Chaperone and antichaperone activities of trigger factorGuo-Chang Huang, Jia-Jia Chen, Chuan-Peng Liu and Jun-Mei Zhou National Laboratory of Biomacromolecules, Institute of Biophysics,
Trang 1Chaperone 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 antichaprone activity, 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 transin 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: zhoujm@sun5.ibp.ac.cn
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)
Trang 2are 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 antichaperone [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
M A T E R I A L S A N D M E T H O D S
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 e280nm¼ 15 930M )1Æcm)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· 107
M )1Æs)1when 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 mMsodium phosphate buffer, pH 8.0, containing 8M
GdnHCl and 150 mMdithiothreitol The reaction mixture
was adjusted to pH 2.0 with 6MHCl, and then dialyzed at
4C, first against 10 mM HCl and then against 100 mM
acetic acid The 200 lM 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
25C The Hepes buffer, 0.1M, pH 7.0, contained 2 mM
EDTA, 5 mMMgCl2and 20 mMNaCl, and the phosphate
buffer, 0.1M, pH 7.5, contained 2 mM EDTA If not
otherwise specified, the refolding buffer contained 1 mM
GSSG and 2 mMGSH (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 lM 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 30C by following the lysis of Micrococcus lysodeikticus[7,31] The decrease in A450of a 0.25 mgÆmL)1 cell suspension in 67 mM sodium phosphate buffer,
pH 6.2, containing 100 mM NaCl was measured in a Shimadzu UV-1601 spectrophotometer Protein concentra-tions were determined by measuring A280using absorbance coefficients of 36 636M )1Æcm)1 for native lysozyme and
33 014M )1Æcm)1for 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 25C by 90 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 lMtrigger 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 0.1M Hepes,
pH 7.0, with or without 150 mMdithiothreitol 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
R E S U L T S
Refolding of lysozyme in phosphate buffer The spontaneous refolding of reduced and denatured lysozyme (10 lM) 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 lM to 20 lM(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, 25C, the spontaneous refolding of reduced denatured lysozyme in a glutathione redox phosphate buffer (1 mM GSSG, 2 mM GSH) is relatively rapid (t1/2¼ 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
Trang 3factor, the recovery of lysozyme activity increases with
increasing molecular ratios of trigger factor to lysozyme
until at 15 lM 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 (t1/2) 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 t1/2of
Fig 2 Effect of trigger factor on lysozyme aggregation in redox phos-phate (j) and redox Hepes (d) bu ffers Aggregation of lysozyme upon dilution was monitored at 25 C by 90 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 C for 5 h before samples
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) and redox Hepes (s) buffer, respectively.
Trang 4lysozyme reactivation in the presence of 20 lMtrigger 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 lMtrigger 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 lM, 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 chaperantichaper-one 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 lM 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 mM
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 lM
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 lM 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.
Trang 5effect 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 lM lysozyme, 5 lM 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 mM
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 lM) was diluted into the
Hepes buffer containing 5 lMBSA as well as 5 lMtrigger
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 mM2-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
D I S C U S S I O N
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 mMNaCl 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) or the same bu ffer containing 100 m M NaCl (,) or 5%
ethylene glycol (r) The experiments were carried out as described in
the legend to Fig 4.
Trang 6improving 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 workindicates 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.
Trang 7mixed 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 lackcompact 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 antichaperchaper-one 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)
A C K N O W L E D G E M E N T S
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-PlankSociety 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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