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Chaperone and antichaperone activities of trigger factor
Guo-Chang Huang, Jia-Jia Chen, Chuan-Peng Liu and Jun-Mei Zhou
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China
Reduced denatured lysozyme tends to aggregate at neutral
pH and competition between productive folding and
aggregation substantially reduces the efficiency of refolding.
Trigger factor, a folding catalyst and chaperone can,
depending on the concentration of trigger factor and the
solution conditions, cause either a substantial increase
(chaperone activity) or a substantial decrease (antichaperone
activity) in the recovery of native lysozyme as compared with
spontaneous refolding. When trigger factor is working as a
chaperone, the reactivation rates of lysozyme are decelerated
and aggregation decreases with increasing trigger factor
concentrations. Under conditions where antichaperone
activity of trigger factor dominates, the reactivation rates of
lysozyme are accelerated and aggregation is increased.
Trigger factor and lysozyme were both released from the
aggregates on re-solubilization with urea indicating that
trigger factor participates directly in aggregate formation
and is incorporated into the aggregates. The apparently dual
effect of trigger factor toward refolding of lysozyme is a
consequence of the peptide binding ability and may be
important in regulation of protein biosynthesis.
Keywords: chaperone; antichaperone; protein folding; trig-
ger factor.
Molecular chaperones assist protein folding by binding
unfolded or misfolded chains and preventing or reversing
misfolding or aggregation [1]. However, in certain cases,
chaperones may also be involved in formation of aggregates
[2–6]. This so-called Ôantichaprone activityÕ, or incitement to

aggregate by a molecular chaperone, has been studied in
most detail for protein disulfide isomerase (PDI) [7–11].
With aggregation-prone substrates and at substoichiometric
concentrations, PDI promotes substrate aggregation ham-
pering productive folding. PDI is involved directly in
aggregate formation and is detected within the aggregates
[7,8,11]. Antichaperone activity has also been observed for
other chaperones, such as heavy chain-binding protein (BiP)
[9]. Similar to PDI, low stoichiometries of BiP induces
lysozyme aggregate formation. Furthermore, the aggregates
formed may act as the intermediates that lead to amyloid
diseases [12]. The participation of chaperones in aggre-
gate formation may present an important physiological
phenomenon [11].
The multifunctional Escherichia coli trigger factor was
originally identified as being involved in the maintenance of
a translocation-competent conformation of the precursor
protein proOmpA (outer member protein A) in a cell free
translation system [13] and stoichiometric complexes of
trigger factor and proOmpA were isolated and studied
[14,15]. Trigger factor was subsequently identified as a
peptidyl-prolyl cis–trans isomerase [16,17] and was detected
in the 50S subunit of functional ribosomes known to
contain the peptidyl transferase center, which covers the exit
domain of the nascent polypeptide chain [17]. Cooperation
of enzymatic and chaperone functions makes trigger factor
more effective than cyclophilins (CyPs), FK506 binding
proteins (FKBPs) and the parvulin family in the catalysis of
prolyl limited protein folding [18]. The groups of Luirink
and Bukau have successfully cross-linked presecretory and

nonsecretory proteins to trigger factor while still associated
with the ribosome [17,19]. Further, trigger factor has been
shown to be an important cofactor in GroEL-dependent
protein degradation in E. coli and to promote binding of
GroEL to unfolded proteins [20,21]. Trigger factor may also
be a rate-limiting component in the degradation of abnor-
mal proteins. Recently, trigger factor from Bacillus subtilis
was reported to catalyze in vitro protein folding and to be
necessary for viability under starvation conditions [22].
Trigger factor from Streptococcus pyogenes contributes
post-transcriptionally to the secretion and processing of
secreted cysteine proteinase (SCP) [23]. There is ample
evidence that trigger factor plays an important and multi-
functional role during protein synthesis in vivo and further
facets to its role remain to be investigated.
We reported that trigger factor could, as a molecular
chaperone, inhibit aggregation and increase the reactivation
yield of
D
-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) [24]. A model for trigger factor assisted refolding
of GAPDH and the conformational states that are prefer-
entially recognized by trigger factor were proposed [24,25].
In order to further investigate how trigger factor influences
the partitioning of an unfolded protein between folding and
aggregation, here we examine the trigger factor assisted
folding of reduced denatured lysozyme, in which folding is
affected by buffer conditions. Lysozyme is a particularly
appropriate substrate to study the chaperone activity in
isolation from the isomerase activity of trigger factor,

because the two prolyl bonds (Pro70 and Pro79) are both
trans in native lysozyme, thus involvement of isomerization
of prolyl bond during refolding is negligible as prolyl bonds
Correspondence to J M. Zhou, National Laboratory of
Biomacromolecules, Institute of Biophysics, Academia Sinica,
15 Datun Road, Chaoyang district, Beijing 100101, China.
Fax: + 86 10 64872026, Tel.: + 86 10 64889859,
E-mail:
Abbreviations: PDI, protein disulfide isomerase; CyP, cyclophilin;
GSH, glutathione; GSSG, glutathione disulfide;
GdnHCl, guanidine hydrochloride.
(Received 19 March 2002, revised 19 July 2002,
accepted 26 July 2002)
Eur. J. Biochem. 269, 4516–4523 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03145.x
are also predominately trans in the unfolded chains [26]. The
results show that in redox phosphate buffer in which
spontaneous refolding of lysozyme is poor, trigger factor
acts as a molecular chaperone that increases the reactivation
yield and decelerates refolding rates. However, in redox
Hepes buffer in which lysozyme refolds well spontaneously,
low concentrations of trigger factor reduce the reactivation
yield significantly and facilitate the formation of aggregates,
behavior that has been described as ÔantichaperoneÕ [7–9]. In
the aggregates, lysozyme is extensively cross-linked by
intermolecular disulfide bonds and trigger factor partici-
pates specifically in the mixed aggregates as an integral
component. The dual effects of trigger factor toward
refolding of lysozyme may be important in regulation of
protein biosynthesis.
MATERIALS AND METHODS

Materials
Hen egg white lysozyme was purchased from Serva, and
GSSG and GSH were from Fluka. Bovine serum albumin
(BSA), ovalbumin, Micrococcus lysodeikticus cell walls
and dithiothreitol were obtained from Sigma. Hepes was
from Merck. Guanidine hydrochloride (GdnHCl) was a
product of ICN Biomedicals (Cosa Mesa, CA, USA), and
urea was purchased from Nacalai tesque Inc. (Kyoto,
Japan). Reagents for gel electrophoresis were from Bio-
Rad. All other chemicals were local products of analytical
grade.
Trigger factor was expressed and purified as described
previously [16]. Final trigger factor preparations were
typically > 90% homogeneous as judged by SDS/PAGE.
An absorbance coefficient of e
280nm
¼ 15 930
M
)1
Æcm
)1
,
calculated using the procedure of Gill and von Hippel [27],
was used for protein concentration determination. Cyclo-
philin (CyP) was prepared from porcine kidney according to
Kofron et al. [28]. The specific constant of the final product
is about 1.9 · 10
7
M
)1

Æs
)1
when assayed using the chymo-
trypsin-coupled method [29].
Reduction and denaturation of lysozyme
Lysozyme at 20 mgÆmL
)1
was completely reduced and
denatured by incubation at room temperature for 4 h in
100 m
M
sodium phosphate buffer, pH 8.0, containing 8
M
GdnHCl and 150 m
M
dithiothreitol. The reaction mixture
was adjusted to pH 2.0 with 6
M
HCl, and then dialyzed at
4 °C, first against 10 m
M
HCl and then against 100 m
M
acetic acid. The 200 l
M
reduced and denatured lysozyme
was divided into aliquots and stored at )20 °C.
Refolding of lysozyme
Oxidative refolding of reduced and denatured lysozyme was
achieved by dilution in various buffers as specified with or

without different concentrations of trigger factor or CyP at
25 °C. The Hepes buffer, 0.1
M
, pH 7.0, contained 2 m
M
EDTA, 5 m
M
MgCl
2
and 20 m
M
NaCl, and the phosphate
buffer, 0.1
M
, pH 7.5, contained 2 m
M
EDTA. If not
otherwise specified, the refolding buffer contained 1 m
M
GSSG and 2 m
M
GSH (as the ratio of GSH to GSSG has
been determined to be 2 in the endoplasmic reticulum) [30].
The final concentration of lysozyme for refolding was
10 l
M
. When GSSG and GSH were not present, the system
was referred to as a nonredox buffer. Recovery of activity
was complete 5 h after dilution and no further change was
observed for at least 24 h. Lysozyme activity was deter-

mined at 30 °C by following the lysis of Micrococcus
lysodeikticus [7,31]. The decrease in A
450
of a 0.25 mgÆmL
)1
cell suspension in 67 m
M
sodium phosphate buffer,
pH 6.2, containing 100 m
M
NaCl was measured in a
Shimadzu UV-1601 spectrophotometer. Protein concentra-
tions were determined by measuring A
280
using absorbance
coefficients of 36 636
M
)1
Æcm
)1
for native lysozyme and
33 014
M
)1
Æcm
)1
for denatured lysozyme. The time course
of reactivation of lysozyme was followed by determining
activities of samples withdrawn at the indicated times. The
half-times were determined by fitted to a single-exponential

function. Lysozyme itself is stable when subjected to the
same treatment without denaturant. Aggregation of lyso-
zyme upon dilution was monitored at 25 °Cby90° light
scattering at 500 nm in a Hitachi F-4500 spectrofluorimeter.
All measurements were repeated several times and the rate
constants obtained were highly reproducible.
Aggregate resolubilization
The insoluble aggregates formed during refolding of lyso-
zyme in the presence of 5 l
M
trigger factor in Hepes buffer
were isolated according to the procedure described by
Sideraki and Gilbert [11] as follows: aggregates were
collected by centrifugation in a bench top centrifuge
(6000 g for 8 min). After washing twice with Hepes
refolding buffer, the pellets were resuspended in various
concentrations of urea in buffer containing 0.1
M
Hepes,
pH 7.0, with or without 150 m
M
dithiothreitol. After four
rounds of vortex mixing, the solution was incubated
overnight at room temperature. After incubation with urea,
the residual insoluble materials were separated from the
supernatant by centrifugation in a bench top centrifuge
(6000 g for 10 min) and then the proteins in the supernatant
were quantified by reducing SDS/PAGE. In another set of
experiments, SDS sample buffers with or without 2-
mercaptoethanol were used to re-solubilize the pellets

instead of urea and samples were examined on both
reducing and nonreducing gels, respectively.
RESULTS
Refolding of lysozyme in phosphate buffer
The spontaneous refolding of reduced and denatured
lysozyme (10 l
M
) in phosphate buffer with no redox
component is only 1.4% (Fig. 1) and shows extensive
aggregation as the native disulfide bonds of lysozyme
cannot form. The presence of trigger factor at a concentra-
tion within the range 5 l
M
to 20 l
M
(molecular ratios of
0.5–2) has no effect on lysozyme refolding in terms of
reactivation yield (Fig. 1) or extent of aggregation under
nonredox conditions. At pH 7.5, 25 °C, the spontaneous
refolding of reduced denatured lysozyme in a glutathione
redox phosphate buffer (1 m
M
GSSG, 2 m
M
GSH) is
relatively rapid (t
1/2
¼ 20.4 min, see later), but only 2.8% of
the lysozyme folds productively (Fig. 1). Upon dilution of
reduced denatured lysozyme in the presence of trigger

Ó FEBS 2002 Chaperone and antichaperone activities of trigger factor (Eur. J. Biochem. 269) 4517
factor, the recovery of lysozyme activity increases with
increasing molecular ratios of trigger factor to lysozyme
until at 15 l
M
trigger factor, 17% of the lysozyme is
productively folded (Fig. 1). Control experiments show that
trigger factor neither affects the lysozyme activity assay
directly nor do trigger factor preparations exhibit any
apparent lysozyme activity. The amount of lysozyme
activity recovered does not increase further during the
24 h after activity determination. Therefore, the partial
recovery of lysozyme activity is due to irreversible misfold-
ing and/or aggregation rather than a biphasic or kinetically
incomplete reaction. The reduced and denatured lysozyme
in the absence of trigger factor aggregated rapidly and to a
significant degree upon dilution, as monitored by light
scattering (Fig. 2). In the absence of trigger factor, light
scattering started to increase within 10 min of dilution and
approached a constant value at about 1 h. Accompanying
the increase in reactivation yield (Fig. 1), the extent of
lysozyme aggregation was inhibited markedly by increasing
concentrations of trigger factor (Fig. 2). CyP, another
peptidyl-prolyl cis–trans isomerase, was used as a compari-
son to dissect out the isomerase and chaperone activities of
trigger factor. Increasing concentrations of CyP showed no
effect on either the extent of lysozyme reactivation (Fig. 1)
or nonproductive aggregation (Fig. 2). There is essentially
the same amount of native lysozyme recovered when
refolding is performed in the absence of CyP (Fig. 1).

The kinetics of reactivation of lysozyme in the presence of
different concentrations of trigger factor or CyP is com-
pared in Fig. 3. The half times (t
1/2
) of lysozyme reactivation
in the presence of trigger factor, as was found earlier for
trigger factor assisted refolding of GAPDH [24], increase
with increasing concentrations of trigger factor. The t
1/2
of
Fig. 2. Effect of trigger factor on lysozyme aggregation in redox phos-
phate (j) and redox Hepes (d)buffers.Aggregation of lysozyme upon
dilution was monitored at 25 °Cby90° light scattering at 500 nm.
Final levels of light scattering were determined 1 h after dilution. The
concentration of lysozyme was 10 l
M
. A CyP control in redox phos-
phate buffer is indicated as (h).
Fig. 1. Effect of trigger factor or CyP on lysozyme re-activation in
phosphate buffer. Refolding of lysozyme was initiated by a 20-fold
dilution into 0.1
M
phosphate buffer, pH 7.5, containing 2 m
M
EDTA.
The reactivation mixtures were kept at 25 °Cfor5hbeforesamples
were taken for assay of activity. Data are presented as the percentage
of lysozyme refolded with respect to nondenatured lysozyme otherwise
treated in exactly the same way. The final concentration of lysozyme
for refolding was 10 l

M
(m)and(j) represent lysozyme in nonredox
and redox buffers, respectively, in the presence of trigger factor. (h)
represents lysozyme reactivation in the redox buffer in the presence of
CyP. The data are fitted to an arbitrary curve.
Fig. 3. Dependence of half times of lysozyme reactivation in redox
phosphate (j) and redox Hepes (d) buffer, respectively, on the con-
centrations of trigger factor. The refolding was followed by the regain
of enzyme activity at a final concentration of lysozyme of 10 l
M
at
25 °C. The kinetic data were analyzed by fitted to a single-exponential
function. The data shown are fitted to an arbitrary curve. CyP is used
as a control at redox phosphate (h)andredoxHepes(s)buffer,
respectively.
4518 G C. Huang et al.(Eur. J. Biochem. 269) Ó FEBS 2002
lysozyme reactivation in the presence of 20 l
M
trigger factor
was 57.5 min, 2.8 times longer than that determined for
spontaneous refolding. Stoichiometric concentrations of
CyP had no effect on the kinetics of lysozyme reactivation.
Thus, in a redox phosphate buffer, trigger factor behaves
like a molecular chaperone that prevents denatured lyso-
zyme from partitioning towards a nonproductive folding
pathway. The chaperone-like activity of trigger factor is
specific, other proteins such as BSA or ovalbumin, at
comparable concentrations, have no effects on lysozyme
refolding under the same conditions (data not shown).
Refolding of lysozyme in Hepes buffer

As in phosphate buffer, refolding of reduced denatured
lysozyme in nonredox Hepes buffer showed almost no
reactivation either in the presence or absence of trigger
factor (Fig. 4). However, the spontaneous refolding of
lysozyme in a glutathione redox Hepes buffer results in a
recovery of activity of 42% (Fig. 4). Intriguingly, the
reactivation of lysozyme decreases significantly with
increasing trigger factor concentration at low molecular
ratios. The reactivation yield of lysozyme decreased from
42% in the absence of trigger factor to 6.6% in the presence
of 5 l
M
trigger factor (Fig. 4), indicating that trigger factor
shows antichaperone activity in lysozyme refolding, similar
to that observed for PDI [7–11]. When the concentration of
trigger factor was greater than 5 l
M
, the reactivation curve
of lysozyme shows a slow upward turn although the
reactivation yields were still much lower than that of
spontaneous refolding (Fig. 4), suggesting that antichaper-
one and chaperone activities of trigger factor operate in
competition to one another. We examined whether the
decrease in lysozyme refolding yield is accompanied by
aggregation by monitoring light scattering. As shown in
Fig. 2, the extent of aggregation was found to increase with
increasing trigger factor when the trigger factor concentra-
tion was below 5 l
M
. Trigger factor alone, in control

experiments, showed no scattered light under the same
conditions. Further increase in trigger factor concentration
resulted in a decrease in light scattering, indicating that
chaperone activity begins to dominate under these condi-
tions. Unlike trigger factor, CyP, as observed in phosphate
buffer, has no influence in either recovery of native lysozyme
(Fig. 4) or the extent of aggregation (Fig. 2).
The kinetics of reactivation of reduced denatured lyso-
zyme in redox Hepes buffer and in the presence of different
concentrations of trigger factor or CyP were also investi-
gated and the results are shown in Fig. 3. The half time of
spontaneous reactivation was 48 min, which is slower than
in phosphate buffer. While stoichiometric quantities of CyP
show no effect on lysozyme refolding, trigger factor at
substoichiometric concentrations accelerates the reactiva-
tion rates to about 1.9 times that of spontaneous refolding
at a molecular ratio of 0.25. However, the accelerated
reactivation results in decreased recovery of activity of
lysozyme (Fig. 4). When the molar ratio of trigger factor in
the refolding buffer is increased above 0.5, the reactivation
yields begin to increase (Fig. 4) and the extents of aggre-
gation to decrease (Fig. 2). This is accompanied by a change
from acceleration of the reactivation rates to deceleration
(Fig. 3). The above results are similar to those reported by
Puig and Gilbert [7,9] for antichaperone and chaperone
activities of PDI except that refolding of lysozyme is
catalyzed by PDI regardless of whether it is the chaperone
or the antichaperone activity that predominates.
Effects of NaCl and ethylene glycol on trigger
factor-assisted lysozyme refolding

Phosphate and Hepes buffers differ greatly in ionic strength
[10], and the different effects of trigger factor on lysozyme
refolding in the two kinds of redox refolding buffers
prompted us to investigate the effects of the refolding buffer,
in terms of ionic strength and hydrophobicity. As shown in
Fig. 5, the spontaneous reactivation of lysozyme in redox
Hepes buffer is dramatically affected by addition of 100 m
M
NaCl, decreasing from 42% to about 6%. On addition of
trigger factor, the recovery of activity increases gradually
with increasing trigger factor concentration and above 5 l
M
trigger factor is essentially the same as in buffer without
added salt. It is interesting to note that under these
conditions where the yield of spontaneous folding is low,
trigger factor shows no detectable antichaperone activity.
When 5% ethylene glycol instead of NaCl was added to the
refolding system, the spontaneous reactivation of lysozyme
reached a maximum of 47%, which is slightly higher than
that in the absence of ethylene glycol. On addition of trigger
factor the reactivation falls dramatically reaching a mini-
mum of 4.8% at a trigger factor concentration of 5 l
M
.This
value is slightly lower than in the absence of ethylene glycol.
These small differences are highly reproducible. This
indicates that addition of ethylene glycol causes a slight
enhancement of the antichaperone effect. It seems that
whether it is the antichaperone or the chaperone activity of
trigger factor that dominates may be determined by the

Fig. 4. Effects of trigger factor or CyP on lysozyme reactivation in
Hepes buffer. The refolding was carried out in 0.1
M
Hepes buffer,
pH 7.0, containing 2 m
M
EDTA, 5 m
M
MgCl
2
and 20 m
M
NaCl. All
other details were the same as for Fig. 1. (m)and(d) represent lyso-
zyme in nonredox and redox buffers, respectively, in the presence of
trigger factor. (s) represents lysozyme in redox buffer in the presence
of CyP.
Ó FEBS 2002 Chaperone and antichaperone activities of trigger factor (Eur. J. Biochem. 269) 4519
effect of the solution conditions on folding of the substrate
itself.
Composition of trigger factor accelerated aggregates
As shown in Fig. 2, maximum formation of insoluble
aggregates during lysozyme refolding under redox condi-
tions occurs at a molecular ratio of trigger factor to
lysozyme of 0.5 (10 l
M
lysozyme, 5 l
M
trigger factor)
indicating that aggregate formation is accelerated by trigger

factor. To understand the mechanism of aggregate forma-
tion, the isolated aggregates were incubated in various
concentrations of urea with or without 150 m
M
dithiothre-
itol and the re-solubilized proteins were analyzed by
reducing SDS/PAGE. As shown in Fig. 6 Aa,b, the
proteins in aggregates were re-solubilized by urea and the
total amount of soluble protein increased with increasing
urea concentration. In each urea concentration, lysozyme
and trigger factor were solubilized to the same extent and in
the same ratio as the original reaction mixture within
experimental error. This suggests that trigger factor and
lysozyme coaggregates and is not present as separate
aggregates. Aggregates formed under conditions that allow
disulfide formation are highly cross-linked, which makes the
trigger factor-lysozyme aggregates more resistant to extrac-
tion with urea unless dithiothreitol is added (Fig. 6A,b). It
should be noted that no covalent bonds between trigger
factor and lysozyme can form, as trigger factor itself
contains no cysteine residues.
In order to understand whether trigger factor, when
acting as an antichaperone, is integrated specifically into the
mixed aggregates or only coprecipitates with rapidly formed
lysozyme aggregates, we carried out experiments using BSA
as a control. When lysozyme (10 l
M
) was diluted into the
Hepes buffer containing 5 l
M

BSAaswellas5l
M
trigger
factor, the refolding was not affected either in recovery of
activity or aggregation formation compared with in the
presence of trigger factor alone (data not shown). The
soluble and insoluble fractions formed in the presence of
trigger factor and BSA were isolated by centrifugation
and the aggregates were then incubated in SDS sample
buffer with or without 200 m
M
2-mercaptoethanol, respect-
ively. The resolubilized proteins were measured by reducing
or nonreducing SDS/PAGE, respectively. As shown in
Fig. 6B,a, in contrast to a clear band of trigger factor
resolubilized together with lysozyme, there is no visible BSA
band on the gel. Clearly, BSA is not present in the mixed
aggregates. This indicates that trigger factor does not
coprecipitate with aggregated lysozyme in a nonspecific
manner. In addition, comparison of electrophoresis under
reducing (Fig. 6B,a) and nonreducing (Fig. 6B,b) condi-
tions shows that aggregated lysozyme is highly cross-linked
by disulfide bonds preventing re-solubilization of lysozyme
from aggregates in the absence of 2-mercaptoethanol. After
treatment in SDS sample buffer containing no 2-mercapto-
ethanol, cross-linked lysozyme, although partially soluble, is
present only as a high molecular weight species, indicating
that intermolecular crosslinking has occurred (not shown).
Under nonreducing conditions, there is also no BSA
detected in the coprecipitated aggregates, although this

result is most clearly seen under reducing conditions where
BSA and trigger factor do not comigrate. It is clear that the
coprecipitation of trigger factor with lysozyme is specific
and is related to its antichaperone function.
DISCUSSION
Upon dilution into refolding buffer, the reduced denatured
lysozyme faces two alternative fates: productive folding to
form active enzyme or aggregation. The relative size of the
populations that partition between productive folding and
aggregation depends to a considerable degree on the
solution conditions, of which the ionic strength and the
nature of the redox reagents are significant factors [10]. In
the absence of GSSG, the spontaneous reactivation of
lysozyme in both phosphate and Hepes buffers is very low
because disulfide formation cannot proceed efficiently in the
absence of redox reagents. In this case, trigger factor shows
no effect on the reactivation yield, indicating that correct
disulfide formation is essential for productive folding of
lysozyme. Increasing the ionic strength in redox phosphate
or Hepes buffers by the addition of 100 m
M
NaCl causes a
marked decrease in the spontaneous reactivation yield due
to increased population of aggregation-prone intermediates
of lysozyme. However, increasing the hydrophobicity of the
refolding buffer by including ethylene glycol, thereby
decreasing the hydrophobic interaction between aggrega-
tion-prone intermediates, results in a slight increase of
spontaneous refolding yield (Fig. 5). Depending on the
conditions, trigger factor shows apparently opposite effects

on lysozyme refolding: as a chaperone, the productive
refolding is enhanced (Fig. 1); or as an antichaperone, the
productive refolding is inhibited (Fig. 4).
As a chaperone
In redox phosphate buffer, trigger factor hinders the
incorrect association of aggregation-prone species and thus
favors the pathway to formation of native lysozyme,
Fig. 5. Effects of trigger factor on lysozyme reactivation in redox Hepes
buffer (d)orthesamebuffercontaining100m
M
NaCl (,)or5%
ethylene glycol (r). The experiments were carried out as described in
the legend to Fig. 4.
4520 G C. Huang et al.(Eur. J. Biochem. 269) Ó FEBS 2002
improving reactivation yield but without being a part of the
final functional structure (Figs 1 and 2). In this case, the
rates of assisted refolding of lysozyme are reduced by trigger
factor compared to the rate of spontaneous refolding
(Fig. 3). Typical chaperone behavior, exhibited by various
members of the stress proteins, involves the interaction of
the nonspecific peptide-binding site of the chaperone with a
denatured protein in such a way as to inhibit aggregation.
Trigger factor possesses a nonspecific peptide/protein-bind-
ing site and a comparison with CyP as a reference foldase
suggests that the high affinity toward unfolded protein
chains is a requisite for the high efficiency of trigger factor in
assisting protein folding [23]. In its efficient binding to
unfolded proteins, trigger factor resembles a chaperone. Our
previous work indicates that trigger factor shows chaperone
activity for GAPDH and strong binding of GAPDH

intermediates appears to decelerate their dissociation from
trigger factor, thus resulting in a decrease in the rate
constant of refolding [24]. Likewise, under conditions where
trigger factor improves the recovery yield of native
lysozyme, it also decreases the rate constant for the folding
reaction. An increase in refolding yields and slowing down
of refolding rates may be a common characteristic of
molecular chaperones [24].
As an antichaperone
In redox Hepes buffer, the productive refolding of lysozyme
is substantially lower in the presence of trigger factor than in
its absence (Fig. 4). At the same time, trigger factor
accelerates the conversion of the denatured lysozyme into
large, disulfide cross-linked aggregates (Fig. 6A,a and b). As
a substantial proportion of the lysozyme would fold
productively in the absence of trigger factor, trigger factor
must intervene early in the folding process to redirect most
of the substrate along an alternative nonproductive pathway
to aggregation. Such behavior of trigger factor is reminis-
cent of PDI, for which chaperone and antichaperone
activities in lysozyme refolding have also been observed
[7–11]. As trigger factor is integrated specifically into the
Fig. 6. (A) Composition of trigger factor accelerated aggregates and (B) reducing (a) and nonreducing (b) SDS/PAGE (15%) analysis of the mixed
aggregates. (A) The aggregates that formed during refolding of 10 l
M
lysozyme in the presence of 5 l
M
trigger factor were separated and then
re-solubilized in increasing concentrations of urea with (a) or without (b) 150 m
M

dithiothreitol. Re-solubilized materials were analyzed on reducing
SDS/PAGE (15%). Lanes labelled 0–7 represent the molar concentration of urea used. L and T indicate native lysozyme and native trigger factor,
respectively, loaded in a molar ratio consistent with the reaction conditions. (B) Insoluble aggregates were formed in redox Hepes buffer at a
lysozyme to trigger factor ratio that ensured maximal aggregation (10 l
M
lysozyme, 5 l
M
trigger factor) in the presence or absence of 5 l
M
BSA.
The isolated aggregates were then incubated in SDS sample buffer with (a) or without (b) 200 m
M
2-mercaptoethanol before analysis by SDS/
PAGE. 1, native trigger factor; 2, native BSA; 3, re-solubilized aggregates formed in the presence of BSA; 4, re-solubilized aggregates formed in the
absence of BSA; 5, native lysozyme.
Ó FEBS 2002 Chaperone and antichaperone activities of trigger factor (Eur. J. Biochem. 269) 4521
mixed aggregates (Fig. 6A), the binding of trigger factor
with folding intermediates must be an essential step in the
antichaperone activity. It is quite possible that antichaper-
one activity is not determined by trigger factor’s third active
site, but probably depends on the folding pathways of the
substrate and the stability and relative populations of
different intermediates, both of which could be dependent
on the solution conditions, such as the ionic strength and
redox state of the solution.
As a chaperone and an antichaperone
The spontaneous folding rate of lysozyme in the redox
Hepes buffer is significantly slower than that in the redox
phosphate buffer (Fig. 3). The intermediates recognized by
trigger factor may differ in conformation in each case and

thus differ in their ability to bind to trigger factor. It has
been reported that trigger factor, in accord with its location
at the ribosome in vivo, binds most strongly to early folding
intermediates which lack compact structure [25,32]. Dena-
tured lysozyme folds more slowly in Hepes buffer than in
phosphate buffer and there may be more availability of
loosely structured intermediates allowing tight binding to
trigger factor. Meanwhile, the strong binding of folding
intermediates also appears to decelerate their dissociation
from trigger factor hence slowing the rate of folding. In
Hepes buffer and at low concentrations of trigger factor
where trigger factor behaves as an antichaperone, each
trigger factor molecule attracts multiple lysozyme mole-
cules to compete for the same peptide/protein-binding site,
thus indirectly facilitating spatial contact between folding
intermediates to form intermolecular disulfide cross-links.
At the same time, decelerated dissociation resulting from
strong binding of trigger factor provides folding interme-
diates with enough time to weave a large, disulfide cross-
linked insoluble network involving trigger factor as an
integral component. The observed relative increase in
folding rate in this region may reflect that it is only the
fastest folding fraction of the population which escapes
interaction with trigger factor and so can fold instead of
aggregating.
As the molecular ratio of trigger factor to lysozyme
increases, contact between aggregation-prone intermediates
of lysozyme is prevented resulting in suppression of
aggregation and an up-turn in the amount of activity
recovered (Fig. 4). The balance between apparent chaper-

one and antichaperone functions of trigger factor in
lysozyme refolding is controlled by the surrounding envi-
ronment and the relative amount of trigger factor to
lysozyme. An apparently similar switch from chaperone to
antichaperone activity was observed in trigger factor
assisted GAPDH refolding when the trigger factor concen-
tration was very high. It seems therefore that the antichap-
erone activity of trigger factor is actually the same as its
chaperone activity, not a distinct function in addition to its
isomerase and chaperone activities. It is a consequence of
the ability of trigger factor to bind folding intermediates
with non-native conformations, depends on the same
peptide-binding site as the chaperone activity and is closely
related to the folding properties of the substrate as
controlled by the conditions. The antichaperone activity
of trigger factor is not specific to lysozyme, as indicated by
our findings with GAPDH [24] and an observation that
trigger factor can also significantly decrease refolding yields
of creatine kinase under conditions where full regain activity
is obtained in spontaneous refolding (C. P. L and J. M. Z,
unpublished results).
ACKNOWLEDGEMENTS
The present study was supported in part by the 973 Project of the
Chinese Ministry of Science and Technology (G1999075608) and the
exchange program between the Max-Plank Society and Chinese
Academy of Sciences. The authors would like to thank Dr S. Perrett
of this department for a critical reading of this paper and helpful
suggestions.
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