Biochemistry 1992, 31, 5705-5717
5705
Disulfide Bond-Coupled Folding of Bovine Pancreatic Trypsin Inhibitor Derivatives Missing One or Two Disulfide Bonds? Phyllis Anne Kosen,*J Cara B. Marks,lJ Arnold M. Falick,$ Stephen Anderson,lJ and Irwin D. Kuntzt Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94143, and Department of Cardiovascular Research, Genentech, South San Francisco, California 94080 Received September 30, 1991; Revised Manuscript Received March 25, 1992 ABSTRACT: The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7,25 OC in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 5 1 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala5 1 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step-rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate-seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J . Mol. Biol. 179, 4971 and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 248 11. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.
D u r i n g the course of protein folding and unfolding reactions, disulfide bond-coupled formation or reduction, when mediated by disulfide and thiol reagents, occurs in two steps as diagramed in Scheme I (Creighton, 1975a, 1986; Creighton & Goldenberg, 1984; Snyder, 1987). The rates of both steps depend on thiol-disulfide exchange chemistry, tempered by solution pH and acidity of the reacting thiol and conjugate thiols of the disulfide (Creighton, 1975a; Szajewski & Whitesides, 1980; Shaked et al., 1980; Snyder et al., 1981; Snyder, 1987).' While both steps also depend on steric accessibility and relative orientations of thiol and disulfide, the second step differs from the first in that, in addition, it reflects the collision rate of two polypeptide cysteine-containing regions. It is this second reaction that is equivalent to a conformational transition in a folding reaction not involving disulfide formation. An apparent first-order rate constant for the intramolecular reaction, khtra,can be extracted from observed reaction rate constants (Creighton & Goldenberg, 1984). Folding and unfolding studies, incorporating disulfide formation and reduction, have an advantage that kinetic intermediates can be trapped by alkylation or protonation of any cysteines not involved in a disulfide. Thus, folding steps preceding the slowest observable step are readily characterized and structures of trapped intermediates can be analyzed. 'Supported by Genentech, Inc., by NSF Grant DMB8606901 and NIH Grant GM19267 (to 1.D.K.k and bv NSF Grant DMB9018707 and NIH Grant ROlAG10462 (t0.S.A.). * Address correspondence to this author. 8 University of California at San Francisco. I Genentech. 11 Present address: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England. Resent address: Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854.
Scheme I
Scheme I1 (14-38, 5-55)
R
I/\
I 6 II
(30-51, 5-55)
=(14-38, 30-51, 5-55)
Bovine pancreatic trypsin inhibitor (BPTI;2 Figure l), a protein of 58 amino acid residues containing three disulfides, is the paradigm for protein folding reactions coupled to di-
'
The reactive species is the thiolate ion. Both steps in Scheme I involve thiolate anions, not thiols. Often, however, these reactions are written as if they were thiol-disulfide exchange reactions (Creighton & Goldenberg, 1984; Goldenberg, 1988). We follow that custom. Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; Ala30/ Ala51 and Ala14/Ala38, mutants of BPTI in which the respective cysteines are replaced by alanines; Ser14/Ser38, a mutant of BPTI in which the respective cysteines are replaced by serines; (Cam14/Cam38; Ala30/Ala5 l), a chemically-modified derivative of Ala30/Ala51 in which the disulfide formed by cysteines 14 and 38 is reduced selectively and the thiols are blocked by carbamoylmethylation; N, native or fully refolded Ala30/Ala51; R, fully reduced Ala30/Ala51, (Cam14/Cam38; Ala30/Ala51), or BPTI; I, one-disulfide bond intermediates of Ala30/ Ala51 excluding the 5-55 disulfide bond intermediates or all of the BPTI one-disulfide bond intermediates (the content clarifies which definition of R or I applies); tetra(carbamoylmethyl)-Ala3O/Ala5 1, fully reduced and carbamoylmethylated Ala30/Ala5 1, Ala30/Ala5 1, (Cam14/Cam38; Ala30/Ala51), BPTI, and kinetic intermediates are also referred to by the residue numbers involved in a disulfide. For example, (Caml4/ Cam38; Ala30/Ala51) is also called (5-55)); DTTZE, dithiothreitol (Cleland's reagent); DTT:, tram-4,5-dihydroxy- 1,2-dithiane (oxidized dithiothreitol); Gdn-HC1, guanidine hydrochloride; GSH, reduced glutathione; GSSG, oxidized glutathione; TFA, trifluoroacetic acid; Tris, tris( hydroxymethy1)aminomethane.
0006-2960/92/043 1-5705$03.00/0 0 1992 American Chemical Society
Kosen et al.
5706 Biochemistry, Vol. 31, No. 25, 1992
FIGURE1: A ribbon diagram of BPTI showing the positions of the three disulfides, modified from Richardson (1 98 1). Disulfides are made by cysteines 14 and 38, cysteines 30 and 51, and cysteines 5 and 55.
sulfide formation (Creighton, 1977a,b; Creighton & Goldenberg, 1984; Weissman & Kim, 1991). Scheme I1 shows a simplified version of the preferred BPTI folding mechanism, characterized at pH 8.7, 25 "C. R is fully reduced protein containing six cysteines. Species I and I1 are mixtures of one- and two-disulfide bond intermediates, respectively. (14-38, 5-55) and (30-51,5-55) are two-disulfide bond intermediates with native-like conformations. Native BPTI is represented as (14-38, 30-51, 5-55). Initial formation of a disulfide from R is most likely a nearly random event with rates of disulfide formation influenced primarily by the number of residues separating two cysteines. However, rapid intramolecular thiol-disulfide exchange among the one-disulfide bond intermediates and stabilizing structural elements present in (30-51), but not in many of the other one-disulfide bond intermediates, cause (30-5 1) to dominate the composition of I (Creighton, 1977c, 1988). Continued productive folding involves (30-5 1). Three two-disulfide bond intermediates, (30-5 1, 14-38), (5-14, 30-51), and (5-38, 30-51), grouped for kinetic purposes as 11, are formed directly and readily from (30-51). For productive folding to continue, thiol-disulfide rearrangements must occur in (5-38,3041) and (5-14,3041) producing (30-51, 5-55). These rearrangements are the rate-limiting steps in folding. The final step in folding is formation of the solvent-accessible 14-38 disulfide. In addition to productive mechanisms generating a folded protein with all three disulfides, a metastable native-like intermediate missing the 30-5 1 disulfide, (14-38, 5-55),3 accumulates to high levels during BPTI folding (Creighton & Goldenberg, 1984; States et al., 1984). For this intermediate, cysteines 30 and 51 are sequestered in the protein interior at the interface of the C-terminal helix and the central 8-sheet. This intermediate arises through thiol-disulfide rearrangements involving other two-disulfide bond intermediates and de novo from the pool of one-disulfide bond intermediates. Since (5-55) has recently been identified as an intermediate of BPTI folding (Weissman & Kim, 1991), it is the logical direct precursor to (14-38, 5-55). The BPTI gene has been cloned into a variety of genetically-engineered expression systems (Altman et al., 1991, and references within). A more detailed picture of BPTI folding and unfolding mechanisms should result from studies using mutants with amino acid substitutions at sites other than those of the cysteines. This approach identifies amino acid residues This intermediate is also known as N(30SH, 51SH) (Creighton & Goldenberg, 1984) and as N* (Staley & Kim, 1990).
key to individual folding and unfolding steps. Such studies have begun (Goldenberg et al., 1989). A complementary strategy employs removal of one or more cysteines. This maneuver eliminates specific intermediates, simplifies the pathway, and clarifies the roles of certain intermediates. Exploration of energetically less favorable pathways becomes possible (Marks et al., 1987a; Goldenberg, 1988). Further, as BPTI is extremely resistant to denaturation, folding and unfolding mechanisms cannot be studied if all disulfides are present. The stability of BPTI is substantially decreased when any one disulfide is removed (Vincent et al., 1971; Schwarz et al., 1987; States et al., 1987; Hurle et al., 1990). With disulfide mutants, it is possible to compare folding mechanisms when disulfides are present (Hurle et al., 1990) and when folding is coupled to disulfide formation. Goldenberg (1988) reported a detailed kinetic analysis of the folding and unfolding of a BPTI mutant in which cysteines 14 and 38 were replaced by serines (Ser14/Ser38). Here we report a similar analysis for a BPTI mutant in which cysteines 30 and 51 are replaced by alanines (Ala30/Ala51) and for a chemically-modified derivative of this mutant in which only the 5-55 disulfide is present (Cam14/Cam38; Ala30/Ala51). The tertiary structure of Ala30/Ala5 1 is nearly identical to that of BPTI (Eigenbrot et al., 1990; Hurle et al., 1991). The disulfide bond pattern is that of the native-like two-disulfide bond intermediate, (14-38, 5-55), identified as a kinetic trap in the BPTI folding mechanism (Creighton & Goldenberg, 1984; States et al., 1984). To the extent that the structural characteristics of Ala30/Ala5 1 are analogous to those of (14-38, 5-55), delineation of Ala30/Ala5 1 folding and unfolding mechanisms clarifies the mechanism of (14-38, 5-55) formation during BPTI folding. While the initial impetus of this study was further characterization of the BPTI folding pathway with Ala30/Ala5 1 as the simplifying model, this mutant protein proves to be an interesting model system for folding in its own right. MATERIALS AND METHODS Materials Protein Preparation and Purification. Plasmid constructs and expression systems for genes encoding the BPTI mutants Ala30/Ala51 and Cys14/Cys38 Cys3O/Cys51 Ala14/Ala38 were described (Marks et al., 1987a,b). Purification and chemical characterization of these proteins were also described (Hurle et al., 1990). The derivative (Cam 14/Cam38; Ala30/Ala5 1) was prepared by incubating 70 pM Ala30/Ala51 with 0.5 mM DTT;; in ice-cold 0.01 M Tris-HC1, pH 8.5, 1 mM EDTA for 30 min under an argon atmosphere. Addition of an equal volume of 0.5 M iodoacetamide, 0.1 M Tris-HC1, pH 8.5, 1 mM EDTA quenched disulfide reduction. After 15 min, salts were removed by chromatography on a column (6 X 34 cm) of Sephadex G-25 equilibrated with 0.05 M ammonium bicarbonate. After lyophilization, (Cam14/Cam38; Ala30/ Ala51) was separated from Ala30/Ala51 by FPLC reversephase PepRPC HR 10/16 chromatography using 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B). The gradient was 28-30.5% B at a flow rate of 4 mL/min for 39 min. Tetra(carbamoylmethy1)-Ala30/51 was prepared by incubation of 3 mg of Ala30/Ala51 with a 10-fold molar excess of DTT;; for 30 min in 6 M Gdn-HC1,0.2 M Tris, pH 8.7, 10 mM EDTA, followed by alkylation with a 20-fold molar excess of iodoacetamide over DTTZ; for 20 min. Salts were removed by chromatography over Sephadex G-25 equilibrated with 10 mM HCl, and the protein solution was lyophilized.
-
-
Folding Kinetics of BPTI Mutants Chemicals. All redox reagents were obtained from Sigma Chemical Co.and used without further purification. Solutions of DTTgfi and GSH prepared by dry weight measurements contained the expected concentration of thiols when assayed by the method of Ellman (1959). Intermolecular disulfide-containing compounds are significantly better oxidizing reagents than DTTS and, if present as contaminants, alter the appearance of DTTs-coupled B folding profiles (Creighton, 1974a, 1977b). Crystalline DTT: was assayed for such impurities using the method of Zahler and Cleland (1968). Impurities, at a level of 0.05% or greater, would have been detected. None were found. We also examined the electrophoretic profiles of reduced Ala14/Ala38 which had been incubated with 0.04 or 0.06 M DTT; for 1 h. (Complete folding conditions are described below.) As expected, for DTT; preparations containing insignificant amounts of impurities, no folded Ala14/Ala38 appeared (Goldenberg, 1988). Finally, we found that 1.7-2.6 pM reoxidized (Cam14/Cam38; Ala30/Ala5 1) accumulated within 40 min in the presence of 0.06 M DTT; with no additional oxidation occurring during the next 20 min. Using the second-order rate constant for 5-55 disulfide formation by DTT; (determined by other m e a n s s e e results) and an initial concentration of 0.06 M DTT;, simulation of reduced (Cam14/Cam38; Ala30/Ala51) folding kinetics suggested that no more than 0.3 pM oxidized (Cam14/Cam38; Ala30/ Ala51) should be present after 40 min. Comparison of the experimental results and the simulation suggests that disulfides, which are less stable than DTT;, were present at a level of 0.005% or less. (Residual air-oxidation might also contribute to the low levels of (Cam14/Cam38; Ala30/Ala51) oxidation in the presence DTT;.) Given the results of the three different experiments described above, it was concluded that insignificant amounts of intermolecular disulfide impurities were present; consequently, DTT;, was not further purified. Methods Identification of the Disulfide Bond in (Cam14/Cam38; Ala30/AIa51). Tetra(carbamoylmethy1)-Ala3O/Ala51and (Cam14/Cam38; Ala30/Ala5 l), repurified by reverse-phase FPLC chromatography after use in folding experiments, were each digested with endoproteinase Lys C (Calbiochem; EC 3.4.99.30) in 0.1 M sodium phosphate, pH 7.5, 2 M urea for 16 h at 37 "C. Protein concentrations were approximately 1 mg/mL, and enzyme to substrate ratios were approximately 0.03 unit:l mg. Peptide fragments were isolated by FPLC reverse-phase PepRPC HR 5/5 chromatography using 0.1% TFA in water (A) and 0.09% TFA in acetonitrile (B). The gradient was 0-40% B at a flow rate of 0.4 mL/min for 110 min. Prior to tandem mass spectrometry experiments, contaminating sodium ions were removed by HPLC reverse-phase chromatography on a Vydac (2-18 analytical column using 0.1% TFA in water (A) and 0.08% TFA in acetonitrile (B). The gradient was 5-50% B for 45 min at a flow rate of 1 mL/min. Molecular masses of endoproteinase Lys C-generated peptides were determined by mass spectrometry using a Kratos MS-50s double-focusing mass spectrometer equipped with a high field magnet (mass range 3000 Da at 8 kV), a cesium ion liquid secondary ion mass spectrometry source (Aberth et al., 1982; Falick et al., 1986a), and a cooled sample introduction probe (Falick et al., 1986b). The matrix was a 1:l mixture of glycerol and thioglycerol which was 0.1 M in HCl. Tandem mass spectrometry experiments were performed on a Kratos Analytical (Manchester, U.K.) Concept IIHH four-sector EBEB tandem mass spectrometer (Walls et al.,
Biochemistry, Vol. 31, No. 25, 1992 5707
1990) fitted with an electro-optical array detector (Cottrell & Evans, 1987), a cesium ion liquid secondary ion source, and
a coolable probe. The same matrix was used, and parent ions were generated with an 18-keV cesium ion primary beam. The collision energy for collision-induced dissociation was 4 keV. The collision gas (He) was used at a pressure sufficient to suppress the parent ion beam to about 30% of its initial value. The instrument was controlled and data were acquired with a DS-90 data system. Analysis and display were carried out with a Mach 3 data system. Folding and Unfolding Experiments. Procedures used for folding and unfolding kinetic studies were those of Creighton and Goldenberg (1984) with modifications. These modifications do not alter folding or unfolding conditions; therefore, our kinetic measurements can be directly compared to previous work. Protein concentrations were determined by ultraviolet spectroscopyusing an extinction coefficient of 5720 cm-' M-' at the wavelength of maximum absorbance in the near-ultraviolet region (Kosen et al., 1981). Solutions of DTT; were filtered through Gilman Acrodisc assemblies. Concentrations of DTT; solutions were determined using an extinction coefficient of 110 cm-' M-' at 310 nm (Creighton, 1975a). Concentrations of DTTgfi, GSH, and GSSG were based on dry weight measurements. Solutions of 10 mM HC1 and 1.0 M Tris-HC1, pH 9.1 (at 25 "C), 2 M KC1, 10 mM EDTA were degassed and saturated with argon. Protein and redox reagents were dissolved separately in 10 mM HC1 and mixed with (one-tenth final volume) Tris/KCl/EDTA buffer plus sufficient HC1 so that final solution conditions were 0.1 M Tris-HC1, pH 8.7 (at 25 "C), 0.2 M KC1, 1 mM EDTA, and 30 pM protein. Reactions of greater than 10-min duration were carried out under an argon atmosphere in septum-capped tubes. Folding and unfolding reactions were quenched by addition of an aliquant of the reaction mixture to 0.91 M iodoacetic acid, 0.45 M Tris (free base), 0.91 M KOH, giving a final iodoacetate concentration of 0.18 M, pH -8.5 (at room temperature), After being mixed, quenched solutions were kept at room temperature for 2 min and then placed on ice. A solution of 0.4% methyl green, 25% glycerol, 75% electrophoresis buffer was added to the quenched solutions. The final glycerol concentration was 3.4%. Samples (40-50 pL) were electrophoresed through 15% nondenaturing polyacrylamide gels (Reisfeld et al., 1962) which contained piperazine diacylamide in place of N,N'-methylenebisacrylamide. The electrophoresis apparatus's glass plates were silanized with Sigmacote (Sigma Chemical Co.) to prevent the gels from adhering to the glass. Electrophoresis was performed at 2-4 "C for 2.5-3 h at 80 V, followed by 4 h at 300 V. Gels were stained and destained as described previously by Creighton and Goldenberg (1984). After being destained, dried gels were scanned with a Hoefer scanning densitometer interfaced to a Macintosh I1 computer (Apple Computer Corp.) running the Hoefer GS-370 data system software. Relative areas of stained protein bands were determined using the software's manual integration mode. Kinetics of folding and unfolding of Ala30/Ala5 1 were simulated using the software package Stella, running on a Macintosh 11. Fourth-order Runge-Kutta numerical integration was performed with a time step of 0.1. Decreasing the time step of 0.01 did not alter the simulation results. Rate constants were adjusted until simulations matched observed time dependencies for the relative amounts of the kinetic species.
Kosen et al.
5708 Biochemistry, Vol. 31, No. 25, 1992 Table I: Summary of Control Alkylation Experiments % of 7% of alkylation conditions (14-38, 5-55) (5-55) % of I Sb of R 15 28 48 8 0.2 M iodoacetate 21 24 46 9 0.4 M iodoacetate 16 24 52 8 1.0 M iodoacetate 17 25 48 10 0.18 M iodoacetate‘ 39 i 2 6.8 f 0.8 23 i 3 31 i 2 0.18 M iodoacetateb “Values obtained from the control experiment. bValues obtained from kinetic experiments following the reduction and unfolding of Ala30/Ala5 1 in the presence of 0.1 mM DTTgi. The 5-min time point is reported. Uncertainties in the measurements are rewrted as standard deviations.
Control Alkylation Experiments. To determine if standard alkylation conditions accurately trapped the kinetic species accumulating during Ala30/Ala5 1 folding and unfolding, the following experiment was performed. Four samples of Ala30/Ala51 were reduced with 0.1 mM DTT:; for 5 min. Then, the usual quench solution was added to one sample, and the following were added to the other three: one-fourth volume of 1 M iodoacetate, 0.5 M Tris-HC1, pH 8.7; one-fourth volume of 2 M iodoacetate, 0.5 M Tris-HC1, pH 8.7; or one-half volume of 2 M iodoacetate, 0.2 M Tris-HC1, pH 8.7. (The pH of the latter three quench solutions was adjusted at room temperature.) After 2 min at room temperature for the first two samples and 1 min at room temperature for the second two, the solutions were frozen until salts could be removed using a FPLC G-25 fast desalting column equilibrated with 10 mM HC1. After lyophilization, a portion of each protein mixture was separated by polyacrylamide gel electrophoresis and the relative amounts of the kinetic species were determined by densitometry. Table I summarizes the experimental results. No systematic differences in the relative amounts of the kinetic species were noted. Differences between control experiments and the average of the kinetic experiments may be ascribed to high concentrations of salts present during electrophoresis which somewhat distort the protein bands’ shapes and thus decrease the accuracy of integration (in kinetic experiments) or to selective loss of unfolded protein on desalting (in control experiments).
RESULTS Characterization of the Disulfide Bond in (Cam14/Cam38; AIa30/Ala5I). Endoproteinase Lys C peptide maps of tetra(carbamoylmethyl)-Ala3O/Ala51 and (Cam14/Cam38; Ala30/Ala51) are shown in Figure 2. Using molecular masses determined by cesium ion liquid secondary ion mass spectroscopy, the labeled peptides in the chromatogram of tetra(carbamoylmethyl)-Ala3O/Ala5 1 (Figure 2; lower tracing) were identified as the five unique fragments expected of endoproteinase Lys C proteolysis. These fragments are peptide 1 (residues 42-46), peptide 2 (residues 47-58), peptide 3 (residues 16-26 and 27-41; two peptide fragments), and peptide 4 (residues 1-15). Peptides 2 and 4, containing res-
.
Time
FIGURE2: Reverse-phase chromatography of endoproteinase Lys C peptides of tetra(carbamoylmethyl)-Ala30/AlaS 1 (lower tracing) and (Cam14/Cam38; Ala30/Ala51) (upper tracing). Identities of the labeled peaks are given in the text. The unlabeled peak following that labeled 3 in the upper tracing was shown to be residues 27-46 by mass spectrometry.
idues 47-58 and 1-15, respectively, were missing from the peptide map of (Cam14/Cam38; Ala30/Ala51). A new peptide appeared in that map, labeled as 4a (Figure 2; upper tracing). Peptides 1 and 3 of the tetra(carbamoylmethy1)Ala30/Ala5 1 and (Cam14/Cam38; Ala30/Ala51) peptide maps were shown to be identical by mass spectrometry. The monoisotopic mass of peptide 4a (2944.4 Da) was that expected for residues 1-15 and 47-58 joined by a disulfide. To determine whether cysteine 5 or cysteine 14 formed the disulfide with cysteine 5 5 , peptide 4a was incubated in 10 pL 0.01% NH,(aq) for 5 min. Then, an of 20 mM DTT:!, aliquant of this mixture was added to the matrix on the probe of the tandem mass spectrometer. A peak was present in the normal mass spectrum of the mixture at 1779.8 Da (monoisotopic mass) corresponding to the protonated peptide consisting of residues 1-15. This peak was selected in the first mass spectrometer and subjected to collision-induceddissociation with He gas. The fragment ions were analyzed in the second mass spectrometer. As seen in Table 11, the collision-induced dissociation spectrum is entirely consistent with a cysteine at position 5 and a (carbamoy1methyl)cysteine at position 14. Therefore, the single disulfide in (Cam14/Cam38; Ala30/Ala51) links cysteines 5 and 5 5 . Reduction of the 5-55 Disulfide in (Cam14/Cam38; Ala30/Ala51) by D T C ; . Figure 3 shows the electrophoretic pattern of reduced and oxidized (Cam14/Cam38; Ala30/
Table 11: Observed Sequence Ions in the High-Energy Collision-Induced Dissociation Mass Spectrum of Residues 1-15 (MH+ = 1779.8 Da) Derived from the Disulfide-Linked Fragments 1-1 5 and 47-58’ a 341 488 591 704 833 930 1190 1291 1348 1445 1605 b 254 369 619 732 861 1055 1218 1319 1633 C 533 749 1235 1336 1490 1650 559 1516 d Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cam Lys W 1102 973 779 x 1437 1187 y 1179 1526 1411 919 725 2 1145 Fragment ion masses in the table are nominal monoisotopic masses in daltons. The data confirm unequivocally that residue 5 is Cys and residue 14 is Cam. The nomenclature used follows that of Biemann (1990). Cam = (carbamoylmethy1)cysteine. ~~~~
Biochemistry, Vol. 31, No. 25, 1992 5709
Folding Kinetics of BPTI Mutants Scheme I11
SSG 'SSG
c-Reduced
GSH
protein
GSSG-'
Rs,SH
+ GSSG
3 1 1 II
kex
S&REr
(5-55)+ GSH
k-I
f
-
GSH
(5-55)
0.5
1
1.5
3
2
4
Time (min) FIGURE 3:
Electrophoretic pattern of species trapped by alkylation with iodoacetate during the reduction of the 5-55 disulfide of (Cam14/Cam38; Ala30/Ala51) by 10 mM DTT;! at 25 "C, pH 8.7. The initial concentration of (Cam1 4/Cam38; Ala30/Ala5 1) was approximately 27 pM. Quench times are indicated in the figure. Positions of (Cam14/Cam38; Ala30/Ala51), as ( 5 - 5 9 , and reduced protein blocked with carbamoylmethyl groups at cysteines 14 and 38 and carboxymethyl groups at cysteines 5 and 55 are indicated in the figure. The unlabeled band, migrating more rapidly than reduced protein, is contaminating protein containing carbamoylmethyl groups at all four cysteines. This material comigrated with (Cam14/Cam38; Ala30/Ala5 1) during reverse-phase chromatography; as it does not interfere with kinetic analysis, further purification was not attempted. 100
2 IE, s -x
am 4
-x
0
200
400
600
800
1000
1200
Time (sec) FIGURE 4: Semi-logarithmicplot of the percentage of (Cam14/Cam38; Ala30/Ala5 1) present versus time after addition of DTT;;, pH 8.7, 25 "C. The concentrations of DTTE; used were 1 mM (a),2 mM (0),4 mM (+), 6 mM (O), and 10 mM (A). Error bars are standard deviations of experimental values. Solid lines are linear least-squares fits of the data, and slopes of these lines are pseudo-first-order rate constants.
Ala51) trapped by carboxymethylation after incubation times were increased with an initial concentrationof 10 mM DTT;;. The rate of reduction of the 5-55 disulfide in the presence of 10 mM DTTEZ followed pseudo-first-order kinetics, and similar kinetics were observed over a 10-fold DTT:; concentration range (Figure 4). When pseudo-first-order rate constants, obtained from slopes of the curves in Figure 4, are plotted against DTT;; concentration, the relationship between the two variables is a straight line with a slope of 1.0 s-l M-l. This value is the second-order rate constant (k1) for reduction of the 5-55 disulfide in (Cam14/Cam38; Ala30/Ala51) by DTT;;. Oxidation of the 5-55 Disulfide in (Cam14/Cam38; Ala30/Ala51) in a Mixed Glutathione Redox Environment.
Table 111: Intramolecular Rate Constants for Folding of (Cam14/Cam38; Ala30/Ala51) in the Presence of Oxidized and Reduced Glutathione [GSHI (M) [GSSGI (M) kintra' (s-') 0.023 0.0 1 0.0001 0.005 0.0073 0.0 1 0.0027 0.01 0.01 0.024 0.015 0.001 0.0005 0.023 0.02 0.014 0.00 1 0.0005 Calculated using ea 1. Calculated using ea 2.
kintrab (8')
0.033 0.029 0.024 0.030 0.028 0.026
Formation of the 5-55 disulfide by DTT: occurs extremely slowly. It is therefore difficult to measure accurately the rate of 5-55 disulfide formation in the presence of DTT:. GSSG is a more powerful oxidizing reagent, with a greater redox potential, than DTT:. It will promote disulfide formation in cases where reaction with DTT; occurs slowly or not at all (Creighton & Goldenberg, 1984; Goldenberg, 1988). The mechanism of formation of the 5-55 disulfide in the presence of GSSG is expected to follow Scheme 111. The rate constants kintraand kl have been defined above. The value for kl is assumed to be identical for reduction of the 5-55 disulfide by DTT;; and GSH (Creighton & Goldenberg, 1984). The rate constant kexreflects an average rate of formation of the mixed disulfide between oxidized glutathione and either one of the two protein thiols. Values of 2 and 1/2 are statistical correction factors for formation and reduction of the initial mixed disulfide. Scheme I11 includes the possibility that RgzE, with mixed disulfides at both cysteines 5 and 55, and/or R';: with a mixed disulfide at either cysteine 5 or 55 accumulate(s). A value for kintracan be determined by following the kinetics of folding of reduced (Cam14/Cam38; Ala30/Ala51) in the presence of a mixed redox buffer containing GSSG and GSH (Goldenberg, 1988), by determining the third-order rate constant for reduction of the 5-55 disulfide by GSH (Creighton & Goldenberg, 1984) or by determining the relative amounts of reduced and oxidized protein present at equilibrium. We used the equilibrium experiment to determine kintra. Reduced (Cam14/Cam38; Ala30/Ala5 1) was incubated with six different mixtures of GSSG and GSH (Table 111) for 2 h under standard folding conditions. Then, iodoacetate was added and a portion of the mixture was subjected to electrophoresis. Fractions of reduced (fRtaI) and oxidized (f&55)) (Cam14/Cam38; Ala30/5 1) were determined by densitometry. For Scheme 111, if khm>> 2kex[GSSG],then at equilibrium, kintra will be related to kl,[GSSG], [GSH], fRldll, and&) as &S-SS)k-l [GSH12 (1) kintra = f ~ , ,x, 4 [GSSGI On the other hand, if kintra
