refolding of bovine pancreatic trypsin inhibitor via non-native disulphide intermediates

J. Mol. Biol. (1995) 249, 463-477

. IMB Refolding of Bovine Pancreatic Trypsin Inhibitor via Non-native Disulphide Intermediates

Nigel J. Darby, Paul E. Morin, Gert Talbo and Thomas E. Creighton*

European Molecular Biology Laboratory, Meyerhofstr. 1 69012 Heidelberg, Germany

*Corresponding author

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species.

The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30--51, 5--14) and (30--51, 5--38). Moreover, the rates of folding through each of these it~termediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathwa)~ and their roles are readily explicable on the basis of their conformations.

Keywords: protein folding; BPTI; disulphide bonds; cysteine residues; protein engineering

Present addresses: Paul E. Morin, Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Ontario, Canada; Gert Talbo, Howard Florey Institute, University of Melbourne, Parkville, Victoria 3052, Australia.

Abbreviations used: BPTI, bovine pancreatic trypsin inhibitor; BPTIR~2, native BPTI produced in Escherichia coli with Met52 replaced by Arg; all the forms of BPTI studied here have this replacement. The disulphide intermediates of BPTI and BPTIR~2 are designated by the residue numbers of tile cysteine residues paired in disulphide bonds. Those forms in which Cysl4 or Cys38 have been replaced by Ser are designated by the superscript "14OH" and the subscript "38OH", respectively. When all tile cysteine residues not paired in disulphide bonds have been replaced by Ser, the subscript "Ser" is used. Disulphide species with the native conformation are designated by the subscript "N"; DT'I'~II and DTT~s, dithiothreitol and its disulphide form, respectively; GSH and GSSG, the thiol and disulphide forms of glutathione, respectively; HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MALDI PSD, MALDI post source decay; NS•, the (30~51, 5--55)

N3soH disulphide intermediate of BPTI and BPTIR52; ~4sH and 1401"I N~s,, tile equivalent forms of NsN in BPTIR,~2 in which

Cys38 and Cys14, respectively, have been replaced by Ser residues; R, reduced BPTI; R t4°" and R_~o,, R in which Cys14 and Cys38, respectively, have been replaced by Ser residues.

Introduction One of the most extensively characterized protein

folding transitions is that of bovine pancreatic trypsin inhibitor (BPTI) coupled to formation of its three disulphide bonds (Creighton, 1978, 1985, 1990, 1992c), summarized in Figure 1. The conformational basis of this pathway is now largely understood, as the three-dimensional structures of the native protein and all tile most important intermediates have been characterized in detail (Kosen et al., 1983; Wlodawer et al., 1984; States et al., 1987; Darby et al., 1992,1993; van Mierlo et al., 1991 a,b, 1992, 1993,1994; Hurle et al., 1992; Kemmink & Creighton, 1993).

Reduced BPTI is largely unfolded, although with local elements of non-random conformation (Kemmink & Creighton, 1993). Consequentl~ it forms a first disulphide bond between any of the 15 possible pairs of the six cysteine residues on a nearly random basis, only varying approximately by the distance between the pair of residues (Darby & Creighton, 1993). The resulting one-disulphide intermediates are in relatively rapid equilibrium by intramolecular thiol-disulphide interchange. They are largely unfolded (Darby et al., 1992), except for the important intermediate (30--51) with a native disulphide bond. This intermediate has a partly- folded conformation in which about two thirds of the polypeptide chain has a native-like conformation

0022-2836/95/220463-15 $08.00/0 ~ 1995 Academic Press Limited

464 Refolding of Bovine Pancreatic Trypsin Inhibitor

Others

SH SH

H <~==C:>" S~H

I ~ S (30-51) HS H

C Reduced

SH

(30-51, 5-38) <:1-===[2> +

S

\ (30-51,5-14)

H (30-51, 14-38)

c (30-51, 5-55) (30-51, 5-55, 14-38)

Native

Figure 1. The productive disulphide folding pathway of BPTI. The major disulphide intermediates at pH 8.7 and their approximate conformations are indicated schematically The non-productive quasi-native species (5--55, 14----38)N is omitted. The relative rates of the most prominent steps are indicated semi-quantitatively by the thickness of the appropriate arrowhead; the wider the arrowhead, the greater the rate in that direction. The rates of disulphide formation refer to the value of k,,, ....

and the remainder is unfolded or very flexible (van Mierlo et al., 1992, 1993). The partly-folded conformation of this intermediate accounts for its lower free energy and predominance amongst the one-disulphide intermediates and for its tendency to form any of the three possible disulphide bonds between the three cysteine residues that are in the unfolded or flexible regions: Cys5, Cys14 and Cys38. Of the resulting two-disulphide intermediates, (30--51,5--14), (30--51, 5--38) and (30--51, 1~1 38)N, the first two have non-native second disulphide bonds. Refolding is completed most readily under normal experimental conditions by intramolecular rearrangement of the non-native disulphide bonds of these intermediates to the native-like (30--51, 5--55),, also designated as NsH. This intermediate can rapidly form the third 14---38 disulphide bond, to complete refolding. Unfolding of native BPTI upon reducing tile disulphide bonds under tile same conditions occurs by the reverse of this pathway (Figure 1).

In addition to the intermediates (30--51), (30--51, 5--14) and (30--51, 5--38), which are all partly folded, other intermediates containing solely native disulphide bonds accumulate primarily because they tend to adopt the native conformation, which is designated by tile subscript "N". The folded colfformation of native BPTI is so stable

(Vincent et al., 1971) that it is still populated when any one of the native disulphide bonds is missing (States et al., 1984; Eigenbrot et al., 1990, 1992; van Mierlo et al., 1991a). The fully folded conformation is even detectable when only tile 5--55 disulphide bond, which stabilizes the native conformation most (Creighton & Goldenberg, 1984), is present (vall Mierlo et al., 1991b). While of interest to protein stability the accumulation of these quasi-native states is not directly relevant to protein folding when disulphide bond formation in them is not coupled to folding. The occurrence of such quasi-native states can be minimized by studying disulphide bond formation at relatively high pH, where disulphide formation is more closely coupled to folding because the thiol groups tend to ionize, decreasing their tendency to be buried in folded structures. Tile productive pathway is the same as at lower pH, but is more efficient and not obscured by the predominance of the quasi-native states.

Tile BPTI folding pathway has been re-investi- gated by Weissman & Kim (1991, 1992b), using alternative methods to trap and to separate tile disulphide intermediates, and at a lower pH where quasi-native species tend to predominate. Their results are consistent with the pathway as originally proposed (Creighton & Goldenberg, 1984; Creighton, 1990, 1992a,b), but it was concluded

Refolding of Bovine Pancreatic Trypsin Inhibitor 465

(Weissman & Kim, 1991, 1992a; Hoffman, 1991) that the pathway should be revised to:

(30--51) ~ (30--51,14--38)N,, N

N (1) - . a

(5--55)N --~ (5--55,14---38)~ 7

The quasi-native states (30--51, 14--38)N and (5--55, 14--38)~ were depicted as obligatory kinetic intermediates. All non-native disulphide intermediates, especially (30--51, 5--14) and (30--51, 5--38), were omitted, even though they were identified by Weissman & Kim (1991) as being present during folding, albeit at somewhat lower levels than previously reported, and were observed to have the kinetic properties expected from the original pathway shown in Figure 1 (Weissman & Kim, 1992b). Non-native disulphide intermediates are a chemical necessity in the intramolecular rearrangement of native disulphide bonds, as occurs in both the original pathway and in that of Weissman & Kim (1991, 1992b), so any such pathway is incomplete without them.

The initial results with the BPTI pathway (Creighton, 1977a, 1978) indicated that a complex kinetic pathway cannot be inferred just on the basis of the levels of accumulation of the intermediates. A species will tend to accumulate to high levels if it is stable and if its further progression along the pathway is slow. An intermediate can be crucial for a reaction, yet not accumulate to a significant extent because it is unstable or because it rapidly progresses further along the pathway. Fortunately, the existence of such crucial but ephemeral intermediates can be detected in disulphide folding pathways by examining the effects of removing the various cysteine thiol group (Creighton, 1977a). For that reason, the rates of disulphide formation were measured in the reduced forms of analogues of the one-disulphide intermediates in which the four free cysteine residues were replaced by serine or alanine residues; they were shown to form individual disulphide bonds at about the expected rates (Darby & Creighton, 1993; Kosen et a/., 1992). Kosen et al. (1992) also studied the equivalent analogue of (5--55, 14--38)x. The two-disulphide intermediates (30--51, 1,1 38)~.r.N, (30--51, 5--38)S,, and (30--51, 5--38)~., with only four cysteine residues and two introduced serine residues, are now examined in the same way.

The roles and disulphide rearrangements of the two-disulphide intermediates with non-native second disulphide bonds, (30--51, 5--14) and (30--51, 5--38), are characterized further by replacing either Cys14 or Cys38. Having a free Cys55 thiol group permits them to rearrange intramolecularly to N~'d, as they are proposed to do in the normal pathway (Figure 1). An important role of the intermediates with non-native second disulphide bonds is their direct formation from (30--51), by-passing the quasi-native (30--51, 14---38)N (Figure 1). This role was indicated initially by the ability of reduced BPTI to refold to the equivalent of N~H when either, but not both, of the Cys14 or Cys38 thiol groups were

irreversibly blocked (Creighton, 1977a). In neither case can the initial two-disulphide intermediates with only native disulphide bonds, (30--51, 14--38)N and (5--55, 14----38)N, be formed, and folding must occur primarily through direct formation of non- native second disulphide bonds. The collective rate of folding of these two derivatives was not greatly diminished from that of normal BPTI (Creighton, 1977a), indicating that the predominant, most favoured kinetic pathway occurred as:

R --, others + (30--51) --, (30--51,5--14)

or (30--51,5--38) --* N~'.I --, N, (2)

by-passing (30--51, 14---38),x and (5--55, 14--38)x. This observation demonstrates that the latter two species are not important kinetic intermediates in BPTI folding, because folding and disulphide formation occur at nearly the normal rate in the absence of both of them (Creighton, 1977a). The pathway described by Weissman & Kim (1991,1992b) is incompatible with this observation, for folding by that pathway should be blocked completely in the absence of both (30--51,14--38)x and (5--55, 14---38),x (see equation (1)).

The objection could be raised to the above experiments that the blocking groups added to Cysl 4 and Cys38 accounted for their rapid rates of refolding. Also, the refolding kinetics of these chemically blocked proteins had not been characterized in detail previously (Creighton, 1977a), and the occurrence of the quasi-native (5--55, 14--38)x had not been recognized at that time (States et al., 1984). For these reasons, and because of the importance of these observations for defining the BPTI folding pathway, the refolding of BPTI in the absence of either Cysl4 or Cys38 has been characterized in greater detail, using more modern methods and engineered proteins in which Cysl4 and Cys38 have been replaced by Ser residues to avoid the necessity of the blocking groups.

The results presented here demonstrate the validity of the original disulphide folding pathway of BPTI (Figure 1) and show directly that the two-disulphide intermediates with non-native disulphide bonds are important kinetic intermediates occurring during disulphide formation in essentially all the molecules and are amongst the most productive kinetic intermediates.

Results

Disulphide formation in reduced analogues of the two-disulphide intermediates

The reduced forms of tile intermediate analogues (30--51, 14 38)S,.r.N, (30--51, 5--14)S,.r and (30--51, 5--38),,,., have only four cysteine residues each and therefore call form only six different disulphide bonds, rather than the fifteen possible with six cysteine residues of normal BPTI. As initial disulphide formation is essentially random, the observed rates of forming the first disulphide bonds

4 6 6 Refolding of Bovine Pancreatic Trypsin Inhibitor

T a b l e 1

Rate and equilibrium constants for disulphide formation and reduction in normal BPTI and in analogues of the two-disulphide intermediates with dithiothreitol as the reagent

kt' k~ b k,,,~' Form of BPTI (s -I M -i) (s -1M-') (s-') K,d

A. BPTI~:~:" R *--. I' 0.027 24 1.4 1.1 x 10 -3 I ~ IIa 0.032 250 1.6 1.3 x 10-*

B. (30--51, 14--38)s+, R~o. *--, I 0.017 42 0.85 4.0 x 10-* l *-* (30--51, 14--38)s~, 0.015 175 0.75 8.6 x 10 -5

C. (30--51,5--14)_~, R~so. *'-* I 0.014 15 0.7 9.3 x 10-* I ~ (30--51, 5--14)_~, 0.004 30 0.2 1.3 x 10-*

D. (30--51, 5--38)~., 14OH f R ~ . ~ I 0.008 25 0.4 3.2 x 10-*

I *--, (30--51, 5---38)s,., 0.005 60 0.2 8.3 x 10 -5 The rate and equilibrium constants were obtained by simulation of kinetic

and equilibrium experiments of disulphide formation and breakage with varying concentrations of the appropriate thiol and disulphide reagents. All measurements were made at pH 8.7 and 25°C.

The rate constant for forming the disulphide bond by reaction with DTT~s. b The rate constant for breaking the disulphide bond by reaction with DTT~II. ' The intramolecular rate constant for forming the protein disulphide bond

from the mixed-disulphide intermediate (see eqn (3)), calculated from k,. d The apparent equilibrium constant for the reaction ( = k~/k,). e Taken from Darby & Creighton (1993). t The collection of all the one-disulphide intermediates. g The two-disulphide precursors of NS~i, primarily (30--51, 14----38)N, (30-~51,

5----14) and (30--51, ~-38).

would be expected to be decreased s o m e w h a t in the four-cysteine ana logues relative to normal BPTI (see Table 2). The normal p r e d o m i n a n t in termediate (30--51) is still possible, bu t each one-d isu lphide intermediate can form only a single second di- su lphide bond. If all the BPTI molecules follow the pa thway shown in Figure 1, forming the (30--51, 14----38)N, (30----51, 5--14) , and (30--51, 5- -38) intermediates directl)4 the combined rates of forming the second d isu lphide bonds in the reduced ana logues would be expected to be the same as the overall rate of the co r respond ing step observed wi th normal BPTI. If in the normal protein they were fo rmed only by in t ramolecular d i su lph ide rea r rangements of other two-d i su lph ide intermediates , they could not be formed in this way in the four-cysteine analogues .

The kinetics of protein d isu lphide b o n d format ion with DTT~s as the d i su lph ide reagent are mos t relevant to protein folding, for the observed rate of each step is then direct ly propor t iona l to the rate constant of the in t ramolecular step, k~ ...... wh ich involves conformat ional t ransi t ions in the p o l y p e p - tide chain (Creighton, 1975b):

S 2K DVr ps. { sH + DTT ~ > PslS-/s DTTsll

k,,b, = 2KDrrki,,r,

kmlra S

, P 1+ DTT~s]~]

(3)

The observed rate constant is direct ly propor t iona l to k~,, .... In the presence of 40 m M DTT~s and va ry ing concentrat ions of DTT~sH", each of the four-cysteine reduced forms formed one-d isu lphide species wi th

about the expected rates and stabilities (Table 1). Second d i su lph ide bonds were fo rmed more slowl)~ and only in the absence of added DTT~s H. They are m u c h less stable than the first d i su lph ide bonds and were rap id ly r educed by even the small quanti t ies of DTT~sH" genera ted by the reaction of fo rming the prote in d i su lph ide bonds. This was conf i rmed by measu r ing direct ly the rates of reduc t ion of the isolated two-d i su lph ide forms by DTT~s~. These rate measu remen t s were used in s imula t ing the disulphide format ion exper iments to obta in the rate and equi l ibr ium constants given in Table 1.

In each of the four-cyste ine analogues , the rates of fo rming the first d i su lph ide bonds were decreased about two- to three-fold relative to normal r educed BPTI (Table 2), and the rates of reduct ion of the one-d isu lphide intermediates were not al tered markedl)4 as expected. Of the second d i su lph ide bonds , 14--38 was formed more rapidly than 5 - - 1 4 and 5- -38 , bu t it was also r educed more rapidly (kr = 175 s -~ M-~). This rapid rate of reduc t ion of the 14---38 d i su lph ide b o n d was not un ique to the analogue , for it is also apparen t dur ing d isu lphide- folding of the normal protein, w h e r e the collective rate constant for all second d i su lph ide bonds is 250 s-' M -~ (Table 1). It was also conf i rmed wi th the ac id- t rapped (30--51, 14----38)N of the normal protein, whe re k, was measu red to be 170 s -~ M -~ (data not shown). The rate constant for reduc ing the 14---38 d i su lph ide b o n d has a normal va lue of about 25 s -~ M -~ in native, th ree-d isu lph ide BPTI and in quasi-nat ive (5--55, 14----38)N (Creighton &


Table 2

Expected and observed relative rates of forming the first disulphide bonds in reduced BPTI with various cysteine residues replaced by serines Form of Rate relative of that of normal R-BPTI BPTP Expected b Observed ~ R 1 1 R~mH 0.60 0.63

14Oil RS~OH 0.56 0.30 38OH Rs~o, 0.39 0.52

R ~°" 0.63 0.52 R~,~, 0.44 0.52

The rates of forming the first disulphide bonds were measured using DT~ as the disulphide reagent at pH 8.7 and 25°C.

The cysteine residues replaced by serine ("OH") are indicated by the super- and subscripts.

b Rate relative to that in normal R-BPTI expected if each pair of cysteine residues forms a disulphide bond at a rate proportional to n ~/2 where n is the number of residues between the 2 cysteine residues (Chan& Dill, 1991); this relationship appeared to hold for the single-disulphide analogues (Darby & Creighton, 1993). The value used for the exceptional strained disulphide bond between Cys51 and Cys55 was that measured (Darby & Creighton, 1993) rather than calculated.

'The relative values of the observed rate constant for R ~ I (Tables 1 and 3).

Goldenberg, 1984; Kosen et al., 1992), so the rate is nearly tenfold greater in both the intermediate (3(3---51, 14 38)N and in the analogue (30--51, 14----38)s~r.N. The similar observations with the analogue, where the thiol groups are not present, indicate that this increased rate is not due to the presence of two ionized, destabilizing thiol groups in the normal intermediate; this was confirmed by finding the same tenfold increase in rate at the lower pH of 7.7, where the thiol groups should not be ionized (data not shown). Although the (30--51, 14--38)s~r,N analogue is very native-like in its structure (van Mierlo et al., 1991a), the 14 38 disulphide bond seems from its reactivity to be somewhat strained.

The sum of the rate constants for forming the (30--51, 14---38)N, (30--51, 5--14) and (30--51, 5--38) species directly from the one-disulphide intermediates (0.024s q M -I) accounts for 75% of the observed rate with BPTIR.~2 (0.032 s-' M-I); this indicates that a corresponding fraction of normal BPTI molecules fold by forming these species directly by the pathway shown in Figure 1. If the discrepancy is real, it would indicate a kinetic contribution of formation of other second disulphide bonds and rapid intramolecular rearrangements of the resulting two-disulphide intermediates.

Disulphide-coupled folding in the absence of Cys14 or Cys38

Replacing either Cys14 or Cys38 of BPTI by Ser residues, or irreversibly modifying their thiol groups (Creighton, 1977a), eliminates all those normal folding intermediates that have these cysteine residues involved in disulphide bonds. The various disulphide forms of these altered proteins will be

designated here by superscript 14OH or subscript 38OH to indicate the serine residue introduced. With only five cysteine residues in R 14°" and R3m., only ten one-disulphide species may be generated in each, rather than the normal fifteen, but the predominant and productive (30--51) is still possible in each case. At the two-disulphide stage, the predominant quasi-native intermediates (30---51, 14---38)N and (5--55, 14 38)N are not possible, thereby greatly simplifying the observed folding process. The normally-occurring two-disulphide intermediates that are possible are the non-native intermediates (30--51,5---14)38o. and (30--51,5--38) 14°" , plus the corresponding forms of the native-like (30--51, 5--55)N, which are designated .~'114s",~o. and N,O. in the altered proteins. This is the most fully 38SH folded and most stable species that is possible in the absence of the 14---38 disulphide bond, and it is the end-point of folding in these modified forms of BPTI. The observed kinetics of disulphide bond formation and folding in the absence of these cysteine residues make it possible to dissect the complex process of folding to NsS. " into some of its individual steps, revealing the roles of the few species that are present (Creighton, 1977a).

14OH Each of the engineered reduced proteins R and R,~OH, with DTT~s as the disulphide reagent (equation (3)), formed one-disulphide intermediates at close to the expected rates (Table 2) with similar stabilities (Table 3). Most importantl)4 each also formed the equivalent of NSs. " at an overall rate not much less than that observed with the normal reduced protein. No other two-disulphide species accumu- lated to substantial levels in these experiments, as expected. Species NSH " and its equivalents are not formed directly at a significant rate (Creighton, 1977a; Goldenberg, 1988), so the kinetic scheme for refolding of both proteins is typified by that for R~OH:

R~OH *-+ I3soH ~ Tivoli --+ NII4SH (4) .L ~-~OH,

where I3so. and II~o. are the one- and two-disulphide intermediates formed initially The latter do not accumulate to significant levels during refolding with DTT~s, as they are reduced back to I~OH by any DTT~sH" present (Table 1), and they are formed more slowly

~,V4SH Consequentljg these than they rearrange to "'.~OH. kinetic data do not give directly the rate constants for formation and reduction of II~oH. The forward rate constant was, however, defined by the data once the rates of rearrangement of the specific intermediates

~,114SH (see below) were determined. The results to .,38OH obtained were virtually identical to those obtained previously with the corresponding species with chemically blocked thiol groups (Creighton, 1977a). Goldenberg (1988) and Kosen et al. (1992) have also noted that the presence of blocking groups has no substantial effects on the kinetics of BPTI disulphide folding.

The two-disulphide intermediates in BPTI refolding accumulate to substantial levels when moderate concentrations of the disulphide form of glutathione (GSSG) are used as the disulphide reagent, in the absence of added reduced glutathione (GSH).


Table 3

Rate constants for disulphide bond formation in reduced BPTI.~2 in which Cys14 or Cys38 have been replaced by Ser

Combined Step R R 14°It R.;,++~. i pathway ~

A. R + D T T ; ~ 1 + D T T ~ ] Forward (s ' M ~) (1.027 0.014 0.014 0.028" Reverse (s ' M ') 24 30 30 30 ~

B. I + D T T " ~ 11 + D T T ~ Forward (s ' M ~) 0.032 0.005 0.007 0.012 d Reverse (s ~ M ') 25/) 60 30 45'

c . H ~ N'II Forward (s ~) 0.005 0.034" 0.013 ~ 0.024¢

R is the reduced protein in which all cysteine res idues are in the thiol form; I is the collection of one-disulphide intermediates, which are in relatively rapid equil ibrium, with (30--51) predominat ing; 11 are the initial two-disulphide intermediates that are in relatively rapid equil ibrium, predominant ly (30--51, 14--38)x, (30--51 ,5- -14) and (30--51 ,5- -38) with normal BPTI. With the Serl4 and Ser38 proteins, the intermediates in which the replaced cysteine residues would be in d isu lphide bonds cannot occur.

The rate constants expected if the protein can refold v& the paths followed by both R '4''" and R~,,,. This includes reactions through both (30--51, 5--141 and (30--51,5--38) , but excludes all species with a 14--38 disulphide bond, especially (30--51, 14~381x and (5--55, 14--381x. The rates of all the s teps are averaged, except for those involved in forming the second disulphide bonds, which are mutual ly exclusive with the 2 proteins and consequent ly are s u m m e d .

b The average rate in R "°" and R~,,,. corrected for the absence of the replaced cysteine residue (Table 2).

• The average of the rate constants with R ~4'''' and R~,,,,. J The sum of the rates with R 'a'" and R~<,,,.

Rate measured directly with the isolated acid-trapped ( 3 0 - - 5 1 , 5 - - 3 8 ) " " ' and (30--51, 5--14),~,, . These values are considerably greater than the apparent value measured with normal R, because species II then includes (30--51, 14--38), which does not rearrange directly to NS]ll.

This disulphide reagent reacts relatively rapidly with the protein thiols, and this is the rate-limiting step in forming those disulphide bonds that are made readily. Consequently, moderate amounts of GSSG cause first and second disulphide bonds to be formed rapidly in reduced BPTI, at about the same rate and almost irreversibl~; so the two-disulphide intermediates accumulate to substantial levels (Figure 2). In addition, cysteine residues that cannot readily form protein disulphide bonds tend to accumulate as the mixed-disulphide with the reagent. The actual levels of the intermediates under these conditions are of only indirect relevance to the folding mechanism, because many of the rates are determined by the chemical reaction between protein thiols and the disulphide reagent, but this makes it possible to detect, identify and characterize the intermediates.

In this wa~ it will be shown that ll~.~o, is primarily (30--51, 5--14)~,OH (see below) and II '~°" is primarily (30--51, 5--38) L4°" (see below). Only small amounts of two-disulphide intermediates other than (30--51, 5--14) and (30---51, 5--38) were detected in these experiments. The rates of rearrangement of these specific intermediates could be measured directly using the acid-trapped species (Table 3). With these rate constants, it was possible to simulate the observed rates at which N t4~ql~,.~ and N ~''~.~,~ were generated from their reduced forms using DTT2~ as the reagent, to obtain all the pertinent rate constants (Table 3). The rate constants for forming the first and

second disulphide bonds are in good agreement with those measured in the species with just four cysteine residues (Table 11, as expected.

Characterization of the disulphide species generated during the refolding of R38o.

The profile of intermediates obtained when GSSG was the disulphide reagent (Figure 2) was more complex than with DTT s, as expected, because two-disulphide and mixed-disulphide species accumulate to significant levels. The intermediates were identified on the basis of their kinetic and other properties. For example, the one-disulphide intermediates were identified on the basis of their rapid accumulation during folding with both GSSG and DTT~s as the reagent and their rapid interconversion when the pH was increased to 8.7. A fraction of these species appeared also to have a mixed-disulphide with glutathione, as they rapidly converted to species with two protein disulphide bonds when the pH was raised. Hs. N~so. was identified because it was an inhibitor of trypsin, was generated by both DTTs and GSSG, and did not change by intramolecular disulphide rearrangements upon increasing the pH; also, it was converted to the mixed-disulphide form N ~4ss~so~ upon the addition of GSSG.

Intermediate (30--51,5--14)~,o. was identified initially from its kinetic behaviour: it did not accumulate substantially when DTT~ was the


.]L 8

(a)

A

m

• m • i

m ~ " m

i i

m o i

I - S S

I - S S

(b)

~, .~ l-SS . ~ ~

i - t m m

m m . • l l I-4

o i r l ¢, i - - i "

in i

T ~ o

v

Time

Figure 2. Disulphide bond formation in (a) R~soH and (b) R '4°H with GSSG as the disulphide reagent. Disulphide bond formation in the presence of 0.3 mM GSSG was allowed to proceed for 3 minutes, when the reaction was quenched by acidification and the trapped species separated by HPLC. The identities of the species were determined as described in the text; an asterisk indicates that the free cysteine residue was present as the mixed-disulphide with glutathione. R is the fully reduced protein, and 1-SS the one-disulphide species.

reagent; treating it with high concentrations of GSSG converted it to a mixed-disulphide with glutathione on its free thiol group and, most importantly, it

N usH upon raising tile converted spontaneously to ~.~OH pH (Table 3). These kinetic observations indicate that this species is the immediate kinetic precursor of N 1 4 S I l

~ 8 O H •

Tile identity of (30--51, 5--14)~.~OH was unequivo- cally confirmed by peptide mapping of the iodoacetamide blocked derivative. Its molecular mass, 6584.7 Da, was consistent with it containing two disulphide bonds and a single --CH2CONH2 group. Digestion with the endopeptidase LysC yielded a peptide of molecular mass 1722.02 Da, which is the expected mass of the peptide of residues 1 to 15 containing a disulphide bond between Cys5 and Cys14. Reduction of this fragment produced the expected increase in mass of 2 Da. Tile mass was calibrated internally against the other peptides in the digest to ensure the required accuracy. The only other major peptide that changed upon reduction was one of molecular mass 2839.1 Da, which yielded reduced peptides of molecular masses 1283.49 Da

and 1559.01 Da; these masses are consistent with the original peptide being residues 47 to 58 with either Cys51 or Cys55 linked in a disulphide bond to Cys30 of residues 27 to 41.

To determine which cysteine residue of peptide 47 to 58 was involved in the disulphide bond, this peptide was isolated from the mixture in the mass spectrometer by fast deflection pulses (Talbo & Mann, 1994). Sequence ions from that peptide were obtained by matrix-assisted laser desorption ionization post source decay (MALDI PSD: Spengler et al., 1992). In this way, the residue covalently blocked by a --CH2CONH~ group was shown to be Cys55, not Cys51. Therefore, this species was identified unambiguously as intermediate (30--51, 5--14)X*OH.

The masses of the remaining peptides were consistent with the sequence of BPTI and the specificity of endopeptidase LysC.

Characterization of the disulphide species generated during the refolding of R '4°H

The major intermediates were identified as above for R~.~o... Species (30--51,5--38) '40" rearranged spontaneously and directly to NI4°H~.s,I upon increasing tile pH (Table 3). Its identity was confirmed by peptide mapping of its iodoacetamide-trapped form, as for (30--51, 5--14)~SOH. Tile expected endopeptidase LysC fragment of molecular mass 4558.9 Da corresponding to peptide 27 to 41 (containing both Cys30 and Cys38) linked through disulphide bonds to both peptides 1 to 15 (containing Cys5) and 47 to 58 (containing both Cys51 and Cys55) was identified. The expected three peptides were identified after reduction of all disulphide bonds. For additional verification, tile fragment of the three peptides linked by the two disulphide bonds was isolated in the mass spectrometer, and fragment ions resulting from fragmentation solely in the disulphide bonds were detected (G. T. & M. Mann, unpublished results). The molecular masses of these fragments identified peptide 27 to 41 as being linked to both peptides 1 to 15 and 47 to 58; a fragment ion corresponding to peptide 1 to 15 linked to 47 to 58 was not detected. Therefore, both Cys30 and Cys38 were linked in disulphide bonds to Cys5 and to either Cys51 or Cys55. To resolve tile latter ambiguity, Cys55 was identified as covalently blocked by a --CH2CONH2 group, not Cys51, using the same procedure as for (30--51, 5--14)~,~OH, above. The results clearly identified this species as (30--51, 5--38) '4°H, as from its kinetic properties it must have one native disulphide bond.

Discussion

The pathway of folding and disulphide formation of reduced BPTI, although relatively simple compared to those of other proteins (Creighton, 1992c; Creighton et al., 1995), involves numerous steps of disulphide formation, rearrangement and breakage. Such a pathway could not be elucidated by just identifying tile major intermediates that


accumulate, for kinetically important intermediates need not accumulate to substantial levels, intermediates that do accumulate may not be of kinetic importance, and the rates of all the possible transitions need to be identified. It cannot be assumed that intermediates differing by one disulphide bond are interconverted directly by making and breaking that disulphide bond. An example is the (30--51) and (30--51,5--55)N intermediates of BPTI, which were initially assumed to be interconverted directly by making and breaking the 5--55 disulphide bond (Creighton, 1975a). That this assumption was incorrect was uncovered by the observation that their interconversion was greatly slowed in the absence of both the Cys14 and Cys38 thiol groups (Creighton, 1977a). Their interconversion normally occurs by the rearrangements of non-native disulphide bonds involving either Cys14 or Cys38 (Figure 1).

In spite of these complications, which are likely to be common in reactions as complex as protein folding, the kinetic pathway can be elucidated almost unambiguously when it is coupled to disulphide formation; the pathway can be dissected by removing or blocking specific cysteine thiol groups and by following disulphide formation, breakage and rearrangement in individual isolated intermediates. Tile conformational basis and feasibility of the pathway can then be determined by studying the properties of the intermediates. The BPTI folding pathway shown in Figure 1 was elucidated in this way.

The initial steps in forming the first disulphide bonds in reduced BPTI have been dissected previously with analogues for five of the fifteen possible one-disulphide species (Darby & Creighton, 1993). Only the overall rate of forming all the various disulphide bonds is normally measured, but only one disulphide bond could be formed in each analogue, so the individual microscopic rates of formation and breakage could be determined. As expected for an unfolded polypeptide chain (Kemmink & Creighton, 1993), the measured rates of forming each disulphide bond were inversely proportional to the number of residues between the cysteine residues, except for forming the strained 51--55 disulphide bond. These observations have been extended here by the effects on the overall rates of replacing just one or two cysteine residues, which diminished the overall rates by about the expected amounts (Table 2). All these results demonstrate directly that formation of all 15 initial disulphide bonds, including the 12 non-native species, contribute in the expected way to the overall rate in normal reduced BPTI; forming the three native disulphide bonds accounts for only about 5% of the total observed rate. Consequently the transient non-native disulphide bonds increase the rate of forming the first disulphide bond markedly and cannot be ignored in any kinetic analysis.

The disulphide bonds formed initially in normal reduced BPTI rapidly rearrange intramolecularly. This probably occurs in the intermediates without stable structure on the same time-scale on which they

were formed intramolecularly, k,~,,,, (Table 1), as very similar conformational transitions are involved in both forming and rearranging these initial disulphide bonds. Only those intermediates with stable non-random conformations have low free energies and tend to predominate, such as the partly-folded (3(M-51) intermediate (van Mierlo et al., 1993) and, at lower pH where the thiol groups are not ionized, the quasi-native (5--55)N intermediate (van Mierlo et al., 1991b). The latter rearranges more slowly and can rapidly form the 14 38 disulphide bond to generate the very stable quasi-native (5--55, 14 38)x (States et al., 1984; Kosen et al., 1992), whereas the partly-folded conformation of (30--51) favours formation of the 14--38, 5--14 and 5--38 disulphide bonds (van Mierlo et al., 1993). This latter, productive part of the pathway was dissected in this study.

The reduced forms of analogues of the resulting two-disulphide intermediates, with only four cysteine residues could form only six possible one-disulphide intermediates, and each of them could form only a single second disulphide bond. Under conditions where the rate of disulphide formation is controlled by the conformational properties of the protein (equation (3)), these reduced proteins were observed to form only the expected two unique disulphide bonds present in tile normal intermediates. The second disulphide bond could be incorporated only directly in each of the analogues, whereas it could conceivably be generated by intramolecular disulphide rearrangements in the normal BPTI intermediates, which have two additional cysteine thiol groups. That the sum of the observed rates approached that of the collective rate of forming second disulphide bonds to generate these intermediates in normal BPTI indicates that they normally are formed directly. With the preponderance of (30--51) amongst the one-disulphide intermediates, it is likely that it formed directly the second disulphide bonds 14--38, 5--14 and 5--38, but the possibility that the 30--51 disulphide bond was formed second in each of the corresponding minor one-disulphide intermediates cannot be strictly excluded. Nevertheless, the predominance of the (30--51) intermediate seems to be important for increasing the rate of forming second disulphide bonds, as the observed rate is 12-fold lower in the presence of 8 M urea, where all the one-disulphide intermediates are unfolded and populated to similar extents (Creighton, 1977c). Therefore, the available data indicate that most of the normal BPTI molecules fold by forming directly the 5--14, 5--38 or 14--38 disulphide bonds in the partly-folded (30--51) intermediate. The 14 38 disulphide bond was formed more rapidly than the others, as would be expected from the close proximity of the two cysteine residues in the conformation of the (30--51) intermediate (van Mierlo et al., 1993), but this disulphide bond was also reduced more rapidly than expected; there appears to be some strain in the quasi-native conformation of (30--51, 14----38)N.

The normal two-disulphide intermediates (30--51, 14---38)N, (30--51, 5--14) and (30--51,


5--38) are in relatively rapid equilibrium by intramolecular disulphide rearrangements during folding of BPTI. The first is t rapped by acidification in somewhat greater levels than the other two (Weissman & Kim, 1991, 1992b), whereas the corresponding analogues were observed here to have similar relative stabilities when measured relative to their respective reduced proteins (Table 1). The reason for the difference is not clear, but is of little significance, for the relative levels of accumulation of these intermediates is not of central importance for the BPTI pathway

These three two-disulphide intermediates do not readily form third disulphide bonds, and they complete refolding and disulphide formation most rapidly under usual experimental conditions by

NSH - rearranging intramolecularly to the native-like SH This occurs directly, in a single step, in both (30--51, 5--14) and (30--51, 5--38) at similar rates (Table 3). Intermediate (30--51, 14----38)N probably rearranges via these particular non-native disulphide intermediates al though the more rapid equilibration be tween these intermediates precludes direct demonstration of this.

The importance of the pathway via the non-native two-disulphide intermediates is demonstrated by the rapid rates of disulphide folding in the absence of just Cys14 or Cys38. Neither form of the protein lacking either Cys14 or Cys38 can form the quasi-native species (5--55, 14----38)N and (30--51, 14--38)N. One folds through (30--51, 5--14), the other through (30--51, 5--38). The rates via these two intermediates could be measured directly, and they could be used to predict the rates through the combined pathway involving both these intermediates (Table 3). Most importantly, the predicted overall rate is found to be virtually identical to that observed with normal BPTI (Figure 3). This illustrates that disulphide folding normally occurs almost exclusively through the two particular non-native two-disulphide intermediates (30--51, 5--14) and (30--51,5--38) and that they are the most productive intermediates preceding ~r~lS~",sH. In their absence, as when both the Cys14 and Cys38 thiol groups are absent, disulphide formation and folding are slowed remarkably (Creighton, 1977a; Goldenberg, 1988).

In contrast, the absence of the quasi-native (5--55, 14 38)Nand (30--51, 14 38)N does not slow the folding process (see Figure 2); this indicates conclusively that they are not kinetically important or productive intermediates. They accumulate to substantial levels during folding of normal BPTI because of their relative stabilities and because they are blocked in forming further disulphide bonds.

There has been considerable confusion about the reasons for the roles of the disulphide rearrangements through the non-native two-disulphide intermediates. The experimental observations indicate clearly that they arise because of the slowness of forming the 5--55 disulphide bond in both the (30--51) and (30--51, 14~38)N intermediates. Forming the 5--55 disulphide bond in (30--51) occurs at about 10-~ times the rate at which the 5--14, 5--38 and 14---38

301(a) ~ 15[ .,-I 0

0 m . . _ . - - - - I 15 30

Time (min)

Figure 3. Comparison of the simulated rates of disulphide folding of reduced BPTI solely through the non-native two-disulphide intermediates (30--51, 5--14) and (30--51, 5--38) with those observed experimentally in normal BPTI. The simulations were performed with the rate constants of the "combined pathway" given in Table 3 in the presence of 40 mM DTT~, with (b) and without (a) added 0.1 mM DTT~s~, and are represented by the continuous curves. The points indicate the appearance of fully refolded BPTIR~a actually measured under the same conditions. The combined pathway rate constants (Table 3) give only the formation of N~s~[, but this species is very rapidly converted to fully native BPTI under the conditions used here. The normal reduced BPTI also forms other species, especially the quasi-native species (30--51, 14----38)N and (5--55, 14~-38)N, which were not included in the simulations. This will affect the simulations only to the extent that it depletes the concentration of the starting reduced BPTI and DTT~£, plus the one-disulphide intermediates; it becomes significant at the greatest extents of folding as is indicated by the fact that the measured results do not maintain the simulated rate.

disulphide bonds are formed (Creighton, 1977a: Goldenberg, 1988). Consequently, the latter disulphide bonds are formed preferentially, and they are converted to the 5--55 disulphide bond by intramolecular rearrangements. The latter step is the slowest intramolecular step in the entire pathway (Creighton, 1977b), with an apparent half-time of about 150 seconds (Table 3). Its transition state is similar in free energy to that involved in tbrming the 5--55 disulphide bond directly in (30--51), suggesting that the same conformational transitions are occurring in each (Creighton, 1978: Mendoza et al., 1994). This high energy transition is overcome more readily by disulphide rearrangement than by disulphide formation simply because the former is an intramolecular process, while the latter is a bimolecular reaction involving the disulphide reagent.

Why is forming the 5--55 disulphide bond so slow in (30--51) and (30--51, 5--55)'? In both cases, the native-like conformation would result, so it might be expected to occur readily. It has been suggested that it is an artefact due to the Cys5 and Cys55 thiol groups being buried in (30--51, 14--38)N and consequently being inaccessible to the disulphide reagent (Goto & Hamaguchi, 1981: Weissman & Kim, 1991, 1992a). This explanation is ruled out by the direct observation that the thiol groups of this species are suitably reactive to the reagent (Creighton, 1975a, 1977a, 1981). The


proposal by Weissman & Kim (1992a) that these mixed-disulplaides of (30--51. 14---38)x are formed initially in the precursor (30--51 ) is implausible, as the high levels of the mixed-disulphides of this intermediate that can be generated (Creighton. 1977b) would require that the 14---38 disulphide bond be formed in (30--51) only ql?er a mixed-disulphide was generated on eithe," Cys5 or Cys55: there is no plausible reason why this should occur. Furthernaore. the disulplfide rearrangement pathway predominates even when the quasi-native (30--51. 14~38)~ is so unstable as not to be populated (Zhang & Goldenberg. 1993).

The experimental data demonstrate that the 5--55 disulphide bond is formed slowly in both (30--51)and (30--51. I ° 38):, for the same reason: the intramolecular step is slow because the native conformation would result. These slow rates of forming the 5--55 disulphide bond mi,'ror the slow rates of reducing it in the opposite direction; both reflect the high energy of the same transition state in both directions. The experimental observations are most cleat in the case of 130--51 ), where the direct formation and breakage of the 5--55 disulphide bond can be measured directly by blocking or removing both the Cysl4 and Cys38 thiol groups (Creighton, 1977a: Goldenberg. 1988). The wtlue o fk ....... tot" forming the 5--55 disulphide bond in (30--51) with a reagent mixed-disulphide on either Cys5 or Cys55 has been measured to be 1.6 x 10 ~ s ', which is only 10 ~ times the corresponding rate lot forming the 14 38.5--14 and 5--38 disulphide bonds {Creighton & Goldenberg. 1984: Darby & Creighton. 1993): consequently, the latter disulphide bonds are formed prefe,-entially. The rate of reducing the 5--55 disulphide bond directly in NS', ', is about 1.2 × 10 as ' M ~,only 10 ~ times the rate at which the 14 38. 5--14 and 5--38 disulphide bonds are reduced in the co,'responding two-disulphide species with the 30--51 disulplaide bond. The much greater difference in the rates of reduction is due to the correspondingly lower flee energy of the N~]', species (Creighton. 1977b). Taking this into account, the values of the forward and reverse rate constants for forming or breaking the 5--55 disulphide bond from. and to. the mixed-disulphide with the reagent, k ....... and k,. respectively, give ahnost exactly the observed equilibrium constant between (30--51) and N~','~ using glutathione as the reagent (53M and 40M, respectively: Creighton & Goldenberg, 1984), when it is assunaed that the Cys5 and Cys55 thiol groups of (30--51} l'orm nfixed-disulplaides with glutathione of the usual stabilities. This consistency of the rate and equilibrium constants indicates that the same high-ene,'gy transition state is being encountered in both directions. The wdue of k ....... for forming the disulphide bonds is small for the same reason that the value of k~ for reducing that disulphide bond is small.

The reason for the high free energy of the transition state becomes apparent by considering reduction of the 5--55 disulphide bond in s,,. Ns.. this disulphide bond is buried in the native-like conformation, and its reduction by thiol reagents requires that it becomes accessible to the reagent by distortion or unfolding of that conformation. This implies that any disulphide

bond that will become buried in a stable conformation will be fornaed more slowly than one that will be accessible (Creightom 1978). This conclusion is independent of the conformations of the disulphide intermediates in which the final disulphide bond is formed: slow disulphide l'ormation does not require that they be folded or that their thiol groups be inaccessible, although disulphide fornaation will be slowed even more in these cases (see below). As expected, slow formation of disulphide bonds that will be buried in a folded cont 'onnation is a general phenomenou that is observed in all known disulphide folding pathways (Creighton, 1992c: Creighton et al., 1995). It is undoubtedly also applicable to forming the 5--55 disulphide bond in the reduced protein (Kosen e t a / . . 1992) and in (30--51, 14 38)x and to forming the 30--51 disulphide bond in the even more stable (5--55, 14-38):` (States et al., 1984: Creighton & Goldenberg, 1984): it is not apparent in the latter case, however, because the thiol groups are buried and t, nreactive in (5--55, 14--38)x.

This high flee energy barrier resulting fi'om the folded conformation that will be formed could seemingly be avoided by first forming the disulphide bond in the unfolded state, and then the protein folding around the disulphide bond (Figure 4). This should occur if the protein without the disulphide bond is unfolded in the region of the cysteine residues, and the rate of forming the disulphide bond should be no slower than in the unfolded protein. The value of/,-, ..... for forming the 5--55 disulphide bond in reduced BPTl has been measured to be l × 10 -'s ' ( D a r b y & Creighton, 1993: Kosen et al., 1992), but the value in (30--51) is lower. 1.6 × 10 ~s ' (Creighton. 1977a: Goldenberg, 1988). Decreased values of /,'+ ...... are predicted if the protein without the disulphide bond is folded into a stable conformation (Figure 4): the more stable the conformation that needs to be tmfolded, the smaller the possible value of k ........ This may be the reason why such small values of/,, ...... are observed for forming the 5 - - 5 5 disulphide bond in (30--51) and (30--51, 14---38)x. which both have stable folded conformations.

The analysis of Figure 4 suggests that forming the 5--55 disulphide bond in (30--51 ) occurs most readily if its partly folded conformation is further unfolded. The reverse step of breaking the 5--55 disulphide bond in N~',',, either by reaction with a thiol reagent or by rearrangement with a Cysl4 or Cys38 thiol group, clearly involves at least some unfolding of the polypeptide chain (Creighton, 1978). Because the same transition state is believed to occur in forming the 5--55 disulphide bond. either directly or by disulphide rearrangements, at least SOlne tmfolding of the (30--51) intermediate is also implied. The experimental observations of Mendoza et a/. (1994) have also led to this conchtsion. Unfolding and then refolding to form a disulphide bond is probably a consequence of the need to undergo interchange with an external distdphide reagent (equations (3) and (4)). The same considerations would be expected to apply, although not so strictly, to protein hydrogen bond formation and breakage, as their interchange between the


~ SSR k intra u Unfo lded " '

/ -SH kr,u

+ R S H

MD K F

SS K F

"-"~S-SR .~" k intra,F~ [ "'s + RSH Folded SHN ' k r,F SN

K~ s _ kintra,u k r,L1

SS

K F K~ s kr,F k intra,F - MD K F

SS u kintra,u + K F K S S kr,F

k intra,obs = MD I + K F

Figure 4. The intramolecular rate constant expected for forming a disulphide bond that will be buried in a folded conformation calculated from its measured rate of reduction. Disulphide formation is postulated to occur in the species with a mixed-disulphide with the reagent, -SR (left), in either the unfolded (top) or folded (bottom) conformations. K~ '"> and K~ ~ are the equilibrium constants for folding of the mixed-disulphide species (left) and the disulphide form (right), respectively; the folding transition is assumed to be rapid relative to the rates of disulphide formation. The mixed-disulphide species is considered here instead of the redtlced protein simply to avoid the complication of a further reaction with the reagent; the two forms of the protein will usually have very similar properties. The values of the parameters will be similar for different reagents with chemically similar aliphatic thiol and distflphide groups, but it is best to measure all the parameters with the same reagent. The rate constant for forming the disulphide bond in the folded conformation, k,,,,~,,.E, can be calculated from the other rate and equilibrium constants due to the requirement that the free energy change around the cycle be zero. The observed rate constant, k~ ......... ~,,, will be the weighted average of the rates in the unfolded and folded conformations. In the case of forming the 5--55 disulphide bond in the (30--51) intermediate of BPTI, the ratio of the measured stabilities of the distflphide bonds in the folded and unfolded states is about 10" (Creighton & Goldenberg, 1984), and the value of K) ~ for the native conformation of (30--51, 5--55)~ is about I × 10 ~ (Hurle et al., 1990). Therefore, the value of K~"' for (30--51) can be calculated to be about 10, which is at least consistent with the experimental observations on an analogue of the partly-folded intermediate in which all other Cys residues were replaced by Ser (van Mierlo et al., 1993); in this case, the folded conformations with and without the disulphide bond are somewhat different. The values of k,,,~., = 0.02 s t and k~ = 7 s ~ M ~ have been measured for cvsteine residues 30 residues apart in an unfolded protein (Darby & Creighton, 1993). The measured value of the rate constant for reduction of the 5--55 distllphide bond in (30--51, 5--55)x using dithiothreitol as the reagent has been measured to be I x l0 4 s ~ M ' (Creighton, 1977a; Goldenberg, 1988), which is close to the value expected for the reaction proceeding through the unfolded state (Mendoza el al., 1994), so the value of k,.v must be less than this. Using all these parameters, the expected value of k,,,~,,.,,b, for forming the 5--55 disulphide bond in (30--51) is calculated to be 1.8 x 10~s ~, close to the measured value of 1.6 x 10 ~s ~ (Goldenberg, 1988), and therefore disulphide formation occurs primarily in the unfolded conformation.

aqueous solvent and other protein groups should be energetically favoured over lbrming and breaking hydrogen bonds in isolation (see Figure 9 of Creighton, 1978).

The main difficulty with this analysis is that the partly-folded conformat ion of (30--51) does not

extend to the Cys5 residue, which is in an unfolded part of the polypeptide chain (van M ierlo el al., 1994). The small wflue of/ , ....... in this case suggests, therefore, that the folded portion of the protein causes a further kinetic block to forming the 5- -55 distilphide bond directly. Perhaps the polypeptide chain would need to


adopt a more folded and strained conformation, including both the Cys5 and Cys55 residues, in the transition state for the reaction in which they form the disulphide bond, which is suggested by considering the reverse reaction, reduction of the 5--55 disulphide bond in NS~ (van Mierlo et al . , 1991a).

The data presented here confirm the importance of the non-native disulphide intermediates in the BPTI pathway. This has been misinterpreted, however, in some instances. The non-native disulphide bonds do not imply non-native stabilizing interactions (Weissman & Kim, 1991, 1992a), as has always been evident from their instabilities (Creighton, 1978) and is shown by the conformations of the intermediates (States et al . , 1987; van Mierlo et al . , 1994). The non-native disulphide bonds do not guide the rearrangement process (Weissman & Kim, 1992b); this has never been suggested, and they occur in relatively unfolded parts of the molecule. They are not "committed" to the rearrangement process (Weissman & Kim, 1992b), which would imply that they occur after the rate-determining step; instead they occur before it (Creighton, 1977b) Nevertheless, they are the most productive initial two-disulphide intermediates through which normal BPTI refolds under the usual experimental conditions. The kinetic importance of going through a limited number of disulphide intermediates is demonstrated by the substantially lower rates of folding in the presence of 8 M urea, where native BPTI and NS~ are folded, but the preceding disulphide intermediates are unfolded; consequently, all of the latter with either one or two disulphide bonds have similar stabilities and accumulate to similar extents (Creighton, 1977c). The BPTI pathway appears to be a compromise between having sufficient stabilizing interactions early in disulphide formation, so that the productive intermediates predominate, while maintaining sufficient flexibility to permit forming the remaining disulphide bonds that will be buried in the final native conformation.

Materials and Methods

Plasmid construction

Genes for expressing the engineered versions of BPTI with either Cys14 and Cys38 replaced by Ser were constructed from a synthetic gene for the BPTI precursor (Creighton et al., 1993) using the polymerase chain reaction. To facilitate purification, the coding sequence was fused to a Met-Gly-His,-Met-sequence that could be removed by CNBr cleavage. As in all the other folding intermediate analogues, the Met52 residue of BPTI was replaced by Arg (Darby & Creighton, 1993), which has been shown to have only very minor effects on the protein structure, stability and folding (van Mierlo et al., 1991a; Darby & Creighton, 1993).

Each gene was ligated into the N c o I / B a m H I sites of the kanamycin-resistant bacteriophage T7 expression vector pET-9d (Studier, 1991). The nucleotide sequences of all the plasmid inserts were confirmed by double-stranded DNA sequencing.

Protein expression and purification

Protein expression was induced in mid-log cultures of BL21(DE3) in terrific broth as previously described (Studier, 1991). After two hours induction, all the expressed peptide was present as inclusion bodies and accounted for approximately 12% of the total cell protein in Coomassie Blue-stained sodium dodecyl sulphate polyacrylamide gels optimized for resolving small proteins (Schagger &von Jagow, 1987).

Cell pellets were thawed in 50 ml of 20 mM Tris-HC1 (pH 8.0), 5mM benzamidine, 0.2mM phenylmethyl- sulphonylfluoride, and 1 mM EDTA, which causes immediate lysis, due to the presence of the lysozyme expressed concurrently from the pLys S plasmid (Studier, 1991). After 30 minutes treatment with deoxyribonuclease I, the material was centrifuged at 10,000 g for 10 minutes. The pellet was washed twice with the same buffer supplemented with 1% (w/v) Triton X-100, and then twice with 20mM Tris-HC1 (pH 8.0), 0.5M NaC1, 5 mM imidazole. The pellet of inclusion bodies resulting from I 1 of original culture was dissolved for 30 minutes at room temperature in approximately 10 ml of the binding buffer supplemented with 6M guanidinium chloride. The resulting solution was clarified by filtration though a 0.44 ~tm filter and loaded onto a metal-chelating Sepharose fast-flow column (Pharmacia, 1 to 1.5 ml of resin per litre of original culture) previously charged with NF* and equilibrated with the binding buffer containing 6 M guanidinium chloride. The column was washed with three volumes of the same buffer, and loosely bound proteins were removed by washing with three volumes of the same buffer supplemented with 45 mM imidazole. The poly-His BPTI fusion protein was eluted by raising the imidazole concentration to 350 mM. The eluant containing the fusion protein was acidified by adding HC1 to 0.1 M and purged with N2. For each original litre of culture, 30 to 60 mg of crystalline CNBr was added; cleavage was allowed to proceed overnight at room temperature in the dark. The resulting solution was dialyzed extensively against 10 mM HC1. It was then made up to 50 mM Tris-HC1 (pH 8.7), 25 mM D T ~ , 1 mM EDTA, and solid urea was added to 8 M. After incubation at room temperature for 30 minutes, the reduced BPTI polypeptide chains were purified as described by Darby & Creighton (1990, 1993), using chromatography on Sephadex SPC-25 under acidic conditions. The reduced proteins were refolded in the presence of 0.5 mM GSSG at room temperature, to generate the species N~Sy~ and N~y and their mixed-disulphide forms with glutathione on the free thiol group, N ~4s~.~o. and N "°".~s~,, respectively The free thiol and mixed-disulphide final products were purified by reverse phase HPLC on a Vydac C-18 column, and both were recovered. The mixed-disulphide could be removed selectively by a brief treatment with DTT~slII in 0.1 M Tris-HCl (pH 8.7), 0.2 M KCI, 1 mM EDTA.

The proteins produced in this way had properties close to those expected from the corresponding species with all six cysteine residues. The fully reduced proteins, plus the .s. N~s.~4°" N ~ . and forms with and without the mixed- disulphide with glutathione, had the expected electrophor-

N38OH etic and HPLC reverse-phase mobilities. Both .4sH and 14OH N3~s. were stoichiometric inhibitors of trypsin, as expected

(Kress & Laskowski, 1967). The molecular mass of the form 14SH N3~o. was determined by mass spectrometry to be the

expected 6521 Da. In its reduced form, the mass was 6526 Da, close to the expected value of 6525 Da, which increased to the expected 6816 Da upon treatment with iodoacetate to introduce five --CH2COOH groups. The


reduced form of the other mutant, R '4°", gave a molecular mass of 6527 Da.

BPTI~_, and its two-disulphide forms (30--51, 14 38)Set.N, (30--51, 5--14)s~, and (30--51, 5----38)S,.r w e r e prepared using another expression system described by Darby & Creighton (1993).

Reduction and disulphide refolding

The procedures used to reduce the protein and to follow disulphide bond formation have been described (Creighton & Goldenberg, 1984; Darby & Creighton, 1993). The fully reduced proteins were generated by reduction with 0.1 M DTT~s~ in 6 M guanidinium chloride, 0.1 M Tris-HC1 (pH 8.7), 1 mM EDTA for 30 minutes at room temperature, followed by acidification with HCI and gel filtration on Sephadex G25 in 10 mM HC1.

Disulphide bond formation and reduction were carried out at 25°C in 0.1 M Tris-HC1 (pH 8.7), 0.2 M KCI, 1 mM EDTA, in the presence of GSSG, D T ~ or DT~] . The species present at different times were trapped by either alkylation with 0.1 M iodoacetamide or iodoacetate for two minutes at 25°C, or by acidification by addition of 0.1 volume of 3 M HC1. The trapped species were separated and quantified either by native gel electrophoresis (Reisfield et al., 1962) for the covalently-trapped species or by reverse-phase HPLC in the case of the acid-trapped samples. HPLC analysis of the refolding of R '4°H used a Vydac C-18 column at 38°C. Elution was with 0.1% (v/v) trifluoroacetic acid at 1 ml/rain, using the following linear gradients in buffer B (0.1% trifluoroacetic acid, 90% (v/v) acetonitrile): 0 minutes, 10% B; 15 minutes, 25% B; 35 minutes, 27% B; 50 minutes, 28% B; 140 minutes, 31% B; 180 minutes, 35% B; 182 minutes, 46% B. Separation of the refolding products of R~,o, was carried out on the same column at 50°C and a flow rate of 1 ml/min, using the following gradients: 0 minutes, 25% B; 5 minutes, 27% B; 15 minutes, 28% B; 30 minutes, 28% B; 60 minutes, 31% B; 70 minutes, 35% B.

The relative amounts of the various species present at different times were quantified by integration of the areas under the peaks measured by densitometry of the stained electrophoresis gels or by the absorbance at 220 mm of the HPLC elution profiles. The kinetics of the appearance and disappearance of the various disulphide species during disulphide formation or rearrangement were simulated by numerical integration, manually fitting the kinetic data to obtain the various rate constants.

Rates of intramolecular rearrangements

The rates at which the various species rearranged intramolectflarly were measured directly by placing the acid-trapped species isolated by HPLC under the normal refolding conditions, but in the absence of any thiol or disulphide reagents (Creighton, 1977c). Species (30--51, 5--14)3.~o, and (30--51, 5--38) "°" were trapped by refolding the corresponding fully reduced proteins for 140 seconds in 0.3 mM GSSG and were isolated by HPLC (see Figure 2). They eluted at 41 minutes and 29 minutes, respectively The intermediates were recovered by lyophilization and dissolved to 40 ~g/ml in 10 mM HCI. Rearrangement was initiated by adding an equal volume of 0.2 M Tris-HC1 (pH 8.7), 0.4 M KC1, 2 mM EDTA. Portions were trapped at various times by addition of 0.1 volume of 3 M HC1, and the various species separated and quantified by reverse-phase HPLC as above.

Identification of disulphide bonds

Intermediates were isolated using HPLC and concen- trated by lyophilization to a volume of about 250 ILl. Free thiol groups were blocked using a saturated solution of iodoacetamide (Gra~ 1993), and the protein purified again by HPLC; rearrangement of disulphide bonds during trapping was minimal. Disulphide assignments were made on endoLysC digests of the protein, using MALDI mass spectrometr)~ as explained in the Results section, above.

Acknowledgements We thank Matthias Mann for advice on the mass

spectrometry measurements and David Goldenberg for discussions. P. E. Morin was supported by a fellowship from the Alexander von Humboldt Stiftung.

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Edited by A. R. Fersht

(Received 14 December 1994; accepted 2 March 1995)

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