J. Biol. Chem., Vol. 275, Issue 25, 18698-18703, June 23, 2000
A Thermodynamic Coupling Mechanism for the Disaggregation of a
Model Peptide Substrate by Chaperone SecB*
Vikram G.
Panse
§,
Pia
Vogel¶,
Wolfgang E.
Trommer¶, and
Raghavan
Varadarajan
**
From the Molecular Biophysics Unit, Indian Institute
of Science, Bangalore 560 012, India, the Chemical Biology
Unit, Jawaharlal Center for Advanced Scientific Research, Jakkur P.O.,
Bangalore 560 004, India, and the ¶ Fachbereich Chemie/Biochemie
der Universitat Kaiserslautern, Erwin Schrodinger Str., 67663 Kaiserslautern, Germany
Received for publication, February 2, 2000, and in revised form, March 17, 2000
 |
ABSTRACT |
Molecular chaperones prevent protein aggregation
in vivo and in vitro. In a few cases,
multichaperone systems are capable of dissociating aggregated state(s)
of substrate proteins, although little is known of the mechanism of the
process. SecB is a cytosolic chaperone, which forms part of the
precursor protein translocation machinery in Escherichia
coli. We have investigated the interaction of the B-chain of
insulin with chaperone SecB by light scattering, pyrene excimer
fluorescence, and electron spin resonance spectroscopy. We show that
SecB prevents aggregation of the B-chain of insulin. We show that SecB
is capable of dissociating soluble B-chain aggregates as monitored by
pyrene fluorescence spectroscopy. The kinetics of dissociation of the
B-chain aggregate by SecB has been investigated to understand the
mechanism of dissociation. The data suggests that SecB does not act as
a catalyst in dissociation of the aggregate to individual B-chains,
rather it binds the small population of free B-chains with high
affinity, thereby shifting the equilibrium from the ensemble of the
aggregate toward the individual B-chains. Thus SecB can rescue
aggregated, partially folded/misfolded states of target proteins by a
thermodynamic coupling mechanism when the free energy of binding to
SecB is greater than the stability of the aggregate. Pyrene excimer
fluorescence and ESR methods have been used to gain insights on the
bound state conformation of the B-chain to chaperone SecB. The data
suggests that the B-chain is bound to SecB in a flexible extended state
in a hydrophobic cleft on SecB and that the binding site accommodates
approximately 10 residues of substrate.
| |
INTRODUCTION |
Molecular chaperones are a class of proteins that prevent
aggregation of partially folded forms of a protein and thus funnel it
to a functional form. (1). They do not bind the native state of
proteins and they are capable of interacting with many different polypeptide chains without apparent sequence preference (2-4). The common property shared by molecular chaperones is the ability to
recognize structural elements exposed in unfolded or partially denatured states, such as hydrophobic surfaces.
SecB is a tetrameric chaperone in Escherichia coli that is
involved in the translocation of polypeptide chains into the
periplasmic space of the cell (5). In vivo, SecB binds to a
subset of precursor proteins and maintains them in an unfolded,
translocation competent state, while in vitro it interacts
with a variety of proteins in the non-native state (6-9). Studies on a
model protein substrate of SecB, barstar, revealed that SecB did not
bind the folded or unfolded state, but trapped a near native-like
molten globule state (8). SecB has also been shown to bind partially
folded states of 
-LA (10). ITC studies with model protein
substrates, which were either partially folded or unfolded, suggested
that between 7 and 29 residues are buried upon substrate binding to SecB (10). The exact nature of the translocation competent state of
SecB's natural substrates is unknown, although it is believed to be a
flexible molten globule state (6, 11). Partially folded species such as
molten globules have exposed hydrophobic surfaces and hence
non-productive interactions leading to aggregation could compete with
translocation events. Growing evidence suggests that protein
aggregation occurs in most cases by mechanisms involving folding
intermediates (12).
Chaperones have evolved to promote the unfolding of trapped
intermediates and then facilitate their correct folding (13). Chaperones such as Hsp70, Hsp104, and Hsp40 are capable of rescuing proteins from an aggregated inactivated state (14, 15). Maltose-binding protein, a natural substrate of SecB aggregates transiently during its
refolding at micromolar concentrations. SecB was shown both to reduce
the extent of aggregation and promote disaggregation during the
refolding reaction.1 The
mechanism and the physical basis of interaction of chaperones with
pre-formed aggregated state of proteins have not been quantitatively investigated in detail. Such studies are complicated by the fact that
it is difficult to separate chaperone binding, folding, and aggregation
kinetics. In the present work, we have investigated the ability of
chaperone SecB to prevent aggregation and also promote dissociation of
pre-formed soluble aggregates. We have used insulin as a model peptide
substrate labeled with pyrene to gain insight into the mechanism of
dissociation of a soluble aggregate of B-chain by chaperone SecB.
Furthermore, we have also investigated the conformational state of the
B-chain bound to SecB using pyrene excimer fluorescence and ESR
spectroscopy. The insulin B-chain is a small 30-residue peptide.
Isolated B-chains have a strong tendency to aggregate in aqueous
solution. In the presence of chaperone, there is competition between
binding and aggregation. In contrast to larger protein systems, these
kinetics are uncomplicated by irreversible folding of the peptide to a native state. In addition, the B-chain has two Cys residues at positions 7 and 19 that can be used to attach spectroscopic probes.
| |
EXPERIMENTAL PROCEDURES |
Materials--
Pyrene maleimide,
DTT,2 and
5,5'-dithiobis(nitrobenzoic acid) were from Sigma. All other chemicals
were of analytical grade.
Protein Purification--
The SecB expression plasmid pJW25 in
strain BL21 (DE3) was obtained from Prof. B. de Kruijff. SecB
purification was done as reported previously (8). The purified protein
was estimated to be 99% pure by SDS-polyacrylamide gel electrophoresis
as detected by silver staining (16) and analytical gel filtration high
Performance liquid chromatography. The extinction coefficient at 280 nm
was taken to be 11,900 M
1 cm1
for monomeric SecB (17). All the SecB concentrations reported here are
monomeric unless otherwise stated.
Light Scattering Assay--
The light scattering assay for
insulin aggregation was performed as described previously (18). To a 50 µM solution of insulin containing varying amounts of SecB
contained in 50 mM potassium phosphate buffer, pH 7.4, 20 mM EDTA, and 4 mM DTT, thioredoxin was added to
a final concentration of 2 µM. The aggregation of the
B-chain was monitored by the increase in scatter at 360 nm.
Pyrene and Spin Labeling of B-chain of Insulin--
5 mg of
insulin was dissolved in 1 ml (1.4 mM) of 50 mM
potassium phosphate buffer, pH 7.4, containing 1 mM EDTA
and 10 mM DTT. This was kept at room temperature for 2 h. The B-chain precipitates in solution, while the A chain is still
soluble. The reaction mixture was centrifuged and the supernatant
discarded. The B-chain precipitate was washed thoroughly with 50 mM potassium phosphate buffer, pH 7.4, to remove A chain
and excess DTT. For spin labeling the precipitate was solubilized in
500 µl of 1% SDS, pH 8, and 50 µl of a 20 mM solution
of
4-(3-iodo-2-oxypropylidene-1)-2,2,3,5,5-pentamethylimidazolidine-1-oxyl in N,N-dimethylformamide was added. For labeling with pyrene
the precipitate was solubilized in 500 µl of 100 mM
sodium carbonate buffer, pH 9, and 50 µl of a 20 mM
solution of pyrene maleimide in N,N-dimethylformamide was
added. The reaction mixtures were again incubated for 2 h in the
dark at room temperature. In the case of the spin-labeled B-chain, the
reaction mixture was desalted on a PD-10 column equilibrated with 1%
SDS to remove excess reagents, while in the case of the pyrene-labeled
B-chain the PD-10 column used was equilibrated with 10 mM
NaOH. Thiol estimation was performed using the
5,5'-dithiobis(nitrobenzoic acid) assay as described previously (19).
The B-chain is soluble in the presence of 1% SDS or at high pH. At
neutral pH in the absence of SDS the B-chain forms a soluble aggregate
at micromolar concentrations.
Kinetic and Steady-state Fluorescence Measurements--
All
fluorescence experiments were carried out using a Jasco FP 777 fluorimeter in 100 mM potassium phosphate buffer, pH 7.4, held in a 1-cm quartz cuvette at 25 °C. For kinetic measurements, the B-chain was first added to the cuvette and allowed to stand for 10 min (final concentration 1 µM). Increasing concentrations of SecB were then added and the increase in fluorescence at 377 nm was
measured as a function of time. Steady-state fluorescence measurements
were carried out using 344 nm as excitation wavelength and the emission
was monitored between 360 and 500 nm. To obtain the excitation spectra,
the excitation wavelength was varied from 320 to 360 nm and the
fluorescence was monitored at 377 and 470 nm for monomer and excimer
fluorescence, respectively. A slit-width of 1.5 nm was used for
measuring both excitation and emission.
Gel Filtration Experiments--
A Superose 6 (Amersham Pharmacia
Biotech) gel filtration column was equilibrated in 50 mM
potassium phosphate buffer, pH 7.4, containing 200 mM KCl.
A complex of B-chain with SecB was prepared by diluting 20 µl of a
100 µM stock solution of B-chain in 20 mM
NaOH, into 180 µl of 10 µM SecB in 100 mM
potassium phosphate buffer, pH 7.4. This mixture was incubated for
1 h before injecting it into the gel filtration column, which was
run at 0.3 ml min1. The soluble aggregate of B-chain was
prepared by diluting 20 µl of 100 µM stock solution of
B-chain contained in 20 mM NaOH, into 180 µl of 100 mM potassium phosphate buffer, pH 7.4. All the solutions
were centrifuged prior to injection. A dual channel detection mode at
280 and 340 nm was used to monitor the co-elution of SecB with the
pyrene-labeled B-chain.
ESR Measurements--
The ESR measurements were carried out
using a Brucker ESP 300 E spectrometer operating in the X-band mode. A
dielectric cavity TE011 (ER 4118) was used for all
experiments. 20-µl aliquots of the spin-labeled B-chain in 1% SDS or
in complex with SecB in 100 mM potassium phosphate buffer,
pH 7.4, were measured in sealed quartz capillaries. The complex of SecB
with the spin-labeled B-chain was prepared by diluting 20 µl of a 1 mM stock solution of B-chain into 990 µl of 300 µM SecB in 100 mM potassium phosphate buffer,
pH 7.4. The sample was then concentrated to 100 µl. 200 µl of
potassium phosphate buffer, pH 7.4, was added and the sample was
concentrated again to 100 µl. This was done 3 times to remove SDS.
Spectra of the samples at room temperature (298 K) were obtained by
averaging 3-5 scans at a scan width of 120 gauss. The microwave power
was set to be 2 mW and the modulation amplitude was optimized to the
line width of the individual spectra. ESR spectra in the frozen state
(183 K) were recorded at a microwave power of 0.05 mW.
| |
RESULTS |
SecB Prevents Aggregation of B-chain--
Reduction of the insulin
interchain disulfides leads to aggregation and precipitation of the
B-chain while the A-chain remains soluble in solution (20). This
reduction of disulfides is catalyzed by thioredoxin, a 10-kDa redox
protein. The aggregation was monitored by an increase in scatter at 360 nm as shown in Fig. 1. The ability of
SecB to prevent aggregation was tested at various ratios of insulin/SecB. As shown in Fig. 1, the presence of SecB significantly delays the onset of aggregation and the final amplitude of scattering. At 1:1 stoichiometry of SecB:B-chain, complete protection of B-chain from aggregation is seen. Thus, SecB monomer binds a single
B-chain.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Aggregation of insulin B-chain in the
presence and absence of SecB. The incubation mixture contained, in
a final volume of 1 ml, 50 mM potassium phosphate buffer,
pH 7, 100 mM NaCl, 1 mM EDTA, 20 mM
DTT, 50 µM insulin, and 2 µM thioredoxin
and increasing concentrations of SecB. From top to
bottom, curve 1, absence of SecB; curve 2 in the
presence of 15 µM SecB; curve 3, 30 µM SecB; curve 4, 40 µM SecB;
and curve 5, 50 µM SecB.
|
|
SecB Forms a Stable Complex with Insulin B-chain--
The B-chain
of insulin is a 30-residue peptide, which has two cysteine residues in
the 7th and 19th positions, 12 residues apart.
The two free thiols were labeled by pyrene maleimide and the
interaction of the labeled B-chain with SecB was investigated by size
exclusion chromatography. At low concentrations (1 µM)
the B-chain forms a soluble aggregate in the absence of the chaperone.
This soluble aggregate of B-chain elutes at 17 ml in 100 mM
potassium phosphate buffer, pH 7.4. This corresponds to a molecular
mass of 40 kDa, suggesting that the aggregate contains approximately 14 B-chains. The elution volume of SecB alone on a Superose 6 column is
13.5 ml. As seen in Fig. 2, in the
presence of SecB, the B-chain co-elutes at 13.5 ml, as shown by the
absorbance of the pyrene chromophore. This demonstrates that the
B-chain forms a stable complex with SecB.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Chromatographic profile of soluble aggregate
of pyrene-labeled B-chain alone and in complex with SecB. The
mixture of SecB (10 µM) and pyrene-labeled B-chain (1 µM) was prepared and chromatography was performed on a
Superose 6 column as described under "Experimental Procedures." The
continuous line ( ) represents the B-chain bound to SecB
and elutes at a position identical to free SecB. The dotted
line ( ... ) is the soluble aggregate of B-chain (1 µM) in the absence of SecB. The elution of the B-chain
bound to SecB and as a soluble aggregate was monitored by the
absorbance of the pyrene chromophore at 340 nm.
|
|
Kinetics of Disaggregation--
The fluorescence emission spectrum
of pyrene (excited at 345 nm) is composed of two bands, a structured
band with peaks near 377 and 390 nm, referred to as the monomer peak
and an unstructured broad excimer peak near 475 nm. An excimer band is
seen when an excited pyrene monomer interacts in a specific manner with
a neighboring pyrene in the ground state. If two pyrenyl groups are
close to each other (3.5 Å) they form an excimer upon excitation. It
has been previously demonstrated that the B-chain alone aggregates in
solution (20). As shown in Fig.
3A, a strong excimer peak centered at 470 nm indicates that the pyrene probes are in close proximity and that at low concentrations (1 µM) the
B-chain is in a soluble aggregated state in 100 mM
potassium phosphate buffer, pH 7.4. The spectrum also shows a small
amount of monomer peaks in the region of 377 nm. Addition of SecB to
the soluble aggregate results in changes in fluorescence intensities of
both monomer and excimer peaks. These changes are due in part to
decreased quenching of pyrene fluorescence by solvent in the SecB bound state. Hence in order to analyze intensities of the excimer peak in the
presence of SecB, the spectra in Fig. 3A were normalized so
as to have identical intensities at 377 nm (Fig. 3B). As
seen in Fig. 3B there is a decrease in the excimer
fluorescence at 470 nm with an increase in SecB concentration.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
A, fluorescence emission spectra of 1 µM pyrene-labeled B-chain alone and in complex with
varying concentrations of SecB. From bottom to
top at 400 nm the spectra represent 1 µM
pyrene-labeled B-chain in complex with increasing concentrations of
SecB: absence of SecB, 0.3, 0.7, 1.5, 3, 6, 9, 12, and 15 µM SecB. B, normalized fluorescence emission
spectra of 1 µM pyrene-labeled B-chain alone and in
complex with varying concentrations of SecB. Each emission spectra was
normalized to the fluorescence intensity at 377 nm. From top
to bottom the spectra represent 1 µM
pyrene-labeled B-chain in complex with increasing concentrations of
SecB: absence of SecB, 0.3, 0.7, 1.5, 3, 6, 9, 12, and 15 µM SecB. C, kinetics of dissociation of
B-chain aggregate (1 µM) in the presence of increasing
concentrations of chaperone SecB. From top to
bottom: kinetic traces with 15, 12, 9, 6, 3, 1.5, 0.7, and
0.3 µM SecB and without SecB. The dissociation of the
B-chain aggregate was monitored by change in fluorescence at 377 nm
after excitation at 344 nm. All the kinetic traces were fitted to a
single exponential function and the fitted lines are shown
along with each raw trace.
|
|
The kinetics of dissociation of the aggregate to individual B-chains
was studied by monitoring the increase in fluorescence in the
pyrene-monomer region of the spectra in Fig. 3A at 377 nm.
This increase in pyrene monomer B-chain fluorescence on interaction with SecB indicates that the aggregate has been disrupted by chaperone SecB. The kinetics of disaggregation was monitored by increase in
monomer fluorescence at 377 nm as a function of time. Fig. 3C shows the effect of increasing SecB concentrations on the
dissociation kinetics of the B-chain aggregate. As the SecB
concentration increases there is an increase in the final fluorescence
amplitude, which is saturable as shown in Fig.
4A. The kinetic traces were
fitted to single exponential and the data are shown in Fig.
4B. Identical values of the rate constant were obtained if
disaggregation was monitored using the excimer peak at 470 nm instead
(data not shown). At the end of each kinetic trace fluorescence
emission spectra were recorded. There is a decrease in excimer
fluorescence on addition of increasing concentrations of SecB. However,
a weak excimer is still present even at high concentrations of SecB as seen in Fig. 3B. This indicates that the pyrenes do come in
close proximity even in the bound state.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of increasing concentrations of SecB
on the dissociation kinetics of 1 µM B-chain aggregate. Kinetic
experiments were carried out as described in the legend to Fig. 3.
A, final fluorescence amplitude at 377 nm after excitation
at 344 nm; B, apparent rate constant obtained from fitting
the kinetic data to a single exponential function.
|
|
ESR Studies--
In order to characterize the insulin B-chain and
its environment in the complex with SecB, the thiols at residues 7 and
19 of the B-chain were labeled with IOPI. Fig.
5A shows the spectrum of
B-chain in 1% SDS. The line shape is characteristic of mobile spin
labels in 1% SDS solutions. The peaks are broader than expected for a
mobile spin label in aqueous solutions because the B-chain is
incorporated into micelles at these SDS concentrations (22). Fig.
5B shows an ESR spectrum of B-chain in complex with SecB. SDS was removed from the sample as described under "Experimental Procedures." Since a large excess of SecB is present, all B-chain is
in the bound state. Furthermore, any free B-chain would be insoluble
under these conditions. The absence of such a precipitate also shows
that all the B-chain is bound to SecB. The ESR line shape is a
characteristic of relatively immobilized labels. The bound signal in
the low field region of the spectrum has 2 spectral components (marked
in arrows). The 2 spin labels are in different environments
with one being in a more rigid environment than the other. The high
field signal is broad, no fine features could be observed due to low
signal to noise ratio. The SecB·B-chain complex can be denatured by
1% SDS and the resulting spectrum is then identical to that of Fig.
5A. In order to measure the distance between spin labels the
solution was frozen and the spectra were recorded at 183 K. As
described previously (23), the extent of magnetic dipolar interaction
between two spin-labeled cysteine side chains can be used to estimate
the distance between the bound radicals. To evaluate the line
broadening due to static dipolar interactions, quantitative analysis of
the interspin distance can be carried out in the absence of motion by
acquiring the spectra in the frozen state (183 K). An estimation of the
extent of broadening due to dipolar interaction is obtained from the
line height ratio d1/d and is in
direct correlation to the average distance of the radicals from each
other. In the absence of interaction (when a distance greater than 20 Å separates the radicals) a value of d1/d of less than 0.4 is expected
(23-27). The d1/d ratio is an indicator of dipolar interactions. A
d1/d of less than 0.4 indicates absence of dipolar interactions, whereas ratios higher than 0.4 shows
that the spin labels are closer than 20 Å in proximity. Fig.
5C shows a frozen spectrum at 183 K of SecB·B-chain
complex. A d1/d of 0.36 is obtained
which indicates that the 2 spin labels are greater than 20 Å apart.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
ESR spectra of insulin B-chain. A,
20 µM
4-(3-iodo-2-oxypropylidene-1)-2,2,3,5,5-pentamethylimidazolidine-1-oxyl
(IOPI)-labeled B-chain in 0.5% SDS. B, 20 µM IOPI-labeled B-chain bound to 300 µM
SecB at room temperature. An ESR spectrum in the low field region was
recorded with a 10-fold higher receiver gain and (averaged over 10 spectra) to confirm the 2 spectral components indicated by
vertical arrows and is plotted above the
vertical arrows. C, 20 µM
IOPI-labeled B-chain bound to 300 µM SecB at 183 K.
|
|
| |
DISCUSSION |
Thermodynamic Coupling Model of B-chain Dissociation by
SecB--
During the course of folding, many proteins reach a compact
state within milliseconds (1). The transition from the compact state to
the final folded structure is a slow process. During this slow process,
the non-native state of the protein can undergo non-productive
interactions leading to formation of aggregates or off pathway
products. The mechanism of interaction of chaperone SecB with such off
pathway dead end products has been investigated in this work, using the
B-chain as a model aggregate. Two kinds of models for the dissociation
of B-chain aggregate can be envisaged.
In one model the chaperone actively dissociates individual B-chains
from the aggregate as shown,
|
(Eq. 1)
|
where An, S, and
A:S represent the peptide aggregate, SecB, and
complex of SecB with peptide. Such a model would predict an increase in
the apparent rate constant of dissociation of the aggregate, with an
increase in concentration of SecB. As shown in Fig. 4B the
apparent rate constant is independent of SecB concentration. Thus, SecB
does not bind to or catalyze dissociation of B-chain aggregate.
In the other model we assume that the aggregate is in equilibrium with
the monomeric B-chains as shown,
|
(Eq. 2)
|
|
(Eq. 3)
|
Here, k1 and k1
are the microscopic rate constants for the dissociation and aggregation
events, respectively. Furthermore, k2 and
k2 represent the on-rate and off-rate of
binding to monomeric B-chain to SecB. It is important to note that the B-chain aggregate is an ensemble of multimeric species, which are at
equilibrium with monomeric B-chains. The slow step in the entire scheme
is assumed to be the reaction that converts the aggregate to monomeric
B-chain. The on-rate for SecB substrate binding is known to be close to
diffusion controlled (7). The free concentration of the unbound monomer
is small and hence both the forward and reverse reactions in Equation 2
are likely to be slow in comparison with the forward reaction in
Equation 3. Such a model would then predict that the observed rate of
disaggregation would be independent of SecB concentration. We indeed
observe that the apparent rate constant for the dissociation of the
aggregate is independent of SecB concentration. Hence, SecB uses the
intrinsic free energy of binding to the B-chain to shift the
equilibrium to the monomeric form.
Bound State Conformation of the B-chain to SecB--
ESR
spectroscopy has been used previously to understand proximity
relationships between spin labels (24-27). The bound state conformations of B-chain and mellitin peptides to chaperone
-crystallins have been investigated using this technique (20). In
addition to ESR, pyrene excimer fluorescence has been used extensively to give insight into the dynamics of labeled proteins and proximity relationships of the attached pyrene probes (21). A large body of work
suggests that proteins that bind a variety of unfolded and partially
folded peptide and protein substrates typically bind to an extended
conformation of the substrate. These systems include MHC molecules, Src
homology domains 2 and 3, and the periplasmic oligopeptide-binding
protein (28). Crystallographic studies on chaperone peptide complexes
have so far shown the only mode of peptide binding to be in an extended
state (29). Chaperones have the ability to bind proteins with no
apparent homology. The spectroscopic data in the present studies
indicate that the bound peptide is in a state in which one part of the
peptide is held more rigidly than the other as reflected from the ESR
data which shows 2 spectral components in the low field region.
However, both components show a substantial degree of immobilization.
This suggests that the binding site on each SecB monomer accommodates on the order of 10-12 residues. In case more than 12 residues were
bound, a greater degree of immobilization would have been expected for
both labels. If substantially fewer residues were bound, then the ESR
spectra should show peaks corresponding to one immobilized and one free
label. Such spectra were indeed observed in ESR studies of unfolded
BPTI mutants in which the spin labels were located greater than 20 residues apart.3 The present
results are also in agreement with a very recent study which suggests
that the binding frame of the substrate is of the order of 9 residues
(30), but not with earlier estimates of as high as 40 residues (31,
32). The low temperature ESR measurements show that in the bound state
the 2 Cys on the B-chain are greater than 20 Å apart. However, we do
observe the presence of a weak excimer for the pyrene-labeled B-chain.
This shows that the more flexible region of the peptide does encounter
the rigidly held pyrene within its excited state lifetime. Thus, the
data is consistent with a model that the B-chain is bound on the
surface of the chaperone SecB in a flexible extended state. In certain cases it has been shown that SecB binds to partially folded
conformations of proteins such as -lactalbumin and barstar (8, 10).
In the present work we suggest that the bound conformation is an extended conformation. The two results are not necessarily
contradictory because of the very short length of the binding frame.
Since SecB binds only small 10-residue regions of the substrate and the
binding site on SecB is solvent accessible (10) it is possible for the bound fragment to be extended even though the remainder of the protein
adopts a relatively compact conformation. Recent NMR studies of the
-lactalbumin molten globule have also shown that some regions of the
molten globule adopt an unfolded conformation (33).
Relevance to the in Vivo Function of SecB--
Protein folding
in vivo occurs at total protein concentrations much higher
than concentrations used for in vitro experiments. The
in vivo concentration of nascent polypeptide chains is
typically 50 µM in the cell (34). Non-productive
interaction among the partially folded proteins leads to aggregation.
The cell has evolved ways to stabilize the aggregation prone state from
engaging in non-productive interactions. Several molecular chaperones
are known to prevent aggregation. However, in only very few cases has
it been possible to show that chaperones can also dissociate pre-formed
aggregates and almost nothing is known about the mechanism of the
process. The Hsp104/Hsp70/Hsp40 and the ClpB/DnaK/DnaJ/GrpE are the
only chaperone systems reported in the literature that are capable of
rescuing dead end products of folding pathways (14, 15) and function by
active dissociation and remodeling of the aggregated species. The
primary function of SecB is believed to be funneling of its protein
substrates to the sec pathway. Since folded proteins are no
longer competent for export, SecB binds to its substrates before
complete folding has occurred and maintains them in an unfolded,
translocation competent state (9, 35). SecB has also been shown to
prevent irreversible aggregation of two of its natural protein
substrates OmpA and PhoE (6, 36). SecB also prevents aggregation and
promotes disaggregation of another natural substrate, maltose-binding
protein.4 However, little is
known at the molecular level about how this occurs with any of the
natural substrates. The present data implicates SecB as a molecular
chaperone whose function on the sec pathway is not only to
maintain substrates in a translocation competent state and prevent
aggregation but also to rescue the aggregated states and direct them to
a productive folding pathway. SecB can promote disaggregation by a
thermodynamic coupling mechanism by binding to free monomer that is in
equilibrium with the aggregated state. This simple and relatively
passive mechanism may be applicable to other instances of
chaperone-mediated protein disaggregation and is in contrast to the
more active role of chaperones such as Hsp104 and ClpB.
| |
ACKNOWLEDGEMENTS |
We thank Prof. B. de Kruijff for kindly
providing the SecB expression plasmid pJW25, Prof. M. Vijayan for
providing insulin for this work, and Dr. M. K. Mathew, C. Ganesh,
and Suvobrata Chakravarty for helpful discussions.
| |
FOOTNOTES |
*
This work was supported by grants from the Department of
Science and Technology and the Department of Biotechnology (to R. V.) and by Bundesministerium für Forschung und Bildung Grant INI-257-95 (to W. E. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of the Journal of Cell Science Traveling
fellowship from the Company of Biologists & Wood-Whelan Research
Fellowship from IUBMB.
**
To whom correspondence should be addressed. E-mail:
varadar@mbu.iisc.ernet.in; Fax: 91-80-3600535 or 3600683.
1
C. Ganesh and R. Varadarajan, unpublished results.
3
V. G. Panse, W. Trommer, P. Vogel, and R. Varadarajan, unpublished results.
4
C. Ganesh, F. Zaidi, J. B. Udgaonkar, and
R. Varadarajan, unpublished results.
| |
ABBREVIATIONS |
The abbreviations used are:
DTT, dithiothreitol;
ESR, electron spin spectroscopy.
| |
REFERENCES |
| 1.
|
Hartl, F. U.
(1996)
Nature
381,
571-580[CrossRef][Medline]
[Order article via Infotrieve]
|
| 2.
|
Gething, M.,
and Sambrook, J.
(1992)
Nature
355,
33-44[CrossRef][Medline]
[Order article via Infotrieve]
|
| 3.
|
Hendrick, J. P.,
and Hartl, F. U.
(1993)
Annu. Rev. Biochem.
62,
349-384[CrossRef][Medline]
[Order article via Infotrieve]
|
| 4.
|
Ellis, R. J.,
and Hartl, F. U.
(1999)
Curr. Opin. Struct. Biol.
9,
102-110[CrossRef][Medline]
[Order article via Infotrieve]
|
| 5.
|
Weiss, J. B.,
and Bassford, P. J.
(1990)
J. Bacteriol.
172,
3023-3029[Abstract/Free Full Text]
|
| 6.
|
Lecker, S. H.,
Driessen, J. M.,
and Wickner, W.
(1990)
EMBO J.
9,
2309-2314[Medline]
[Order article via Infotrieve]
|
| 7.
|
Fekkes, P.,
Blaauwen, T.,
and Driessen, A. J. M.
(1995)
Biochemistry
34,
10078-10085[CrossRef][Medline]
[Order article via Infotrieve]
|
| 8.
|
Panse, V. G.,
Udgaonkar, J. B.,
and Varadarajan, R.
(1998)
Biochemistry
37,
14477-14483[CrossRef][Medline]
[Order article via Infotrieve]
|
| 9.
|
Hardy, S. J.,
and Randall, L. L.
(1991)
Science
251,
439-443[Abstract/Free Full Text]
|
| 10.
|
Panse, V. G.,
Swaminathan, C. P.,
Surolia, A.,
and Varadarajan, R.
(2000)
Biochemistry
39,
2420-2427[CrossRef][Medline]
[Order article via Infotrieve]
|
| 11.
|
Bychkova, V.,
Krummeck, G.,
and Ptitsyn, O. B.
(1988)
FEBS Lett.
238,
231-234[CrossRef][Medline]
[Order article via Infotrieve]
|
| 12.
|
Schuler, J.,
Frank, J.,
Saenger, W.,
and Georgalis, Y.
(1999)
Biophys. J.
77,
1117-1125[Abstract/Free Full Text]
|
| 13.
|
Zahn, R.,
Perrett, S.,
and Fersht, A. R.
(1996)
J. Mol. Biol.
261,
43-61[CrossRef][Medline]
[Order article via Infotrieve]
|
| 14.
|
Glover, J. R.,
and Lindquist, S.
(1998)
Cell
94,
73-82[CrossRef][Medline]
[Order article via Infotrieve]
|
| 15.
|
Goloubinoff, P.,
Mogk, A.,
Zvi, A. P.,
Tomoyasu, T.,
and Bukau, B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13732-13737[Abstract/Free Full Text]
|
| 16.
|
Laemmli, U.
(1970)
Nature
227,
680-685[CrossRef][Medline]
[Order article via Infotrieve]
|
| 17.
|
Fasman, G. D.,
Park, K.,
and Randall, L. L.
(1995)
J. Prot. Chem.
14,
595-600[CrossRef][Medline]
[Order article via Infotrieve]
|
| 18.
|
Holmgren, A.
(1979)
J. Biol. Chem.
254,
9627-9632[Abstract/Free Full Text]
|
| 19.
|
Creighton, T. E.
(1990)
Protein Structure: A Practical Approach
, pp. 155-166, IRL Press, Oxford University, Oxford
|
| 20.
|
Farahbakhsh, Z. T.,
Huang, Q. L.,
Ding, L. L.,
Altenbach, C.,
Steinhoff, H. J.,
Horwitz, J.,
and Hubbell.
(1995)
Biochemistry
34,
509-516[CrossRef][Medline]
[Order article via Infotrieve]
|
| 21.
|
Lehrer, S. S.
(1997)
Methods Enzymol.
278,
286-295[Medline]
[Order article via Infotrieve]
|
| 22.
|
Stopar, D.,
Spruijt, R. B.,
Wolfs, C. J.,
and Hemminga, M. A.
(1996)
Biochemistry
3,
15467-15473
|
| 23.
|
Rabenstein, M. D.,
and Shin, Y. K.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
8239-8243[Abstract/Free Full Text]
|
| 24.
|
Hubbell, W. L.,
Mchaourab, H. S.,
Altenbach, C.,
and Lietzow, M. A.
(1996)
Structure
4,
779-783[Medline]
[Order article via Infotrieve]
|
| 25.
|
Hubbell, W. L.,
Gross, A.,
Langen, R.,
and Lietzow, M. A.
(1998)
Curr. Opin. Struct. Biol.
8,
649-656[CrossRef][Medline]
[Order article via Infotrieve]
|
| 26.
|
Anthony-Cahill, S. J.,
Benfield, P. A.,
Fairman, R.,
Wasserman, Z. R.,
Brenner, S. L.,
Stafford, W. F.,
Altenbach, C.,
Hubbell, W. L.,
and DeGrado, W. F.
(1992)
Science
255,
979-983[Abstract/Free Full Text]
|
| 27.
|
Zhao, M.,
Zen, K. C.,
Hernandez-Borrell, J.,
Altenbach, C.,
Hubbell, W. L.,
and Kaback, H. R.
(1999)
Biochemistry
38,
15970-15977[CrossRef][Medline]
[Order article via Infotrieve]
|
| 28.
|
Stanfield, R. L.,
and Wilson, I. A.
(1995)
Curr. Opin. Struct. Biol.
5,
103-113[CrossRef][Medline]
[Order article via Infotrieve]
|
| 29.
|
Joachimiak, A.
(1997)
Nature Struct. Biol.
4,
430-434[CrossRef][Medline]
[Order article via Infotrieve]
|
| 30.
|
Knoblauch, N. T.,
Rudiger, S.,
Schonfeld, H. J.,
Driessen, A. J.,
Schneider-Mergener, J.,
and Bukau, B.
(1999)
J. Biol. Chem.
274,
34219-34225[Abstract/Free Full Text]
|
| 31.
|
Topping, T. B.,
and Randall, L. L.
(1994)
Protein Sci.
3,
730-736[Abstract]
|
| 32.
|
Khisty, V. J.,
Munske, G. R.,
and Randall, L. L.
(1995)
J. Biol. Chem.
270,
25920-25927[Abstract/Free Full Text]
|
| 33.
|
Redfield, C.,
Schulman, B. A.,
Milhollen, M. A.,
Kim, P. S.,
and Dobson, C. M.
(1999)
Nat. Struct. Biol.
6,
948-952[CrossRef][Medline]
[Order article via Infotrieve]
|
| 34.
|
Morimoto, R. I.,
Tisseres, A.,
and Georgopoulos, C.
(1994)
The Biology of Heat Shock Proteins and Molecular Chaperones
, pp. 1-30, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
|
| 35.
|
Khisty, V. J.,
and Randall, L. L.
(1995)
J. Bacteriol.
177,
3277-3282[Abstract/Free Full Text]
|
| 36.
|
Breukink, E.,
Kusters, R.,
and De Kruijff, B.
(1992)
Eur. J. Biochem.
208,
419-425[Medline]
[Order article via Infotrieve]
|
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
T. Hornung, O. A. Volkov, T. M. A. Zaida, S. Delannoy, J. G. Wise, and P. D. Vogel
Structure of the Cytosolic Part of the Subunit b-Dimer of Escherichia coli F0F1-ATP Synthase
Biophys. J.,
June 15, 2008;
94(12):
5053 - 5064.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Motz, T. Hornung, M. Kersten, D. T. McLachlin, S. D. Dunn, J. G. Wise, and P. D. Vogel
The Subunit b Dimer of the FoF1-ATP Synthase: INTERACTION WITH F1-ATPase AS DEDUCED BY SITE-SPECIFIC SPIN-LABELING
J. Biol. Chem.,
November 19, 2004;
279(47):
49074 - 49081.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Sheluho and S. H. Ackerman
An Accessible Hydrophobic Surface Is a Key Element of the Molecular Chaperone Action of Atp11p
J. Biol. Chem.,
October 19, 2001;
276(43):
39945 - 39949.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. G. Panse, K. Beena, R. Philipp, W. E. Trommer, P. D. Vogel, and R. Varadarajan
Electron Spin Resonance and Fluorescence Studies of the Bound-state Conformation of a Model Protein Substrate to the Chaperone SecB
J. Biol. Chem.,
August 31, 2001;
276(36):
33681 - 33688.
[Abstract]
[Full Text]
[PDF]
|
|
|
Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
TOP
ABSTRACT
INTRODUCTION
