Biochem. J. (2011) 438, 505–511 (Printed in Great Britain)
505
doi:10.1042/BJ20110264
Interaction between bacterial outer membrane proteins and periplasmic
quality control factors: a kinetic partitioning mechanism
Si WU*1 , Xi GE†1 , Zhixin LV*, Zeyong ZHI*, Zengyi CHANG†2 and Xin Sheng ZHAO*2
*Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of
Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing 100871, China, and †State Key Laboratory for Protein and Plant Gene
Research, School of Life Sciences, Center for Protein Science, Peking University, Beijing 100871, China
The OMPs (outer membrane proteins) of Gram-negative bacteria
have to be translocated through the periplasmic space before
reaching their final destination. The aqueous environment of the
periplasmic space and high permeability of the outer membrane
engender such a translocation process inevitably challenging.
In Escherichia coli, although SurA, Skp and DegP have been
identified to function in translocating OMPs across the periplasm,
their precise roles and their relationship remain to be elucidated. In
the present paper, by using fluorescence resonance energy transfer
and single-molecule detection, we have studied the interaction
between the OMP OmpC and these periplasmic quality control
factors. The results of the present study reveal that the binding rate
of OmpC to SurA or Skp is much faster than that to DegP, which
may lead to sequential interaction between OMPs and different
quality control factors. Such a kinetic partitioning mechanism
for the chaperone–substrate interaction may be essential for the
quality control of the biogenesis of OMPs
INTRODUCTION
SurA, a major chaperone protein, is also located in the
periplasm, assisting the folding of OMPs in the periplasmic space
[13,14]. The level of OMPs in a strain with single deletion of
surA is decreased in the outer membrane, demonstrating the
participation of SurA in the translocation and folding of OMPs
[14]. Although SurA has two peptidyl-prolyl domains, these
domains are dispensable for the chaperone function of SurA [15].
SurA interacts with the Ar-X-Ar (Ar is an aromatic amino acid,
and X is any amino acid) amino acid motif, which is prevalent in
the OMPs, with affinities in the micromolar range as revealed by
phage display and isothermal titration calorimetry studies [16,17].
The periplasmic protein DegP has been reported to exhibit both
protease and chaperone activities [18], and is also suggested to be
involved in the biogenesis of OMPs [19–22]. Double-deletion of
surA and degP leads to a lethal phenotype at normal temperature,
suggesting that DegP may play a role in the translocation of OMPs
[22,23]. Structural studies indicate that DegP alone exists as an
inactive hexamer [24]. Previous work from our laboratory has
shown that DegP is activated via formation of large cages, both 12and 24-mers, upon its binding to the unfolded substrate proteins
[25]. The formation of complexes of 12- and 24-meric DegP
with substrates was independently reported by Krojer et al. [19],
and suggestions on the mechanism for the protease-chaperone
regulation of DegP were given based on the structural information.
These studies are undoubtedly very important in understanding
the function of these quality control factors in the biogenesis of
OMPs. However, the debate continues regarding the pathways
in which SurA, Skp and DegP are involved, and the exact
roles they play in safeguarding OMPs as they traverse the
periplasmic space [1–3]. Revealing these aspects of the proteins
will be greatly helped by looking into the interactions of these
periplasmic quality control factors with OMPs. The present study
In the Gram-negative bacterium Escherichia coli, the biogenesis
of the OMPs (outer membrane proteins) involves many steps,
including the translocation of OMPs through the cytosol, the
inner membrane and the periplasmic space, before the OMPs
are folded and assembled into the outer membrane [1–3]. Among
these steps, going across the periplasmic space is considered to
be highly challenging owing to the aqueous environment and the
fluctuating nature of the periplasm as a consequence of the high
permeability of the outer membrane. At present, the translocation
and protection of the nascent OMPs in the periplasmic space
is not well understood. In vitro investigations have shown that
many of the OMPs are capable of folding and inserting into
the lipid bilayers spontaneously with different folding kinetics
and efficiencies [4–6]. However, the efficient transportation of
OMPs across the periplasmic space needs assistance from some
facilitators in vivo [1–3].
Skp has been identified as a periplasmic chaperone that
participates in the translocation of OMPs through the periplasmic
space of E. coli [7,8]. The Skp protein binds to OMPs
with remarkable selectivity [7] and interacts with the nascent
OMPs at an early stage when OMPs are translocated through
the inner membrane [9]. Skp prevents the nascent OMPs
from aggregating in the periplasmic space by forming soluble
periplasmic intermediates with them [8]. In vitro studies have
shown that the Skp trimer forms a 1:1 complex with unfolded
OmpA, OmpG and YaeT, and the dissociation constant was
reported to be in the range of tens of nanomolar [10]. Skp
was also found to accelerate the folding kinetics of OmpA
and to increase its membrane insertion efficiency under in vitro
conditions [11,12].
Key words: DegP, fluorescence resonance energy transfer
(FRET), outer membrane protein, single-molecule detection, Skp,
SurA.
Abbreviations used: AF488, Alexa Fluor® 488; BODIPY 493/503, boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s -indacene) 493/503; Cy3,
indocarbocyanine; Cy5, indodicarbocyanine; FRET, fluorescence resonance energy transfer; LB, Luria–Bertani; OMP, outer membrane protein.
1
These authors contributed equally to this work.
2
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
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S. Wu and others
was conducted in an attempt to reveal the interaction between
OMPs and the three quality control factors involved in the
translocation of OMPs across the periplasmic space. For this
purpose, we used immunoblotting to analyse the level of OMPs
in cells with two of these three genes deleted and probed the
protein interactions, especially the binding kinetics between
the unfolded OMP OmpC and SurA, Skp and DegP by using FRET
(fluorescence resonance energy transfer) and single-molecule
detection. Our observations reveal that OMPs may interact with
these periplasmic quality control factors via a kinetic partitioning
mechanism, in which SurA and Skp interact with OMPs in a
fast step and protect the newly synthesized unfolded OMPs by
forming protease-resistant structures and conveying them through
subsequent quality control steps.
EXPERIMENTAL
Bacterial strains, cell growth and immunoblotting analysis
All bacterial strains were grown in liquid LB (Luria–Bertani)
medium at 37 ◦ C with antibiotics added according to their
resistance. For subculturing, the JGS276 [MC4100 skp
degP] strain was diluted 1:1000-fold into LB medium
after overnight culture. The JGS199 [MC4100 surA degP;
λ(pBADsurA)], JGS200 [MC4100 skp surA; λ(pBADsurA)]
and JGS272 [MC4100 skp surA degP(S210A); λ(pBADsurA)]
strains were first grown in the presence of 0.2 % Larabinose overnight and washed twice with fresh LB medium
before being diluted 1:2000, 1:10000 or 1:2000 into LB
medium without arabinose to get surA degP and skp
surA strains respectively. All cells were grown further for
6 h (MC4100 and JGS276) or 8 h (JGS199, JGS200, and
JGS272) prior to immunoblot analysis. To overexpress DegP
and DegP(S210A) in the MC4100 cells, the pACYC184derived plasmids pACYC184p-DegP-His6 and pACYC184pDegP(S210A)-His6 (see Supplementary Experimental section
at http://www.BiochemJ.org/bj/438/bj4380505add.htm for the
plasmid construction) were transformed by electroporation.
Samples containing an equal amount of cells with different
genotypes were heated at 95 ◦ C for 10 min in 2× loading
buffer before being subjecting to SDS/PAGE analysis. The
immunoblotting analysis was performed according to standard
protocols [26]. Rabbit anti-OmpC, anti-OmpA, anti-DegP and
mouse anti-OmpF polyclonal antibodies, and a mouse anti-GroEL
monoclonal antibody were used as the primary antibodies. AP
(alkaline phosphatase)-conjugated rabbit anti-mouse and goat
anti-rabbit secondary antibodies were used and visualized by
adding NBT (Nitro Blue Tetrazolium; Sigma) and BCIP (5bromo-4-chloroindol-3-yl phosphate; Promega) according to the
manufacturer’s instructions.
Protein expression and purification
The pET-28a vector carrying the His-tagged DegP(S210A),
Skp, SurA and OmpC were transformed into BL21(DE3)
cells. Cells were grown in LB medium containing 50 μg/ml
kanamycin at 37 ◦ C. IPTG (isopropyl β-D-thiogalactopyranoside,
0.5 mM) was added after the D600 reached 0.6. All His-tagged
proteins were purified by affinity chromatography using NiNTA (Ni2 + -nitrilotriacetate) resin (GE Healthcare) according to
the manufacturer’s protocol. The proteins were finally dialysed
into 50 mM sodium phosphate buffer containing 100 mM NaCl
(pH 7.0). Urea (8 M) was included in the lysis buffer for the
purification of OmpC in order to dissolve the inclusion bodies
containing the unfolded OMP.
c The Authors Journal compilation c 2011 Biochemical Society
Fluorescence labelling
Fluorescence labelling was performed by incubating the
proteins with a 5-fold molar excess of monoreactive NHS
(N-hydroxysuccinimide) ester-modified Cy3 (indocarbocyanine;
GE Healthcare), Cy5 (indodicarbocyanine; GE Healthcare),
BODIPY 493/503 [boron dipyrromethene (4,4-difluoro-4-bora3a,4a-diaza-s-indacene) 493/503; Invitrogen] or AF488 (Alexa
Fluor® 488; Invitrogen) in a 0.1 M NaHCO3 /Na2 CO3 buffer
(pH 8.3) at 25 ◦ C for 3 h. Urea was also added to the reaction
mixture to a final concentration of 8 M for labelling OmpC to
prevent its aggregation. The free dye was removed using a PD10 desalting column (GE Healthcare), using a 50 mM sodium
phosphate buffer containing 100 mM NaCl (pH 7.0) to elute
the labelled proteins (with 8 M urea added in the buffer for
eluting unfolded OmpC). The dye-to-protein molar ratios of the
eluted protein samples were all determined by dividing the molar
concentration of the dye by that of the particular protein after
correction according to the product instructions. The labelling
ratios are 1.2 for the SurA–AF488 monomer, 1.5 for the Skp–
AF488 trimer, 4.0 for the DegP(S210A)–AF488 hexamer, 2.7
for the OmpC–Cy3 monomer, 4.1 for the DegP(S210A)–Cy5
hexamer, 1.5 for the SurA–BODIPY monomer and 2.0 for Skp–
BODIPY trimer. The secondary structures of the labelled proteins
were found to be similar to the unlabelled proteins, as indicated
by their highly comparable far-UV (195–260 nm) CD spectra
(Supplementary Figure S1 at http://www.BiochemJ.org/bj/438/bj
4380505add.htm). The labelled proteins retained their function
of preventing the unfolded OmpC from precipitation, as was
demonstrated by the light scattering assay and the size-exclusion
chromatography (Supplementary Experimental section, and
Supplementary Figures S2 and S3 at http://www.BiochemJ.org/
bj/438/bj4380505add.htm).
Stopped-flow fluorescence measurement
The kinetics of interactions between chaperones and unfolded
OmpC were measured by using stopped-flow equipment (Biologic) consisting of an SFM-300 mixer and a MOS450/AF-CD
optical detection system equipped with a 150 W mercury/xenon
lamp. The fluorescence donor (AF488) intensity was recorded
with 488 nm excitation and the emission band pass filter was
510 +
− 15 nm. The AF488-labelled SurA, Skp or DegP(S210A)
and Cy3-labelled OmpC in 8 M urea were mixed to a volume
ratio of 20:1 to dilute the urea and to achieve a nondenaturing environment. The final concentration of each labelled
protein component was 0.3 μM in the reaction system. In the
three-component kinetic measurement, the unlabelled 5-fold
excess of DegP(S210A) was first mixed with AF488–SurA or
AF488–Skp before being loaded into the sample chamber. A
similar experiment was carried out in order to directly monitor
the interaction between OmpC–Cy3 and DegP(S210A) in the
presence of SurA or Skp, in which AF488-labelled DegP(S210A)
and unlabelled SurA or Skp were used. The normalized binding
curves were then fitted by using a second-order reaction equation
to obtain the kinetic parameters. The kinetic curves representing
the slow binding process were fitted to a single exponential
function to obtain the half time. The errors presented in the
kinetics data are the fitting errors of the curves. The relative error
was found to be 19 % by repeated experiments on different days.
Single-molecule FRET measurement
Single molecule FRET measurements were carried out by using
a home-built confocal microscope that has been described
Kinetic partitioning mechanism of the chaperone–OMP interaction
previously [27]. An inverted fluorescence microscope (TE2000U, Nikon) was equipped with a ×100 objective [N.A. (numerical
aperture) = 1.4, oil; Nikon] and a 532 nm laser for excitation.
The fluorescence emission from each sample was collected
via the same objective and split into a donor and an acceptor
channel by utilizing a Di650 dichroic mirror (Semrock) before
being focused on to two avalanche photon diodes (SPCM-AQR15; PerkinElmer Optoelectronics) with appropriate fluorescence
filters. The sample was prepared by directly mixing Cy3-labelled
DegP and Cy5-labelled OmpC, or by initially mixing Cy3-labelled
DegP(S210A) and SurA before adding Cy5-labelled OmpC, or
by initially mixing Cy3-labelled DegP(S210A) and Cy5-labelled
OmpC before adding SurA, in which the final concentration
of DegP(S210A)–Cy3 and OmpC–Cy5 were both 1×10 − 7 M,
whereas that of SurA was 5×10 − 7 M. Such samples were
subsequently diluted 2000-fold before being subjected to singlemolecule FRET measurement. Surface absorption of proteins
for the above measurements was prevented by adding 0.01 %
Tween 20 (Sigma) into the sample buffer (50 mM phosphate
containing 100 mM NaCl, pH 7.0). The fluorescence of the
labelled proteins or complex that freely diffused across the focal
volume (approximately 1 fl) was simultaneously collected in the
donor channel (565–650 nm) and the acceptor channel (670–
735 nm) with the bin time chosen as 1 ms. The FRET efficiency
was calculated as I acceptor /(I donor + I acceptor ) of each selected burst, and
was used to generate the statistical FRET distribution histograms.
Single-molecule fluorescence coincidence detection
The setup for single-molecule fluorescence coincidence detection
was similar to that of single-molecule FRET measurements,
except that different fluorescence filters were used in each
channel and a 488 nm laser (Spectra-Physics) was used as the
excitation light source. For coincidence detection, BODIPY
493/503–SurA (or BODIPY 493/503–Skp), Cy5–DegP(S210A)
and Cy3–OmpC were mixed at submicromolar concentrations,
incubated for 10 min to reach equilibrium, and diluted to 10–
50 pM. The fluorescence coincidence bursts of BODIPY and
Cy5 were recorded in parallel by 510/30 filter and 692/40 filter
(Semrock) after the fluorescence emission of the sample was
split via the utilization of a Di555 dichroic mirror (Semrock).
The simultaneous fluorescence bursts detected in both the
BODIPY and Cy5 channels were counted. In this experimental
design, fluorescence energy was also transferred from OmpC–
Cy3 to DegP(S210A)–Cy5 under the direct excitation of Cy3
by the 488 nm laser. However, such a fluorescence burst
in the Cy5 channel could be excluded if no signal was
simultaneously detected in the BODIPY channel. The total
number of real coincidence events, representing the formation
of the SurA–OmpC–DegP(S210A) or Skp–OmpC–DegP(S210A)
ternary complex, was calculated according to a method described
previously [28]. The control experiment was performed by
replacing BODIPY–SurA (or BODIPY–Skp) with BODIPY–
BSA, or by using the two-component system in the above
measurement.
RESULTS
SurA or Skp prevents the cleavage of the translocated OMPs
by DegP
In an attempt to reveal the functional relationship of SurA, Skp
and DegP in translocating OMPs through the periplasmic space,
we started by examining via immunoblotting the level of OMPs
507
Figure 1 The periplasmic chaperones SurA and Skp prevent the
degradation of OMPs by DegP
Immunoblot analysis on the level of the OMPs OmpA, OmpC, and OmpF in whole cells with the
indicated genotypes grown at normal temperature. The level of GroEL was monitored to indicate
equal sample loading in each lane. Wt, wild-type.
in the whole cell of three different strains with double-deletion
of the skp, surA and degP genes. The results showed that the
level of OMPs (as represented by OmpA, OmpC and OmpF) was
decreased in the skp surA double-deletion cells (Figure 1, lane 4),
whereas those in the skp degP or surA degP double-deleted strains
were highly comparable with that in the wild-type cells (Figure 1,
lanes 1, 2 and 3). On the other hand, the strain with the surA
skp double-deletion and with the mutation of the chromosomal
degP gene to protease-deficient DegP(S210A) did not exhibit
an obvious decrease in the level of OMPs (Figure 1, lane 5),
indicating that it was the protease activity of DegP that caused
the decrease in the level OMPs in the surA skp double-deleted
mutant strain. To show that the decrease in OMPs in the surA
skp double-deletion mutant was due to the lack of SurA and Skp
instead of an up-regulated expression of DegP, we overexpressed
DegP using a pACYCp-DegP vector in the wild-type cells, and
no obvious decrease of OMPs was observed (Figure 1, lane 6),
even though the DegP level in this strain was much higher than
that in the strain with both surA and skp deleted (Figure 1, lane
4). As a control, OMPs in the wild-type cells overexpressing the
protease-deficient DegP(S210A) by the pACYCp-DegP(S210A)
vector were also analysed (Figure 1, lane 7). These results indicate
that DegP exercises its protease activity to degrade the nascent
unfolded OMPs that are not well protected by SurA and Skp
during the translocation of OMPs across the periplasm.
The binding of unfolded OMPs to SurA or Skp exhibits
a kinetic advantage
To discover the molecular mechanism for the interaction
between OMPs and the chaperones, we carried out FRET
measurements between OMPs (as represented by OmpC) and
SurA, Skp or DegP(S210A) (a protease-defective mutant of
DegP), in which SurA, Skp and DegP(S210A) were labelled with
AF488, and unfolded OmpC was labelled with Cy3. The CD
spectra, light scattering and the size-exclusive chromatography
c The Authors Journal compilation c 2011 Biochemical Society
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Figure 2
S. Wu and others
Unfolded OmpC preferentially binds to Skp or SurA and is subsequently bound to DegP
(A) Time-dependent fluorescence curves of AF488-labelled SurA, Skp or DegP(S210A) proteins upon binding to the unfolded OmpC–Cy3. All of the fluorescence curves in this Figure were
recorded utilizing a stopped-flow instrument. (B) Time-dependent fluorescence curves of AF488-labelled SurA upon binding to unfolded OmpC–Cy3 in the absence or presence of DegP(S210A).
(C) Time-dependent fluorescence curves of AF488-labelled Skp upon binding to unfolded OmpC–Cy3 in the absence or presence of DegP(S210A). (D) Time-dependent fluorescence curves of
AF488-labelled DegP(S210A) upon binding to unfolded OmpC–Cy3 in the presence of SurA or Skp. The fit is shown as dark grey lines, from which the reaction half times (t 1/2 ) were obtained.
analyses indicated that the labelling process affected neither the
secondary structure of the chaperones (Supplementary Figure
S1) nor their chaperone activities (Supplementary Figures S2
and S3). We then performed stopped-flow studies to measure
the kinetics of the interaction between OmpC and the three
chaperones. The direct binding of Cy3-labelled OmpC to AF488labelled SurA, Skp or DegP(S210A) was monitored via the
time-dependent change of the donor (AF488) fluorescence. The
results clearly demonstrated that unfolded OmpC bound to
SurA (t1/2 = 58.5 +
− 0.7 ms) or Skp (t1/2 = 18.8 +
− 0.3 ms) at a rate
approximately 1000-fold higher than to DegP (t1/2 = 28.3 +
− 0.1 s)
(Figure 2A). When unfolded OmpC–Cy3 was added into a
mixture containing SurA–AF488 and DegP(S210A), or Skp–
AF488 and DegP(S210A), we observed a sudden initial decrease
followed by a gradual increase in donor fluorescence. This most
probably reflects the initial binding of unfolded OmpC–Cy3 to
either SurA–AF488 or Skp–AF488 and the subsequent slow
binding to DegP(S210A), with the half time of the processes
measured to be 6.92 +
− 0.06 s and 26.1 +
− 0.3 s respectively
(Figures 2B and 2C). Additional evidence supporting this
conclusion is the observation of a time-dependent decrease
in donor fluorescence when DegP(S210A), instead of Skp
or SurA, was labelled with AF488 in the above experiment.
We found that the reaction half times were 7.28 +
− 0.03 s and
0.3
s
respectively,
consistent
with
the
above
observations
27.7 +
−
(Figure 2D). These results indicated that OmpC–Cy3 was
captured by SurA or Skp and then bound to DegP(S210A), rather
than dissociating from SurA/Skp into solution. These revelations
imply that the binding of unfolded OMPs to SurA or Skp
(rather than to DegP) is under kinetic control, and this kinetic
advantage prevents their direct binding to and degradation by
DegP.
c The Authors Journal compilation c 2011 Biochemical Society
Ternary complexes containing OMP, SurA or Skp, and DegP
are formed
The observations that unfolded OmpC captured by SurA or Skp
can bind to DegP (Figure 2) and that there exists an interaction
between DegP and SurA or Skp (Supplementary Figure S4 at
http://www.BiochemJ.org/bj/438/bj4380505add.htm) imply the
formation of SurA–OmpC–DegP or Skp–OmpC–DegP ternary
complexes during this process. Therefore we designed a threecolour FRET experiment, in which SurA (or Skp), OmpC
and DegP(S210A) were each labelled with BODIPY 493/503,
Cy3 and Cy5 respectively. FRET between SurA–BODIPY (or
Skp–BODIPY) and OmpC–Cy3, and between OmpC–Cy3 and
DegP(S210A)–Cy5 were observed in the three-component system
by ensemble fluorescence spectroscopy (Supplementary Figure
S5 at http://www.BiochemJ.org/bj/438/bj4380505add.htm). To
directly observe the ternary complexes, we then carried out
FRET detection at the single-molecule level, envisioning that
a coincident burst of BODIPY and Cy5 in the single-molecule
coincidence detection would be possible only when the SurA–
OmpC–DegP(S210A) or Skp–OmpC–DegP(S210A) ternary
complex was formed (Figure 3A). We mixed the fluorescence
labelled SurA (or Skp), OmpC and DegP(S210A) and counted
the rate of coincidence occurrence in the BODIPY and Cy5
channels. The coincidence rate detected for the three-component
samples (Figure 3B, bars 1 and 4) was revealed to be significantly
higher than that of either the two-component systems,
i.e. SurA–BODIPY/OmpC–Cy3, Skp–BODIPY/OmpC–Cy3 or
Skp–BODIPY/DegP(S210A)–Cy5 (where coincidence signals
came from the cross-talk of the two components) or the
three-component negative control of BSA–BODIPY/OmpC–
Cy3/DegP(S210A)–Cy5 (Figure 3B, bars 2, 3, 5 and 6). These
Kinetic partitioning mechanism of the chaperone–OMP interaction
509
Figure 4 The distribution of single-molecule FRET efficiency for the
interaction between DegP(S210A)–Cy3 and unfolded OmpC–Cy5
Figure 3 Capture of ternary complexes of OmpC, DegP and SurA (or Skp)
by single-molecule coincidence detection
(A) Schematic diagram indicating the three-colour single-molecule FRET detection. (B)
Coincidence occurrence rate in: 1, SurA–BODIPY + OmpC–Cy3 + DegP(S210A)–Cy5; 2,
SurA–BODIPY + DegP(S210A)–Cy5; 3, BSA–BODIPY + OmpC–Cy3 + DegP(S210A)–Cy5;
4, Skp–BODIPY + OmpC–Cy3 + DegP(S210A)–Cy5; 5, Skp–BODIPY + DegP(S210A)–Cy5;
and 6, Skp–BODIPY + OmpC–Cy3. (C) Distributions of the ratio factor R in the mixture of
Skp–BODIPY, OmpC–Cy3 and DegP(S210A)–Cy5 (dark grey), the mixture of Skp–BODIPY and
OmpC–Cy3 (light grey), and the mixture of Skp–BODIPY and DegP(S210A)–Cy5 (white).
observations demonstrated the existence of the SurA–OmpC–
DegP(S210A) and Skp–OmpC–DegP(S210A) ternary complexes
in the three-component system. In addition, we calculated the ratio
factor R, which was defined as the coincidence burst intensity in
the Cy5 channel divided by the total intensity in the Cy5 and
BODIPY channels. The result in Figure 3(C) shows that, besides
the lower coincidence occurrence, the R value distribution of the
control systems were also lower than that of the three-component
complex, further confirming that the coincidence signals came
from the ternary complex.
Presence of SurA or Skp alters the interaction pattern between
OMPs and DegP
We next examined whether there is any difference between the
interaction of OmpC and DegP in the presence or absence of SurA
(or Skp). For this purpose, the distribution of single-molecule
FRET efficiency was measured, which can provide information
about the conformational changes in protein–protein interactions.
The histogram of FRET efficiency distribution of adding unfolded
(A) In the absence of SurA. (B) SurA addition before unfolded Cy5–OmpC was added. (C) SurA
addition after unfolded OmpC–Cy5 was added. The distribution histograms are fitted (grey lines)
by a Gaussian function for the right peak and a γ -function for the left ‘zero’ peak in each panel,
which results from donor-only-labelled protein complex or photobleached acceptor.
OmpC–Cy5 into a solution containing DegP(S210A)–Cy3 alone
(with the peak at 0.53) (Figure 4A) differed significantly from that
of adding unfolded OmpC–Cy5 into a mixture containing SurA
and DegP(S210A)–Cy3 (with the peak at 0.39) (Figure 4B). By
contrast, the distribution histogram of adding SurA protein into a
pre-mixed solution of unfolded OmpC–Cy5 and DegP(S210A)–
Cy3 (Figure 4C) was very similar to that of adding unfolded
OmpC–Cy5 into DegP(S210A)–Cy3 alone (Figure 4A). The
common peak seen here on the left of all of the histograms
primarily resulted from the fluorescence signal of the unbound
Cy3-labelled DegP(S210A), as well as that of the Cy3-labelled
DegP(S210A) bound to the photobleached Cy5-labelled OmpC.
A similar effect was also observed when SurA was replaced by
Skp in the above analysis (results not shown). Our single-molecule
detection demonstrated that the FRET efficiency distribution of
unfolded OmpC directly bound to DegP differs from that of
unfolded OmpC pretreated with SurA, indicating that SurA (or
Skp) induces an alternation in the conformation of OmpC, which
may allow OmpC to avoid being acted upon by the protease
activity of DegP.
DISCUSSION
The translocation of newly synthesized OMPs through the
periplasmic space in Gram-negative bacteria is regarded as
c The Authors Journal compilation c 2011 Biochemical Society
510
S. Wu and others
an extremely challenging process [1,29,30]. Although several
periplasmic chaperones have been identified as playing a role
in the translocation of OMPs across the periplasmic space, little
is known about how they work and collaborate in assisting
the folding of OMPs. To understand more about their working
mechanism, biophysical studies that probe the direct interactions
in this process between the three related periplasmic quality
control factors SurA, Skp, and DegP and their substrate OMPs
are very helpful. One of the novel discoveries in the present
study is that OmpC binds to SurA and Skp with dramatically
different kinetics compared with its binding to DegP. In view
of this novel observation, we suppose that the three chaperones
interact with the OMPs in a sequential fashion, such that SurA
and Skp function at an early stage when the unfolded OMPs
are secreted into the periplasm, whereas DegP, as a bifunctional
protease-chaperone, functions subsequently after SurA and Skp.
Furthermore, the binding affinities between OmpC and the three
chaperones, measured using FRET, reveal that the interaction
between unfolded OmpC and DegP(S210A) is more stable than
that between OmpC and SurA or Skp (Supplementary Figure
S6 at http://www.BiochemJ.org/bj/438/bj4380505add.htm). The
kinetic preference of OmpC binding to SurA and Skp and
the thermodynamic advantage of OmpC binding to DegP together
depicts a flow chart of sequential interactions that may be crucial
for the translocation of OMPs across the periplasm.
The role of DegP in Gram-negative bacteria is known to be
essential for cells to grow at high temperatures, and DegP exhibits
both chaperone and protease activities in vitro [10]. Previously,
DegP was found to form stable complexes with OMPs in their
folded state, providing new clues for unveiling its quality control
function [19]. Our immunoblotting analysis demonstrates that the
OMPs in the surA skp double-deletion strain could be degraded
by DegP via a direct interaction. Previous genetics studies have
shown a decreased amount of the OMPs in the whole cell, even
with a single deletion of either surA or skp [7,22]. It is conceivable
that chaperones SurA and Skp may act as folding factors that bind
OMPs immediately after the OMPs are secreted from the inner
membrane through the Sec machinery. The kinetic partitioning
of OMPs to the three quality control factors ensures the
protection and partial folding of OMPs, to prevent their excessive
degradation by DegP, facilitating effective OMPs biogenesis. Our
single-molecule FRET results indicate that SurA may vary the
interaction pattern between OMPs and DegP, and that DegP can
form a ternary complex with SurA–OMPs or Skp–OMPs. These
revelations suggest that, as a quality control factor, DegP may
act upon OMPs subsequent to SurA and Skp, either to safeguard
the partially folded OMPs for further folding and assembly, or to
degrade the misfolded OMPs, depending on whether the OMPs
have been properly protected by SurA/Skp or not.
One formerly proposed model regarding the roles of these three
quality control factors in the biogenesis of OMPs revealed by
genetic evidence is that they work as two parallel pathways:
one involves Skp and DegP, whereas the other involves only
SurA [22,23]. This hypothesis was primarily proposed in view
of the fact that the skp surA and degP surA double-knockout
strains show lethal phenotypes, whereas the skp degP doubleknockout strain remains viable at normal temperatures [22,23].
However, this model has been questioned by other researchers
[3]. The results of the present study imply that DegP not only
collaborates with Skp, but also with SurA, which is demonstrated
by the observation of an interaction between SurA and DegP,
and by the formation of the SurA–OmpC–DegP ternary complex.
Nevertheless, the interaction between SurA and DegP is weaker
in comparison with that between Skp and DegP (Supplementary
Figure S4), suggesting that the Skp protein somehow works more
c The Authors Journal compilation c 2011 Biochemical Society
closely with DegP than does SurA, which is consistent with the
previous genetic studies [22,23].
An issue that remains undefined is whether SurA and Skp have
any preference in the OMPs that they bind. A broad range of
substrate OMPs have been identified by proteomics methods to
interact with Skp in vivo [31]. By comparing the abundance of
OMPs in wild-type and surA-deleted strains, SurA was found
to be involved in the biogenesis of FhuA and LptD, as well
as in the biogenesis of the major OMPs OmpA, OmpC and
OmpF [32]. There seems to be some overlap in OMP substrates
for SurA and Skp, and the underlying purpose for having
evolved two molecular chaperones upstream of DegP warrants
further investigation. Undoubtedly, safeguarding the OMPs going
through the periplasmic space is an extremely challenging and
complicated task, exploration of which would greatly enhance
our understanding of this paradigm of protein quality control.
AUTHOR CONTRIBUTION
Si Wu, Xi Ge, Zengyi Chang and Xin Sheng Zhao designed the experiments; Si Wu
performed the FRET and single-molecule detection experiments; Xi Ge constructed
the plasmids and carried out the biochemical experiments; Zhixin Lv contributed to the
chaperone activity detection; Zeyong Zhi set up the instrument for single-molecule
detection; and Si Wu, Xi Ge, Zengyi Chang and Xin Sheng Zhao analysed the data
and wrote the paper.
ACKNOWLEDGEMENTS
We thank Professor Pengye Wang (Institute of Physics, Chinese Academy of Sciences,
Beijing, China) and Professor Luhua Lai (Institute of Physical Chemistry, Peking University,
China) for allowing us to use their stopped-flow instruments; Professor Thomas J.
Silhavy (Department of Molecular Biology, Princeton University, Princeton, NJ, U.S.A.)
for providing us with the knockout strains of JGS199, JGS200, JGS272 and JGS276;
Professor Xuanxian Peng (School of Life Sciences, Sun Yat-Sen University, China) for
providing us with the anti-OmpC antibody; and Professor Sarah Perrett (Institute of
Biophysics, Chinese Academy of Sciences, Beijing, China) for the critical reading of our
paper prior to submission.
FUNDING
This work was supported by the National Natural Science Foundation of China [grant
numbers 20733001, 20973015 (to X.S.Z.), 30570355, 30670022 (to Z.Y.C.)]; and the
National Key Basic Research Foundation of China [grant numbers 2010CB912302 (to
X.S.Z.), 2006CB806508 (to Z.Y.C.), 2006CB910300 (to X.S.Z. and Z.Y.C.)].
REFERENCES
1 Ruiz, N., Kahne, D. and Silhavy, T. J. (2006) Advances in understanding bacterial
outer-membrane biogenesis. Nat. Rev. Microbiol. 4, 57–66
2 Knowles, T. J., Scott-Tucker, A., Overduin, M. and Henderson, I. R. (2009) Membrane
protein architects: the role of the BAM complex in outer membrane protein assembly. Nat.
Rev. Microbiol. 7, 206–214
3 Walther, D. M., Rapaport, D. and Tommassen, J. (2009) Biogenesis of β-barrel membrane
proteins in bacteria and eukaryotes: evolutionary conservation and divergence. Cell. Mol.
Life Sci. 66, 2789–2804
4 Burgess, N. K., Dao, T. P., Stanley, A. M. and Fleming, K. G. (2008) β-Barrel proteins that
reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior
in vitro . J. Biol. Chem. 283, 26748–26758
5 Kleinschmidt, J. H. and Tamm, L. K. (1996) Folding intermediates of a β-barrel
membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism.
Biochemistry 35, 12993–13000
6 Kleinschmidt, J. H. and Tamm, L. K. (2002) Secondary and tertiary structure formation of
the β-barrel membrane protein OmpA is synchronized and depends on membrane
thickness. J. Mol. Biol. 324, 319–330
7 Chen, R. and Henning, U. (1996) A periplasmic protein (Skp) of Escherichia coli
selectively binds a class of outer membrane proteins. Mol. Microbiol. 19, 1287–1294
8 Schafer, U., Beck, K. and Muller, M. (1999) Skp, a molecular chaperone of Gram-negative
bacteria, is required for the formation of soluble periplasmic intermediates of outer
membrane proteins. J. Biol. Chem. 274, 24567–24574
Kinetic partitioning mechanism of the chaperone–OMP interaction
9 Harms, N., Koningstein, G., Dontje, W., Muller, M., Oudega, B., Luirink, J. and de Cock,
H. (2001) The early interaction of the outer membrane protein phoe with the periplasmic
chaperone Skp occurs at the cytoplasmic membrane. J. Biol. Chem. 276, 18804–18811
10 Qu, J., Mayer, C., Behrens, S., Holst, O. and Kleinschmidt, J. H. (2007) The trimeric
periplasmic chaprone Skp of Escherichia coli forms 1:1 complexes with outer membrane
proteins via hydrophobic and electrostatic interactions. J. Mol. Biol. 374, 91–105
11 Bulieris, P. V., Behrens, S., Holst, O. and Kleinschmidt, J. H. (2003) Folding and insertion
of the outer membrane protein OmpA is assisted by the chaperone Skp and by
lipopolysaccharide. J. Biol. Chem. 278, 9092–9099
12 Patel, G. J., Behrens-Kneip, S., Holst, O. and Kleinschmidt, J. H. (2009) The periplasmic
chaperone Skp facilitates targeting, insertion, and folding of OmpA into lipid membranes
with a negative membrane surface potential. Biochemistry 48, 10235–10245
13 Lazar, S. W. and Kolter, R. (1996) SurA assists the folding of Escherichia coli outer
membrane proteins. J. Bacteriol. 178, 1770–1773
14 Rouviere, P. E. and Gross, C. A. (1996) SurA, a periplasmic protein with peptidyl-prolyl
isomerase activity, participates in the assembly of outer membrane porins. Genes Dev.
10, 3170–3182
15 Behrens, S., Maier, R., de Cock, H., Schmid, F. X. and Gross, C. A. (2001) The SurA
periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone
activity. EMBO J. 20, 285–294
16 Bitto, E. and McKay, D. B. (2003) The periplasmic molecular chaperone protein SurA
binds a peptide motif that is characteristic of integral outer membrane proteins. J. Biol.
Chem. 278, 49316–49322
17 Bitto, E. and McKay, D. B. (2004) Binding of phage-display-selected peptides to the
periplasmic chaperone protein SurA mimics binding of unfolded outer membrane
proteins. FEBS Lett. 568, 94–98
18 Spiess, C., Beil, A. and Ehrmann, M. (1999) A temperature-dependent switch from
chaperone to protease in a widely conserved heat shock protein. Cell 97, 339–347
19 Krojer, T., Sawa, J., Schafer, E., Saibil, H. R., Ehrmann, M. and Clausen, T. (2008)
Structural basis for the regulated protease and chaperone function of DegP. Nature 453,
885–890
20 Misra, R., CastilloKeller, M. and Deng, M. (2000) Overexpression of protease-deficient
DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants
in a degP background. J. Bacteriol. 182, 4882–4888
511
21 CastilloKeller, M. and Misra, R. (2003) Protease-deficient DegP suppresses lethal effects
of a mutant OmpC protein by its capture. J. Bacteriol. 185, 148–154
22 Sklar, J. G., Wu, T., Kahne, D. and Silhavy, T. J. (2007) Defining the roles of the
periplasmic chaperones SurA, Skp, and DegP in Escherichia coli . Genes Dev. 21,
2473–2484
23 Rizzitello, A. E., Harper, J. R. and Silhavy, T. J. (2001) Genetic evidence for parallel
pathways of chaperone activity in the periplasm of Escherichia coli . J. Bacteriol. 183,
6794–6800
24 Krojer, T., Garrido-Franco, M., Huber, R., Ehrmann, M. and Clausen, T. (2002) Crystal
structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature 416,
455–459
25 Jiang, J. S., Zhang, X. F., Chen, Y., Wu, Y., Zhou, Z. H., Chang, Z. and Sui, S. F. (2008)
Activation of DegP chaperone-protease via formation of large cage-like oligomers
upon binding to substrate proteins. Proc. Natl. Acad. Sci. U.S.A. 105,
11939–11944
26 Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Struhl, K. and Smith, J. A. (1995)
Short Protocols in Molecular Biology, John Wiley & Sons, Chichester
27 Chen, X. D., Zhou, Y., Qu, P. and Zhao, X. S. (2008) Base-by-base dynamics in DNA
hybridization probed by fluorescence correlation spectroscopy. J. Am. Chem. Soc. 130,
16947–16952
28 Orte, A., Clarke, R., Balasubramanian, S. and Klenerman, D. (2006) Determination of the
fraction and stoichiometry of femtomolar levels of biomolecular complexes in an excess
of monomer using single-molecule, two-color coincidence detection. Anal. Chem. 78,
7707–7715
29 Koebnik, R., Locher, K. P. and Van Gelder, P. (2000) Structure and function of bacterial
outer membrane proteins: barrels in a nutshell. Mol. Microbiol. 37, 239–253
30 Danese, P. N. and Silhavy, T. J. (1998) Targeting and assembly of periplasmic and
outer-membrane proteins in Escherichia coli . Annu. Rev. Genet. 32, 59–94
31 Jarchow, S., Luck, C., Gorg, A. and Skerra, A. (2008) Identification of potential substrate
proteins for the periplasmic Escherichia coli chaperone Skp. Proteomics 8,
4987–4994
32 Vertommen, D., Ruiz, N., Leverrier, P., Silhavy, T. J. and Collet, J. F. (2009)
Characterization of the role of the Escherichia coli periplasmic chaperone SurA using
differential proteomics. Proteomics 9, 2432–2443
Received 10 February 2011/2 June 2011; accepted 14 June 2011
Published as BJ Immediate Publication 14 June 2011, doi:10.1042/BJ20110264
c The Authors Journal compilation c 2011 Biochemical Society
Biochem. J. (2011) 438, 505–511 (Printed in Great Britain)
doi:10.1042/BJ20110264
SUPPLEMENTARY ONLINE DATA
Interaction between bacterial outer membrane proteins and periplasmic
quality control factors: a kinetic partitioning mechanism
Si WU*1, Xi GE†1, Zhixin LV*, Zeyong ZHI*, Zengyi CHANG†2 and Xin Sheng ZHAO*2
*Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology,
College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing 100871, China, and †School of Life Sciences,
Center for Protein Science, and State Key Laboratory for Protein and Plant Gene Research, Peking University, Beijing 100871, China
EXPERIMENTAL
Plasmid construction
Plasmids expressing DegP(S210A), Skp, SurA or OmpC with or
without the His tag were constructed using standard recombinant
DNA techniques, as described below.
Plasmids expressing DegP under the control of the natural
degP promoter were constructed as follows: the DNA fragment
containing both the promoter and the coding region of the degP
gene was PCR-amplified using the genomic DNA of E. coli K12 as
the template and the respective oligonucleotides complementary
to the region 500 bp upstream of its start codon and the region
of its stop codon as the 5 and 3 primers (see Supplementary
Table S1), and was directly ligated on to the pEASY-T1-Simple
TA cloning vector (TransGen Biotech). After being verified by
DNA sequencing, the DNA fragment was removed and inserted
into the pACYC184 plasmid, after being cleaved with HindIII
and BamHI. The resulting construct, denoted as pACYC184pDegP-His6 , was verified by PCR-amplification using appropriate
primers (primers 2 and 5, Supplementary Table S1). The plasmid
pACYC184p-DegP(S210A)-His6 was constructed by overlapping
PCR for generating the serine-to-alanine replacement (using two
pairs of primers: 1 and 4 and 2 and 3, Supplementary Table S1) at
position 210 in DegP. The plasmid overexpressing DegP(S210A)–
His6 was constructed by inserting the coding sequences without
promoters into pET-28a (Nonagen), using the NcoI and HindIII
cloning sites.
The pET28a-derived plasmids overexpressing the SurA–His6 ,
His6 –Skp and unfolded His6 –OmpC were also constructed in a
similar fashion as pET28a-DegP-His6 , but without their signal
peptides.
CD experiment
The secondary structure of unlabelled chaperones and
fluorescently labelled chaperones was compared by CD
spectroscopy. CD spectra were recorded in a 1-mm path quartz
cuvette using a Jasco J-815 spectrometer. All spectra were
measured at 25 ◦ C in 50 mM sodium phosphate buffer containing
100 mM NaCl (pH 7.0). The final concentrations of labelled and
unlabelled proteins for CD experiments were 1.1 μM for Skp,
1.1 μM for SurA and 4.7 μM for DegP(S210A).
Light scattering is a convenient method to check whether protein
precipitation is occurring and whether a chaperone is functioning
normally [1–3]. The unfolded OMPs form aggregates when
diluted from 8 M urea to an aqueous environment, and the
chaperone activity of the periplasmic quality control factors can
prevent the aggregation of unfolded OmpC. These processes
can thus be detected by the light scattering assay [1–3]. In the
present study, the light scattering assay was performed on a UV
absorbance spectrometer (U3900; Hitachi). The samples were
prepared by directly diluting unfolded OmpC in 8 M urea into
PBS in the absence or presence of different chaperones. The
final concentration of OmpC was 1.5 μM. The concentrations
of DegP(S210A), Skp and SurA were 3 μM, 7.5 μM and 30 μM
respectively, which were the minimum concentrations necessary
for the wild-type chaperones to prevent OmpC aggregation in
vitro. The time-dependent light scattering curves were recorded
immediately after rapid mixing of OmpC and the different
chaperones. The light scattering caused by the aggregation of
OmpC was monitored by measuring light absorption at 380 nm.
The rapid precipitation of OmpC in PBS buffer decreased the
transparency of the solution and gave rise to the increase in
the absorbance which is maintained for hundreds of seconds,
whereas, in the presence of SurA, Skp or DegP(S210A), the light
scattering intensity remained at a near zero level over this time
period (Supplementary Figure S2). These results demonstrated
that the labelling did not have any noticeable influence on the
chaperone activity in protecting OmpC from aggregation.
Size-exclusive chromatography
Size-exclusive chromatography was performed using an AKTA
FPLC system (Amersham Biosciences) with a Superdex 200
10/300 GL column (GE Healthcare). A 100 μl protein sample
was loaded into the column and eluted with 50 mM sodium
phosphate buffer containing 100 mM NaCl (pH 7.0). The final
concentrations of each component were 5 μM for OmpC, 25 μM
for DegP(S210A) and Skp, and 100 μM for SurA. The absorbance
at 280 nm of the elution component was recorded. The formation
of the soluble complex of OmpC and fluorescently labelled SurA,
Skp or DegP(S210A) indicated that labelling did not change the
function of chaperones in forming soluble complex with OmpC,
which was comparable with the action of the unlabelled proteins
(Supplementary Figure S3).
Light scattering assay
Protein precipitation causes the intensity of light scattering to
increase. This can be prevented by the formation of a soluble
complex between the molecular chaperones and the substrates.
1
2
Fluorescence spectroscopy measurement
The FRET between fluorescently labelled proteins was measured
using a microscopic spectrometer (Renishaw) with a 488 nm
These authors contributed equally to this work.
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
S. Wu and others
Table S1
Primers used in the present study
Primer number
Brief description
DNA sequence
1
2
3
4
5
6
7
8
9
10
11
12
13
5 at 500 bp upstream of the start codon of the coding sequence of degP
3 of the coding sequence of degP
5 at Ser210 of the coding sequence of degP
3 at Ser210 of the coding sequence of degP
5 upstream of HindIII site on pACYC184p
5 of the coding sequence of degP
3 of the coding sequence of degP
5 at the signal cleavage site of the coding sequence of surA
3 of the coding sequence of surA
5 at the signal cleavage site of the coding sequence of skp
3 of the coding sequence of skp
5 at the signal cleavage site of the coding sequence of ompC
3 of the coding sequence of ompC
5 -AAGCTTAAAATGTCGCTGTAAAACATGTGTT-3
5 -GGATCCTTAGTGGTGGTGGTGGTGGTGCTGCATTAACAGGTAGATGGTG-3
5 -CGATGCAGCGATCAACCGTGGTAACGCGGGTGGTGCGCTGGTTAACCTGAACG-3
5 -CGTTCAGGTTAACCAGCGCACCACCCGCGTTACCACGGTTGATCGCTGCATCG-3
5 -TCATGTTTGACAGCTTATCATCGAT-3
5 -ATATCCATGGGCATGAAAAAAACCACATTAGCACTGA-3
5 -ATTAAGCTTTTAGTGGTGGTGGTGGTGGTGCTGCATTAACAGGTAGATGGTG-3
5 -ATATCCATGGGCATGAAGAACTGGAAAACGCTGCTTC-3
5 -ATTAAGCTTTTAGTGGTGGTGGTGGTGGTGGTTGCTCAGGATTTTAACGTAG-3
5 -ATATCATATGGCTGACAAAATTGCAATCGTCAACATGG-3
5 -AAGCTTTTATTTAACCTGTTTCAGTACGTCG-3
5 -CATATGGCTGAAGTTTACAACAAAGACGGCAACA-3
5 -ATTAAGCTTTTAGAACTGGTAAACCAGACCCAGAG-3
Figure S2 The comparison of the chaperone activity between fluorescence
labelled and unlabelled proteins shown by light scattering assay
The aggregation of unfolded OmpC is prevented by the addition of both labelled and unlabelled
chaperones with the same extent. The components are indicated in the Figure.
Binding affinity assay
The binding affinity between OmpC and chaperones was detected
by FRET, in which OmpC was labelled by the donor Cy3
and the chaperones were labelled by the acceptor Cy5. A 5 μl
aliquot of the stock solution of unfolded OmpC–Cy3 (8.8 μM)
in 8 M urea was diluted to 500 μl with PBS containing different
concentrations of Cy5-labelled SurA, Skp or DegP(S210A) in
order to reduce the urea concentration 100-fold. The samples
were equilibrated for 5 min at 25 ◦ C and excited at 532 nm. The
fluorescence spectra were recorded from 540 nm to 700 nm. The
nominal FRET efficiency was calculated by:
E =1−
Figure S1
indicated
CD spectra of fluorescent-labelled and unlabelled proteins as
The final concentrations of labelled and unlabelled proteins are 1.1 μM for Skp, 1.1 μM for
SurA and 4.7 μM for DegP(S210A).
argon laser (Spectra-Physics) as the excitation light source. The
fluorescence spectra for the mixture of SurA–Cy3 (or Skp–
Cy3) and DegP(S210A)–Cy5 were collected from 520 nm to
800 nm (Supplementary Figure S4). The spectra of three-colour
FRET sample of SurA–BODIPY (or Skp–BODIPY), OmpC–Cy3
and DegP(S210A)–Cy5 was recorded from 495 nm to 800 nm
(Supplementary Figure S5).
c The Authors Journal compilation c 2011 Biochemical Society
FDA
FD
(S1)
where F D and F DA are the donor fluorescence (Cy3-labelled
OmpC) intensities in the absence or presence of the acceptor (Cy5labelled chaperone) in the solution respectively. The fluorescence
signal is linearly dependent on the concentration of chaperonebound or free OmpC.
FD = f F [C]
(S2)
FDA = f B [B] + f F ([C] − [B])
(S3)
where [C] and [B] are the total and chaperone-bound
concentrations of OmpC–Cy3, and f F and f B are the coefficients
Kinetic partitioning mechanism of the chaperone–OMP interaction
Figure S4 Fluorescence spectra of the mixture of SurA–Cy3 (or Skp–
Cy3) and DegP(S210A)–Cy5 after subtraction of the direct excitation of
DegP(S210A)–Cy5 by the excitation laser (red lines)
The fluorescence spectrum of SurA–Cy3 or Skp–Cy3 is shown as the donor-only control (black
lines). All of the spectra are normalized according to the maximum of the donor fluorescence.
Figure S3 Fluorescent-labelled chaperones prevent the aggregation of
OmpC via forming soluble complexes between them
Size-exclusive chromatography of the mixture of OmpC and each fluorescence labelled (blue)
or unlabelled chaperones (red). The position of chaperone alone is also shown (grey).
between concentration and fluorescence intensity of free and
bound OmpC–Cy3 respectively. Putting eqn S2 and eqn S3 into
eqn S1, we have:
E=
[B]
fF − fB
×
fF
[C]
(S4)
The chaperone-bound concentration of OmpC is obtained
according to the binding equilibrium equation:
[B] = [C] ×
R+n+
Kd
[C]
−
R+n+
2n
Kd
[C]
2
− 4n R
(S5)
where R is the molar ratio of Cy5-labelled chaperone to
OmpC–Cy3. K d is the dissociation constant. n is the binding
number. The expression of E can be rearranged to be a
function of R. K d is then obtained from fitting the titration
curve. The K d values obtained from the fits were 106 +
− 84 nM,
15.9 +
− 7.2 nM and 8.63 +
− 0.37 nM for the binding of OmpC to
Figure S5 FRET is observed in the fluorescence spectra of the mixtures of
OmpC and the quality control factors
The fluorescence spectra of the three-component mixture of SurA–BODIPY (or Skp–BODIPY),
OmpC–Cy3 and DegP(S210A)–Cy5 (red lines). The fluorescence spectra of SurA–BODIPY
(or Skp–BODIPY) (black lines) and the mixture of SurA–BODIPY (or Skp–BODIPY) and
DegP(S210A)–Cy5 (grey lines) are plotted as controls. The decrease in BODIPY fluorescence
and the increase in Cy5 fluorescence in the three-component samples demonstrate that FRET
occurred between SurA–BODIPY (or Skp–BODIPY) and OmpC–Cy3 and between OmpC–Cy3
and DegP(S210A)–Cy5.
c The Authors Journal compilation c 2011 Biochemical Society
S. Wu and others
Figure S6 Binding affinity assay of the interaction between OmpC–Cy3 and
Cy5-labelled SurA, Skp or DegP(S210A)
The donor Cy3 fluorescence intensity was plotted against the molar ratio of the Cy5-labelled
chaperone to OmpC–Cy3. The units of OmpC, SurA, Skp and DegP(S210A) are monomer,
monomer, trimer and hexamer respectively. The data were fitted (grey curve) according to eqn
+
+
S4. The K d values obtained from the fit are 106 +
− 84 nM, 15.9 − 7.2 nM and 8.63 − 0.37 nM
for the binding of OmpC to SurA, Skp and DegP(S210A) respectively.
SurA, Skp and DegP(S210A) respectively. The binding numbers
were 0.99 +
− 0.02, 0.45 +
− 0.14 and 0.024 +
− 0.007 respectively,
reflecting the fact that either the true binding units may not be
that pre-assumed or they may vary with the variation of the
concentrations, or both.
REFERENCES
1 Behrens, S., Maier, R., de Cock, H., Schmid, F. X. and Gross, C. A. (2001) The SurA
periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone
activity. EMBO J. 20, 285–294
2 Walton, T. A. and Sousa, M. C. (2004) Crystal structure of Skp, a prefoldin-like chaperone
that protects soluble and membrane proteins from aggregation. Mol. Cell 15, 367–374
3 Jiang, J. S., Zhang, X. F., Chen, Y., Wu, Y., Zhou, Z. H., Chang, Z. and Sui, S. F. (2008)
Activation of DegP chaperone-protease via formation of large cage-like oligomers upon
binding to substrate proteins. Proc. Natl. Acad. Sci. U.S.A. 105, 11939–11944
Received 10 February 2011/2 June 2011; accepted 14 June 2011
Published as BJ Immediate Publication 14 June 2011, doi:10.1042/BJ20110264
c The Authors Journal compilation c 2011 Biochemical Society