Folding disulfide-containing proteins faster with an aromatic thiol

Article abstract of DOI:10.1021/ja016938p

The traditional method for in vitro folding of disulfide-containing proteins is slow and involves a redox buffer of glutathione and glutathione disulfide. To increase the folding rate and to gain insight into the folding process, we replaced glutathione, an aliphatic thiol, with a commercially available aromatic thiol, 4-mercaptobenzeneacetate (1). Aromatic thiol 1 was selected due to its enhanced nucleophilicity and its enhanced leaving-group ability relative to glutathione at pH 7.7. To demonstrate the advantages of 1, the folding of reduced and scrambled RNase A at pH 7.0 and 7.7 in the presence of 1 and glutathione was investigated. For each set of folding conditions, the optimum concentration of each thiol was initially determined and then the folding rates in the presence of each thiol were measured concurrently. In all cases examined, the folding rate enhancement with the aromatic thiol was 5- 6-fold. Furthermore, under similar conditions folding rates were almost identical with either reduced or scrambled RNase A. In addition the 5-6-fold folding rate enhancement varied only slightly with pH, 7.0 vs 7.7.

Full text of DOI:10.1021/ja016938p

Published on Web 03/22/2002
Folding Disulfide-Containing Proteins Faster with an Aromatic
Thiol
Jonathan D. Gough, Rhondye H. Williams, Jr., Anthony E. Donofrio, and
Watson J. Lees*
Contribution from the Department of Chemistry, Syracuse UniVersity, Syracuse, New York 13244
Received August 25, 2001. Revised Manuscript Received February 9, 2002
Abstract: The traditional method for in vitro folding of disulfide-containing proteins is slow and involves a
redox buffer of glutathione and glutathione disulfide. To increase the folding rate and to gain insight into
the folding process, we replaced glutathione, an aliphatic thiol, with a commercially available aromatic
thiol, 4-mercaptobenzeneacetate (1). Aromatic thiol 1 was selected due to its enhanced nucleophilicity
and its enhanced leaving-group ability relative to glutathione at pH 7.7. To demonstrate the advantages of
1, the folding of reduced and scrambled RNase A at pH 7.0 and 7.7 in the presence of 1 and glutathione
was investigated. For each set of folding conditions, the optimum concentration of each thiol was initially
determined and then the folding rates in the presence of each thiol were measured concurrently. In all
cases examined, the folding rate enhancement with the aromatic thiol was 5- 6-fold. Furthermore, under
similar conditions folding rates were almost identical with either reduced or scrambled RNase A. In addition
the 5-6-fold folding rate enhancement varied only slightly with pH, 7.0 vs 7.7.
Scheme 1
Introduction
Almost all pharmaceutically relevant proteins and many
extracellular proteins contain disulfide bonds. To obtain these
proteins in biologically active form, it is often necessary to
employ in vitro folding of inclusion bodies or synthetic
peptides.1-4 A significant challenge for the in vitro folding
process is the rapid formation of the correct disulfide bonds
between cysteine residues. Disulfide bonds are the only covalent
bonds formed during in vitro folding and their formation is
usually the rate-determining step. Formation of native disulfide
bonds involves thiol-disulfide interchange reactions. These
interchange reactions occur within the protein itself (Scheme
1) or between the protein and the redox buffer (Scheme 2),
which is a mixture of small-molecule thiols (RSH) and disulfides
(RSSR). In vitro, the small molecule thiol is usually dithio-
threitol (DTT, pKa ) 9.2, 10.1), mercaptoethanol (ME, pKa )
9.6), or a cysteine derivative such as glutathione (pKa ) 8.7).
Surprisingly, very few other thiols have been investigated as
redox buffers.
Scheme 2
a low pKa value, 6.7, and is exceptionally reactive with disulfides
compared to small molecule aliphatic thiols, such as cysteine
derivatives.7,8 However, the use of PDI for in vitro folding is
usually prohibitive as it is expensive, requires the use of
approximately stoichiometric amounts relative to the protein of
interest, and requires an additional protein purification step after
catalysis.9 The dithiol nature of PDI has recently been modeled
with a small molecule, which was shown to increase the yield
of active protein but not the rate constant for folding.10
To increase the rate of in vitro folding of disulfide-containing
proteins, we envisioned utilizing a small-molecule thiol with a
thiol pKa value similar to that of the solvent-exposed thiol in
PDI. Furthermore, our rationally designed thiol should have
enhanced reactivity toward disulfides at pH 7 relative to
In vivo, the thiol-disulfide interchange reaction is catalyzed
by protein disulfide isomerase (PDI). Each active site of PDI
contains two cysteine thiol groups in a CXXC motif (two
cysteines separated by two amino acids);5 one thiol is solvent-
exposed and the other is buried.6 The solvent-exposed thiol has
(1) Rudolph, R.; Lilie, H. FASEB J. 1996, 10, 49-56.
(2) Chaudhuri, J. B. Ann. N.Y. Acad. Sci. 1994, 721, 374-85.
(3) De Bernardez Clark, E. Curr. Opin. Biotechnol. 1998, 9, 157-63.
(4) Woycechowsky, K. J.; Raines, R. T. Curr. Opin. Chem. Biol. 2000, 4, 533-
39.
(5) Edman, J. C.; Ellis, L.; Blacher, R. W.; Roth, R. A.; Rutter, W. J. Nature
(London) 1985, 317, 267-70.
(6) Kemmink, J.; Darby, N. J.; Dijkstra, K.; Nilges, M.; Creighton, T. E.
Biochemistry 1996, 35, 7684-91.
(7) Hawkins, H. C.; Freedman, R. B. Biochem. J. 1991, 275, 335-9.
(8) Darby, N.; Creighton, T. E. Biochemistry 1995, 34, 16770-80.
(9) Lyles, M. M.; Gilbert, H. F. Biochemistry 1991, 30, 613-19.
(10) Woycechowsky, K. J.; Wittrup, K. D.; Raines, R. T. Chem. Biol. 1999, 6,
871-9.
9
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J. AM. CHEM. SOC. 2002, 124, 3885-3892
3885

A R T I C L E S
Gough et al.
Figure 1. Rate constant, k, and maximum percent activity, A, for the folding of scrambled RNase A versus varying concentrations of thiol, RSH: glutathione
(squares) and 1 (circles). Assays were performed at pH 7.0 or 7.7 (as indicated on the graph) and 25 °C in the presence of 0.2 mM GSSG and 1 mM EDTA.
Maximum percent activity, A, was normalized to 100% for each compound.
glutathione. To accomplish our goal we prepared aromatic thiols,
which despite advantageous properties have never been inves-
tigated previously for protein folding. Aromatic thiols have
lower pKa values (pKa ) 3-7) than aliphatic thiols (pKa )
7-11) and are more reactive in thiol-disulfide interchange
reactions than aliphatic thiols with similar pKa values.11-14
Herein, we report the ability of an aromatic thiol to increase
the folding rate of reduced and oxidized RNase A relative to
glutathione. Initial studies determined the optimum concentration
of aromatic thiol and glutathione required for protein folding.
The protein folding rates of the two thiols (aromatic thiol and
glutathione) at their optimum concentrations were then measured
side by side. The effect of pH on the relative and absolute
folding rates was then examined.
by Konishi and Scheraga.17,18 In the assay detailed herein, the
wavelength of observation was shifted from 286 to 292 nm to
lower the background absorbance of aromatic thiol and 2′,3′-
cyclic CMP (cCMP). In some cases, reduced RNase A was
replaced with scrambled RNase A, an oxidized form of RNase
A with a random distribution of four disulfide bonds.19
A
continuous assay was not practical due to the large background
absorbance of the aromatic thiol.9 Protein folding rate constants
are illustrated in Figures 1 and 2 and were determined at either
pH 7.0 (0.10 M Bis-tris propane acetate) or 7.7 (0.10 M Tris
acetate).
To determine the folding rate constant of scrambled RNase
A (Figure 1), aliquots were withdrawn at prescribed times from
a mixture at 25 °C containing 0.025 mM scrambled RNase A,
0.2 mM glutathione disulfide (GSSG), 1.0 mM EDTA, and
varying concentrations of reduced glutathione (GSH) or 1. The
concentration of 0.2 mM glutathione disulfide was chosen
because Lyles and Gilbert9 had shown it to be the optimum
concentration for PDI-catalyzed folding of fully reduced RNase
A at pH 8. The EDTA was added to suppress metal-catalyzed
oxidation of the thiols by oxygen. The aliquots were immediately
assayed for RNase A activity by following the hydrolysis of
cCMP at pH 5 for 2 min. The solution was lowered to pH 5
and the redox reagents were diluted in an effort to minimize
any folding during the 2 min of the assay.9,20,21 A plot of RNase
A activity versus time was fitted to a single exponential defined
as percent activity ) A(1 - e-kt), where percent activity is
proportional to the activity of fully folded native RNase A
(Figure 3). The constants A and k represent the maximum
Results
The selection of the aromatic thiol was based upon its thiol
pKa value and water solubility. The pKa value of the aromatic
thiol should be similar to that of PDI (6.7).7 Because most
protein folding assays are performed in aqueous media at close
to neutral pH, the aromatic thiol chosen must be water-soluble
at pH 7 in both the thiol and disulfide forms. Compound 1,
4-mercaptobenzeneacetate (pKa ) 6.6),14 was selected on the
basis of these criteria and was purchased or synthesized
following literature procedures.15,16 The effect of 1 on the rate
of protein folding compared to glutathione (pKa ) 8.7) was
determined by a variation of the discontinuous assay developed
(11) Wilson, J. M.; Bayer, R. J.; Hupe, D. J. J. Am. Chem. Soc. 1977, 99, 7922-
6.
(12) Szajewski, R. P.; Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 2011-
(17) Crook, E. M.; Mathias, A. P.; Rabin, B. R. Biochem. J. 1960, 74,
234-8.
26.
(13) Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P. J. Org. Chem. 1977,
42, 332-8.
(18) Konishi, Y.; Scheraga, H. A. Biochemistry 1980, 19, 1308-16.
(19) Hillson, D. A.; Lambert, N.; Freedman, R. B. Methods Enzymol. 1984,
107, 281-94.
(14) DeCollo, T. V.; Lees, W. J. J. Org. Chem. 2001, 66, 4244-4249.
(15) Aultz, D. E.; Helsley, G. C.; Hoffman, D.; McFadden, A. R.; Lassman, H.
B.; Wilker, J. C. J. Med. Chem. 1977, 20, 66-70.
(16) Tagawa, H.; Ueno, K. Chem. Pharm. Bull. 1978, 26, 1384-93.
(20) Scheraga, H. A.; Konishi, Y.; Rothwarf, D. M.; Mui, P. W. Proc. Natl.
Acad. Sci. U.S.A. 1987, 84, 5740-4.
(21) Creighton, T. E. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5082-6.
9
3886 J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002

Folding Disulfide-Containing Proteins Faster
A R T I C L E S
Figure 2. Rate constant, k, and maximum percent activity, A, for the folding of reduced RNase A versus varying concentrations of thiol, RSH: glutathione
(squares) and 1 (circles). Assays were performed at pH 7.0 or 7.7 (as indicated on the graph), and 25 °C in the presence of 0.5 mM GSSG and 1 mM EDTA.
Maximum percent activity, A, was normalized to 100% for each compound.
percent activity and the folding rate constant, respectively. The
percent activity should correspond closely to the percent native
protein, as was indicated previously for glutathione-folding
studies.18 Some of the folding intermediates do possess catalytic
activity,22-25 but these intermediates are likely to be poorly
populated during the folding process and/or possess low specific
activity in the enzymatic assay.22,26,27
Figure 3. Folding of scrambled RNase A (0.025 mM) at the optimum
concentration of glutathione (squares) or 1 (circles). Assays were performed
at pH 7.7 and 25 °C in the presence of 0.2 mM GSSG and 1.0 mM EDTA.
For the second batch of scrambled RNase A, the optimum concentrations
of glutathione and 1 were 2.0 and 6.5 mM, respectively (Table 1).
The same general procedure was used to determine the folding
and 1 were then measured side by side at each pH value (Tables
1-3, Figure 3). In addition, at pH 7.7 the folding rates were
measured side by side at a constant thiol concentration (2 mM)
which is the optimum concentration of glutathione for folding
scrambled RNase A at pH 7.7 (Table 4). The complete process,
including determining the optimum concentrations, was repeated
for each batch of protein investigated. The results obtained from
the folding experiments with glutathione at pH 7.7 were
consistent with published values for scrambled (0.010 or 0.006
rate constant of reduced RNase A (0.025 mM) (Figure 2) with
the exception of increasing the concentration of GSSG to 0.5
mM. Because folding of reduced RNase A to native protein
results in the net consumption of 4 equiv of GSSG, a higher
initial concentration of GSSG was employed to maintain relative
uniformity.
To elucidate the advantage of using aromatic thiols to fold
proteins relative to traditional methods, the optimum concentra-
tions of glutathione or 1 for protein folding at pH 7.0 and 7.7
were determined (Figures 1 and 2). The optimum concentration
was defined as the concentration of thiol at which the initial
rate of protein folding, A × k, was at a maximum. The rates of
protein folding at the optimum concentrations of glutathione
min-1 10,28
)
or reduced RNase A (0.007 or 0.02 min-1 9,29
)
obtained at or near pH 7.7.
To establish the validity of the data, several control experi-
ments were performed. (1) To ensure that the rate increase was
independent of the batch of scrambled RNase (Tables 1 and 2),
several batches were tested, including one from commercial
sources (batch 1). The relative rates of protein folding were
always within error of each other, although the absolute rate
(22) Laity, J. H.; Shimotakahara, S.; Scheraga, H. A. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 615-19.
(23) Klink, T. A.; Woycechowsky, K. J.; Taylor, K. M.; Raines, R. T. Eur. J.
Biochem. 2000, 267, 566-572.
(24) Sperling, R.; Burstein, Y.; Steinberg, I. Z. Biochemistry 1969, 8, 3810-
20.
(25) Creighton, T. E. FEBS Lett. 1980, 118, 283-8.
(26) Konishi, Y.; Ooi, T.; Scheraga, H. A. Biochemistry 1981, 20, 3945-55.
(27) Creighton, T. E. J. Mol. Biol. 1979, 129, 411-31.
(28) Walker, K. W.; Gilbert, H. F. J. Biol. Chem. 1997, 272, 8845-8.
(29) Hantgan, R. R.; Hammes, G. G.; Scheraga, H. A. Biochemistry 1974, 13,
3421-31.
9
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A R T I C L E S
Gough et al.
Table 1. Folding of Three Separate Batches of Scrambled RNase
Table 4. Folding of Reduced and Scrambled RNase A at pH 7.7
A at pH 7.0a
and 2.0 mM Thiola
concn
(mM)
concn
(mg/mL)
k × 103
(min-1
concn
(mM)
concn
(mg/mL)
k × 103
(min-1
additive
A (%)
Batch 1
)
relative rate
additive
A (%)
)
relative rate
Scrambled RNase A
glutathione
1
1.0
6.0
0.31
1.0
105 ( 15
97 ( 21
2.1 ( 1.0
14 ( 2
1
glutathione
1
2.0
2.0
0.61
0.34
102 ( 5
98 ( 4
6.7 ( 1.8
14 ( 6
1
6.9 ( 2.7
2.2 ( 0.4
Batch 2
Reduced RNase A
glutathione
1
1.5
5.0
0.46
0.84
98 ( 19
100 ( 15
2.8 ( 0.7
13.8 ( 0.9
1
glutathione
1
2.0
2.0
0.61
0.34
115 ( 7
93 ( 5
7.0 ( 1.3
18 ( 2
1
5.2 ( 1.4
2.6 ( 0.5
Batch 3
117 ( 13
107 ( 4
a See footnote a in Table 2. The error was determined from three assays,
three side-by-side runs.
glutathione
1
1.5
3.0
0.46
0.50
2.0 ( 0.1
11.2 ( 0.3
1
5.7 ( 0.3
Table 5. Folding of SRNase A with Various Disulfides at pH 7.7a
Overall Average
glutathione
1
1.33
4.67
0.41
0.78
107 ( 10
101 ( 5
2.3 ( 0.4
13 ( 2
1
disulfide
additive
1
(mM)
disulfide
(mM)
k × 103
(min-1
5.8 ( 0.9
A (%)
)
relative rate
a For the results from each batch the error corresponds to the 95%
confidence limit, ts/N0.5, where N is the number of data points, s is the
standard deviation, and t is from the t-test table. The error was determined
from three assays for batch 1 and six assays (three duplicate side by side
runs comparing glutathione to 1) for batches 2 and 3. The error for the
overall average is the standard deviation when each batch is treated as a
single point.
GSSG
2
no disulfide
4.0
4.0
4.0
0.2
0.2
0.0
102 ( 28
91 ( 32
26 ( 6
25 ( 6
28 ( 6
1
1.0 ( 0.1
1.1 ( 0.1
102 ( 28
a See footnote a in Table 2. The error was determined from three assays,
three side-by-side runs.
Table 6. Folding of Reduced RNase A with Various Disulfides at
Table 2. Folding of Scrambled RNase A at pH 7.7a
pH 7.7a
concn
(mM)
concn
(mg/mL)
k × 103
(min-1
disulfide
additive
1
(mM)
disulfide
(mM)
k × 103
(min-1
additive
A (%)
Batch 2
)
relative rate
A (%)
)
relative rate
GSSG
GSSG
2
4.0
4.0
4.0
0.5
0.2
0.2
113 ( 9
112 ( 20
104 ( 22
26 ( 11
33 ( 5
33 ( 3
0.8 ( 0.2
1
1.0 ( 0.2
glutathione
2.0
6.5
0.61
1.1
90 ( 18
94 ( 15
8.2 ( 0.5
40 ( 4
1
1
4.9 ( 0.4
Batch 3
a See footnote a in Table 2. The error was determined from three assays,
three side-by-side runs.
glutathione
1
2.0
5.0
0.61
0.84
94 ( 3
84 ( 14
5.0 ( 0.6
25 ( 1
1
5.0 ( 0.8
Average
glutathione
1
2.0
5.8
0.61
1.0
92
89
6.6
32
1
5.0
not stimulating or inhibiting the native enzyme in the cCMP
assay, aliquots were removed from a mixture containing native
RNase A (0.025 mM), 1 (15 mM), and glutathione disulfide
(0.2 mM) at prescribed times and the enzyme’s activity was
determined with the cCMP assay. No measurable change in
activity was observed with time. The activity was the same as
that of a 0.025 mM solution of native RNase A. (4) In fitting
the data to the equation percent activity ) A(1 - e-kt), it is
assumed that scrambled RNase A itself has little or no activity.
To verify this assumption, the activity of a scrambled RNase A
solution (0.025 mM) containing no thiols was measured.
Essentially no activity was found.
a The error corresponds to the 95% confidence limit, ts/N0.5, where N is
the number of data points, s is the standard deviation, and t is from the
t-test table. The error was determined from six assays, three duplicate side
by side runs comparing glutathione to 1. Batch 1, which was obtained from
commercial sources, became unavailable from the vendor during the course
of this work.
Table 3. Folding of Reduced RNase A at pH 7.0 and 7.7a
concn
(mM)
concn
(mg/mL)
k × 103
(min-1
additive
A (%)
pH 7.0
114 ( 21
98 ( 4
)
relative rate
glutathione
1
2.0
4.0
0.61
0.67
2.0 ( 0.4
11.7 ( 1.2
1
5.9 ( 1.3
To investigate the importance of the disulfide on protein
folding rates, glutathione disulfide was replaced with aromatic
disulfide 2, the oxidized form of 1. Protein folding rates were
determined in the presence of 0.2, 0.5, and 1.0 mM aromatic
disulfide. The optimum concentration of aromatic disulfide was
0.2 mM. At higher aromatic disulfide concentrations the A value
(maximum percent refolded) decreased. When 0.2 mM aromatic
disulfide was employed, the optimum concentration of aromatic
thiol was 4 mM. The folding of RNase A in the presence of
glutathione disulfide or aromatic disulfide 2 was then measured
side by side (Tables 5 and 6). The concentration of 1 in these
experiments was 4.0 mM, as it is close to the optimal value for
each disulfide. The folding of scrambled RNase A in the absence
of disulfide was also investigated. Scrambled RNase A, unlike
reduced RNase A, does not require oxidation to form native
RNase A. No significant difference in k or A was detected for
any of these variations in disulfide.
pH 7.7
101 ( 4
93 ( 5
glutathione
1
1.0
7.5
0.31
1.3
7.5 ( 1.2
33 ( 4
1
4.5 ( 0.7
a See footnote a in Table 2.
constants did vary somewhat. (2) To demonstrate that the
enhanced folding rate constant was due to the aromatic thiol
group, we measured protein folding rates at the optimum
concentration of glutathione (determined in the absence of
additives) in the presence of p-hydroxyphenylacetate (5 mM,
pH 7.7, for reduced and scrambled RNase A, and 12 mM, pH
7.7, for scrambled RNase A). p-Hydroxyphenylacetate is an
analogue of 1 in which the thiol group is replaced with a
hydroxyl group. No appreciable change relative to glutathione
alone was observed at either concentration. The folding rate
constant, k, decreased less than 15% and the maximum percent
activity, A, decreased less than 10%. (3) To confirm that 1 was
9
3888 J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002

Folding Disulfide-Containing Proteins Faster
A R T I C L E S
Scheme 3
Discussion
Explanation of the enhanced folding rate constants achieved
with aromatic thiols requires consideration of the reactions that
take place during protein folding. During the in vitro folding
of disulfide containing proteins, eight variations of the thiol-
disulfide interchange reaction can occur (Scheme 3). The first
four reactions in Scheme 3 involve the protein thiolate acting
as the nucleophile. The last four reactions involve the small-
molecule thiolate acting as the nucleophile. These thiolates can
nucleophilically attack a protein disulfide (reactions I and V),
a small-molecule disulfide (reactions II and VI), or a mixed
disulfide between the small-molecule thiol and a protein thiol
(reactions III, IV, VII, and VIII). As detailed below, aromatic
thiols are better nucleophiles, better leaving groups, and better
central thiols than glutathione at pH 7.0 or 7.7.12,14 Therefore,
the observed rate constants for reactions II-VIII are expected
to be greater with aromatic thiol than with glutathione. We
confine our comparison to glutathione, but very similar com-
parisons can be made with either mercaptoethanol or DTT.
The inherent nucleophilicity of aromatic thiolate 1 and
glutathione thiolate is approximately the same, but at pH 7.0
or 7.7 the bulk of the glutathione (pKa ) 8.7) is in the unreactive
thiol form while the bulk of aromatic thiol (pKa ) 6.6) is in the
reactive thiolate form.14 Therefore, at pH 7.0 or 7.7 compound
1 will be a better nucleophile than glutathione.14 Prior work
has shown that, at pH 7.0, the observed rate constant for reaction
with a small-molecule disulfide, 2-pyridyldithioethanol (2-PDE),
is 32 times greater for 1 than for glutathione.14 At pH 7.7, the
observed rate constant is expected to be 9 times greater for 1
than glutathione. The expected observed rate constant at pH
7.7, kobsd(7.7), is obtained from the observed rate constant at
pH 7.0, kobsd(7.0), and the proportion of the thiol in the reactive
Scheme 4
thiolate form, [1/(1 + 10pK pH], at pH 7.0 and 7.7. Thus, at
a-
pH of the solution. If the nucleophile is an aromatic thiol, then
the constant in eq 1 is increased by 0.9 from 6.3 to 7.2 to account
for the greater nucleophilicity of aromatic thiols relative to
aliphatic thiols of similar thiol pKa values.12 From eq 1, rate
constants with compound 1 as the leaving group are calculated
to be 17 times greater than those with glutathione as the leaving
group, all else being equal.
both pH 7.0 and 7.7 compound 1 is expected to react faster
with disulfides than glutathione.
The leaving-group ability of thiols is inversely correlated to
the pKa of the thiol, and 1 is thus a better leaving group than
glutathione because 1 has a lower pKa value.12,14 The relative
leaving group ability of the two thiolates can be qualitatively
compared. Equation 1 represents a formula developed to predict
the rate of thiol-disulfide interchange reactions.12 In eq 1, k is
log k ) 6.3 + 0.59pKanuc - 0.40pKac - 0.59pKalg (1)
nuc
the rate constant of the reaction, pKa
is the pKa of the
c
log kobsd ) log k - log(1 + 10pK -pH
)
(2)
a
conjugate acid (thiol) of the nucleophile (thiolate), pKa is the
pKa of the conjugate acid (thiol) of the central sulfur, and pKa
lg
is the pKa of the conjugate acid (thiol) of the leaving group
(thiolate); see Scheme 4. The rate constant k can be converted
to kobsd by taking into account the proportion of the nucleophile
in the reactive thiolate form, eq 2. In eq 2, pKa corresponds to
the pKa of the nucleophile (pKanuc) and pH corresponds to the
The reaction rates of thiol-dusulfide interchange reactions
are also inversely correlated with the pKa of the central thiol
(Rc, Scheme 4).12 Again according to eq 1, the rate constant of
thiol-disulfide interchange reactions in which compound 1
corresponding to the central sulfur should be approximately 7
9
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A R T I C L E S
Gough et al.
Scheme 5
The portion of species in the preequilibrium mixture that can
be directly and rapidly converted to native RNase A is critical
for the overall rate of protein folding. We will refer to these
species as productive intermediates. For the folding of RNase
A with DTT, the relative concentration of the 3S species,
excluding des[40-95] and des[65-72], within the preequilib-
rium mixture is crucial. For the folding of RNase A with DTT
or monothiols, such as glutathione, the relative portion of
productive intermediates will vary with the composition of the
redox buffer. If the concentration of the small molecule thiol
(RSH) is higher than optimal, then the portion of productive
intermediates is reduced, presumably by inhibiting the formation
of disulfide bonds. As a result, at high glutathione concentrations
folding rates of RNase A decrease even though the rates of many
of the reactions involved actually increase. Therefore, increasing
the rate of the eight thiol-disulfide interchange reactions in
Scheme 3 is likely very desirable but may not result in an overall
increased rate of protein folding due to changes in the composi-
tion of the preequilibrium mixture.
The composition of the preequilibrium mixture will be
dependent on the concentration of the redox buffer thiol in the
protonated SH form. The equilibrium constant for the formation
of mixed disulfide with the protein is shown in eq 3, where
PSSR is the mixed disulfide between protein and small-molecule
thiol, RSH is the small-molecule thiol, RSSR is the small-
molecule disulfide, and PSH is a free thiol group on the protein.
The redox potential is proportional to ln([RSH]2/[RSSR]). In
both cases it is the concentration of the small-molecule thiol in
the protonated form that is important (Scheme 5). Thus, in
comparing results at the optimum concentrations of 1 and
glutathione, we report below both the total concentration of thiol
and the concentration of thiol in the protonated form. The
concentration of the thiol in the protonated form will be a
function of the total thiol concentration, the thiol pKa value,
and the pH of the solution.
Figure 4. Folding pathway of RNase A in the presence of DTT proposed
by Scheraga et al.32-34 The folding pathway involves reduced RNase A
(R); RNase A with one (1S), two (2S), three (3S), or four diuslfide (4S)
bonds; native RNase A (N); and RNase A with three native disulfide bonds
but lacking the disulfide bond between amino acids 40 and 90 (des[40-
95]) or between amino acids 65 and 72 (des[65-72]. A minor pathway
between the 2S species and the two des species exists but is not shown.
times faster than those with glutathione, all else being equal.
The rate constant for reaction I in Scheme 3, which does not
involve the small-molecule thiol, should be unaltered by the
choice of aromatic thiol or glutathione.
On the basis of the above analysis, aromatic thiol 1 is
predicted to increase the observed rate constants for seven of
the eight thiol-disulfide interchange reactions (reactions II-
VIII) relative to glutathione. Thus, aromatic thiol 1 might be
expected to enhance the folding rate of disulfide-containing
proteins considerably relative to glutathione at pH 7.0 or 7.7.
However, increasing the folding rate of disulfide-containing
proteins is much more complex than increasing the rates of these
eight reactions in isolation.9,30,31 For example, increasing the
concentration of glutathione beyond a certain point only
decreases the folding rate of RNase A, although it should
increase the rate of many of the eight reactions.9 This result
can be understood on the basis of the mechanism of protein
folding. The formation of native RNase A, which contains four
correctly matched disulfide bonds, from reduced RNase A in
the presence of a redox buffer has been well studied.27,32-34
The work has concentrated on the folding pathway in the
presence of glutathione or DTT, a less efficient catalyst for
folding RNase A. In the presence of oxidized DTT, reduced
RNase A is rapidly converted into a mixture containing many
different protein species such as reduced RNase A (R) and
RNase A with one (1S), two (2S), three (3S), or four (4S)
disulfide bonds (Figure 4).26,27,32-34 The species within the
mixture reach a quasi-equilibrium state (preequilibrium mixture).
The 3S species, excluding des[40-95] and des[65-72], are then
transformed via rate-determining steps to des[40-95] and
des[65-72], which are native RNase A lacking either the
disulfide bond between amino acids 40 and 95 or between amino
acids 65 and 72, respectively. The two des species are then
converted to native RNase A. The folding pathway of RNase
A in the presence of glutathione, a monothiol, is similar to that
with DTT, a dithiol. A preequilibrium mixture is formed and
then via rate-determining steps native RNase A is formed.
However, the rate-determining steps may be different with
monothiols. Also, the preequilibrium mixture probably will
contain a greater proportion of mixed disulfides between protein
and redox buffer. Mixed disulfides between proteins and DTT
usually have a fleeting existence due to an intramolecular
displacement by the second thiol of DTT.
K(mixed disulfide) ) [PSSR][RSH]/([RSSR][PSH]) (3)
On the basis of the preequilibrium analysis it is predicted
that protein folding rates should increase with the concentration
of small molecule thiol, reach a plateau, and then decrease. As
expected, the protein folding rate constants of both reduced and
scrambled RNase A as a function of glutathione concentration
increase to an optimum concentration and then decrease (Figures
1 and 2). The optimum concentration was between 1 and 2 mM,
similar to what had been observed previously at pH 8 with
reduced RNase A.9 With the aromatic thiol the rate constants
increased and then remained uniform as the concentration of
thiol was increased (Figures 1 and 2). However, the initial rate
of protein folding, A × k, in the presence of an aromatic thiol
increased, plateaued, and then decreased. On the basis of the
initial rate of protein folding, the optimum concentrations of
aromatic thiol were approximately 4.5 (pH 7.0) and 6.5 mM
(pH 7.7).
(30) Konishi, Y.; Ooi, T.; Scheraga, H. A. Biochemistry 1982, 21, 4734-40.
(31) Saxena, V. P.; Wetlaufer, D. B. Biochemistry 1970, 9, 5015-23.
(32) Wedemeyer, W. J.; Welker, E.; Narayan, M.; Scheraga, H. A. Biochemistry
2000, 39, 4207-4216.
Comparing the optimum concentrations of thiols in terms of
the concentration of protonated (SH) form results in the optimum
glutathione concentration being greater than that of the aromatic
(33) Welker, E.; Narayan, M.; Wedemeyer, W. J.; Scheraga, H. A. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 2312-2316.
(34) Narayan, M.; Welker, E.; Wedemeyer, W. J.; Scheraga, H. A. Acc. Chem.
Res. 2000, 33, 805-812.
9
3890 J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002

Folding Disulfide-Containing Proteins Faster
A R T I C L E S
thiol. As mentioned previously (Scheme 5), the concentration
of thiol in the protonated form is important in establishing
equilibria. At pH 7.0, the aromatic thiol (pKa ) 6.6) exists
mainly in the deprotonated thiolate form (72%), while gluta-
thione (pKa ) 8.7) exists almost exclusively (97%) in the
protonated form. Thus at pH 7.0, when the concentration of
the protonated form is compared, the optimum concentrations
of aromatic thiol (1.3 mM SH form) and glutathione (1-2 mM)
are very similar. At pH 7.7, the bulk of the aromatic thiol is in
the thiolate form (93%) but glutathione still exists almost
exclusively in the protonated form (91%). The optimum
concentration of aromatic thiol in the protonated form (0.5 mM
) 6.5 mM × 0.07) is slightly less than that of glutathione (0.9-
1.8 mM). However, results obtained at high aromatic thiol
concentrations are affected by the decrease in the maximum
percent refolded, A, which may result in a lowering of the
optimum aromatic thiol concentration.
pH. The folding rate enhancement of aromatic thiols is likely
the result of pH-independent effects such as the enhanced
leaving-group ability of aromatic thiols or the enhanced reactiv-
ity of disulfides in which an aromatic group is attached to the
central thiol (Rc, Scheme 4).
Having increased the folding rate constant of proteins by
replacing glutathione with an aromatic thiol, we investigated
replacing glutathione disulfide with an aromatic disulfide 2
(Table 6). The replacement of glutathione disulfide with 2 in
the protein folding reaction has little to no effect. Therefore,
we recommend the use of glutathione disulfide since it is
commercially available. The lack of an effect is likely the result
of glutathione disulfide being rapidly converted to aromatic
disulfide in the presence of aromatic thiol (4.0 mM). A lower
limit can be placed on the reaction rate. At 30 °C the rate
constant for glycol dimercaptoacetate, which is generally less
reactive than the aromatic thiol, reacting with glutathione
disulfide is 500 M-1 min-1 11-14
Assuming a similar rate
.
At higher concentrations of 1 the folding rate constant was
relatively uniform up to 15 mM (1.1 mM protonated form) but
the maximum percent folding, A, decreased. A decrease in
RNase A activity was not observed when native RNase A was
incubated with the refolding mixture, suggesting that the
decrease in activity is taking place during refolding. The most
likely cause is aggregation of the RNase A folding intermediates
at high aromatic thiol or aromatic disulfide concentrations.
Aggregation is supported by the fact that a white precipitate is
observed in protein folding mixtures containing high concentra-
tions of aromatic thiol but not those containing glutathione at
its optimal concentration.
constant for the aromatic thiol (4.0 mM) reacting with gluta-
thione disulfide (much less than 4.0 mM), the half-life of
glutathione disulfide would be 20 s, in the absence of equilib-
rium effects. The initially formed mixed disulfide between
glutathione and aromatic thiol is most likely more reactive than
glutathione disulfide itself.12 Thus, even in reactions initially
containing glutathione disulfide, the concentration of aromatic
disulfide very quickly becomes significant.35
Combining all the data together, we found that for a similar
set of conditions the folding rate constants of reduced and
scrambled RNase A are almost identical. The similarity in rates
can be explained on the basis of a similarity of preequilibrium
mixtures. Since oxidation of reduced RNase A occurs much
more rapidly than formation of the native form, reduced RNase
A very quickly reaches its preequilibrium state.9,26,29 Presumably,
scrambled RNase A rapidly reaches a similar preequilibrium
state under comparable redox conditions. The folding rate
constants from these two similar preequilibrium states are alike.
In conclusion, we have demonstrated a simple method to
dramatically increase the rate of in vitro folding of disulfide-
containing proteins relative to traditional methods. Improved
folding rates are observed by replacing glutathione with an
aromatic thiol. Aromatic thiols were originally selected because
of their enhanced nucleophilicity and enhanced leaving-group
ability relative to glutathione at pH 7.7. With aromatic thiol 1
the folding rate enhancement is 5-6-fold at either pH 7.0 or
7.7, even though the protein folding rates increase by a factor
of approximately 3 with the increase in pH. The relative
invariance of the folding rate enhancement with pH suggests
that pH-dependent effects such as the concentrations of thiol in
the deprotonated thiolate form and the enhanced nucleophilicity
of the aromatic thiol are not dominant factors in enhancing the
protein folding rate. The enhanced folding rate was not increased
further by replacing glutathione disulfide, the traditional reagent,
with aromatic disulfide 2. Under similar conditions, folding rate
constants and enhanced folding rates were nearly the same for
both reduced and scrambled RNase A.
Once the optimum concentrations of glutathione and aromatic
thiol for protein folding were determined, the relative folding
rates were measured side by side. Aromatic thiol 1 significantly
enhances the folding rate for both reduced and scrambled RNase
A at pH 7.0 and 7.7 relative to glutathione. At both pH values
the rate enhancement is between a factor of 5 and 6 (Tables
1-3). Surprisingly, there is very little variation in the rate
enhancement with pH. For comparable sets of experiments, the
folding rate constant at pH 7.7 is almost 3 times that at pH 7.0.
Explanations for the increase in folding rate constant with pH
include an increase in the concentration of protein thiolate or
small-molecule thiolate, a change in the conformation of the
protein folding intermediates, or a change in the equilibrium
between the folding intermediates. An increase in thiolate
concentration would be expected to increase the reaction rate,
as the thiolate form of the thiol is the active species in thiol-
disulfide interchange reactions (Scheme 3). Since the concentra-
tion of glutathione thiolate increases more dramatically (360%)
than aromatic thiolate (29%) with the change in pH (7.0 to 7.7),
the folding rate constant with glutathione might be expected to
increase considerably more than that with aromatic thiol.
However, our experiments showed this is not the case. There-
fore, an increase in the concentration of small-molecule thiolate
is not the lone reason for the increased rate of protein folding
with pH. Furthermore, these results suggest that it is not the
enhanced nucleophilicity of the aromatic thiol relative to
glutathione that is most important. As previously outlined, the
enhanced nucleophilicity of aromatic thiols relative to gluta-
thione changes dramatically with pH (32-fold at pH 7.0 and
9-fold at pH 7.7). However, the enhanced folding rates (6-fold
at pH 7.0 and 5-fold at pH 7.7) are relatively independent of
Experimental Section
General Information. NMR spectra were recorded at 300 MHz (1H)
and at 75 MHz (13C) on a Bruker spectrometer. Chemical shifts were
internally referenced to the solvent. Thin-layer chromatography (TLC)
(35) Lees, W. J.; Whitesides, G. M. J. Org. Chem. 1993, 58, 642-7.
9
J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002 3891

A R T I C L E S
Gough et al.
was conducted on Aldrich general-purpose silica gel on polyester plates
with UV indicator. All reagents purchased were used without purifica-
tion. UV-vis spectra were recorded on a Cary 1 UV-visible
spectrophotometer. All proteins were purchased from Sigma unless
otherwise noted. Compound 1 was obtained from Toronto Research
Corporation or synthesized.15,16 E & R Microanalytical Laboratory Inc.
performed elemental analysis.
Reduced RNase A (0.27 mM)18,36 and scrambled RNase A (0.27
mM),19,37 which is RNase A with a random distribution of disulfide
bonds, were stored at -5 °C in an aqueous solution (0.6% AcOH and
1 mM EDTA). All buffers were prepared by the addition of base to a
0.6% solution of AcOH. Tris, [tris(hydroxymethyl)aminomethane], was
used in the preparation of all pH 7.7 buffers. Bis-tris, propane [1,3-
bis[tris(hydroxymethyl)methylamino]propane], was used in the prepara-
tion of all pH 7.0 buffers. All pH 7.0 and 7.7 buffers contained 1 mM
EDTA. Solutions were deoxygenated by bubbling Ar through the
solution for 30 min.
deoxygenated for use in the refolding reactions. The refolding reaction
was initiated by the addition of the pH-adjusted scrambled or reduced
RNase A. The refolding reaction (total volume 500 µL) contained
protein (25 µM), GSSG (0.5 mM for reduced RNase A and 0.2 mM
for sRNase A), thiol (GSH or ArSH-1), and buffer. The protein was
allowed to refold until no further increase in activity was observed (up
to 2 days). The concentration of active protein was determined by the
assay for native RNase A.
Assay for Native RNase A. An aliquot (20-50 µL) of the refolding
reaction was diluted to 1 mL in a cuvette by the addition of pH 5.0
buffer such that the final concentration of active protein was between
5 × 10-7 and 5 × 10-8 M. The pH 5.0 buffer was prepared from the
same base that was used in the refolding mixture. To the cuvette was
added 10 µL of a 20 mg/mL cCMP solution. The rate of the change in
absorbance at 292 nm was used to determine concentration of native
RNase A; see calibration of assay.
Refolding Rate. The refolding rate was determined by plotting the
concentration of active protein versus time. The concentration of active
protein was expressed as percent refolded, where percent refolded )
([active protein]/(2.5 × 10-5 M)) × 100. The plot was fit by least
squares to percent refolded ) A(1 - e-kt), where A is maximum percent
activity achieved, k is the refolding rate, and t is time in minutes.
4,4′-Dithiobisbenzeneacetic acid, Disulfide 2. An aqueous solution
(125 mL) of 1 (1.00 g, 6.0 mmol) and Na2CO3 (3.05 g, 29 mmol) was
vigorously stirred under air until the solution tested negative for thiols
with Ellman’s reagent (36 h). The solution was then acidified to pH 1
with concentrated HCl and filtered. The solid was washed with water
and then dried in a lyophilizer to yield 0.894 g (90% yield) of disulfide.
1H NMR (300 MHz, CD3OD) δ 7.44 (d, J ) 8.4 Hz, 4H), 7.24 (d, J
) 8.5 Hz, 4H), 3.58 (s, 4H); 13C NMR (75 MHz, CD3OD) δ 175.33,
136.80, 135.87, 131.47, 129.26, 41.44. Anal. Calcd for C14H14O2S2:
C, 57.47; H, 4.22; S, 19.18. Found: C, 57.42; H, 4.19; S, 19.12.
Calibration of Assay for Native RNase A.17,18 A stock solution of
native RNase A (≈0.25 mM) was prepared by dissolving native protein
in buffer. Protein concentration was determined from the absorbance
at 275 nm (ꢀ275 ) 9300 for native RNase A).18 From the stock solution
of RNase A, solutions varying in concentration from 0.5 to 25 µM
were prepared. Aliquots of these solutions, varying in volume from 20
to 50 µL, were diluted to 1 mL in a cuvette by the addition of 0.1 M
Tris-acetate buffer (pH 5.0). To the cuvette, 10 µL of a cytidine 2′,3′
-cyclic monophosphate (cCMP) (20 mg/mL) solution was added. After
the solution was mixed, the change in absorbance at 292 nm was
measured over the course of 2 min. The rate of change in absorbance
with time was determined by a linear least-squares fit (initial rate
analysis). These values were plotted vs native protein concentration.
The rate was found to vary linearly (y ) mx + b) with respect to protein
concentration when the concentration of active protein inside the cuvette
was between 5 × 10-8 M and 5 × 10-7 M.
Acknowledgment. We thank the donors of the Petroleum
Research Fund, administered by the American Chemical Society,
for partial support of this research (34902-G4). Financial support
from the National Science Foundation (CHE9975076 to W.J.L.
and an REU summer fellowship to R.H.W., Grant NSF-
CHE9987838), and the Camille and Henry Dreyfus Foundation
(New Faculty Award) are gratefully acknowledged. We thank
Dr. Rajeeva Singh of Immunogen for helpful comments.
Protein Folding.17,18 Refolding experiments for reduced and scrambled
RNase A were conducted at 25 °C. Protein aliquots were adjusted to
the pH of the refolding experiment by the addition of 0.1 M base: Tris
for pH 7.7, Bis-tris propane for pH 7.0. Solutions of 5 mM glutathione
disulfide (GSSG), 10 mM glutathione (GSH), and 20 mM aromatic
thiol (ArSH-1) were prepared individually in deoxygenated buffer and
(36) Lundstroem-Ljung, J.; Holmgren, A. J. Biol. Chem. 1995, 270, 7822-8.
(37) Hawkins, H. C.; Blackburn, E. C.; Freedman, R. B. Biochem. J. 1991, 275,
349-53.
JA016938P
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3892 J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002