylol ‘$).

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