 |
INTRODUCTION |
Studies on the mechanistic principles of insertion and folding of
integral membrane proteins have been mostly performed in vitro with two model proteins, the
-helical bundle protein
bacteriorhodopsin of Halobium salinarium (1-4) and the
8-stranded
-barrel outer membrane protein A
(OmpA)1 of Escherichia
coli (5-12) (for recent reviews, see Refs. 1 and 13-15). In case
of bacteriorhodopsin, the folding kinetics into lipid membranes were
studied after mixing denatured bacteriorhodopsin in SDS micelles with
preformed lipid bilayers (1). Surrey and Jähnig (11, 16)
demonstrated the oriented folding and insertion of completely unfolded
OmpA into dimyristoylphosphatidylcholine bilayers upon dilution of the
denaturant urea in absence of detergent. It has been observed that OmpA
folds relatively fast into micelles but with rather slow kinetics into
phospholipid membranes (7, 11, 12). Previous studies have also shown
that urea-unfolded OmpA inserts and folds into phospholipid bilayers by
a highly concerted mechanism that takes place via at least three
structurally distinct membrane-bound folding intermediates (8-10). The
folding kinetics and the yields of folded OmpA depended strongly on the properties of the lipid bilayer (6, 11, 16), on temperature (8-10),
and on pH (11). It was further demonstrated that secondary and tertiary
structure formation in OmpA are synchronized and coupled to membrane
insertion (6). However, in these previous studies, the OmpA folding
kinetics were relatively slow, suggesting that folding in
vivo might be facilitated by folding catalysts.
The folding of soluble proteins in the cytoplasm is assisted by
molecular chaperones (for recent reviews, see Refs. 17-20). It is not
known how molecular chaperones facilitate the folding process of
membrane proteins. Periplasmic and OMPs of Gram-negative bacteria are
translocated across the inner membrane in a mostly unfolded form by the
SecA/E/Y/G export system (see Refs. 21, 22). In the periplasm, a signal
peptidase cleaves off the signal sequence. It has been suggested that
periplasmic proteins assist in the assembly of OMPs, because reduced
concentrations of some OMPs in the outer membrane of E. coli
were observed after deletion of the genes for the periplasmic proteins
Skp or SurA (23-25). Skp binds to the NH2-terminal part of
OmpA and is required for the release of incompletely folded OmpA into
the periplasm (26, 27). The conditions that OMPs require for subsequent
folding and membrane insertion have not yet been described.
The skp gene maps at the 4-min region on the chromosome and
is located upstream of genes that encode proteins involved in lipid A
biosynthesis (28-30). For example, the gene firA, which codes for
UDP-3-O-[3-hydroxymyristoyl]glucosamine-N-acyltransferase is located only four bases downstream of the stop codon of
skp (31). Lipid A is an essential component of
lipopolysaccharide (LPS) that is present in the periplasm after
biogenesis (32) and the major component of the outer leaflet of the
outer membrane. Pulse-labeling and biochemical reconstitution
experiments suggested that LPS is required for efficient assembly of
OMPs such as trimeric PhoE (33) and monomeric OmpA (34) into outer
membranes. In these previous studies, refolding was performed with
micelles of LPS and Triton X-100 instead of phospholipid bilayers. It
is not clear whether the LPS concentration in the periplasm is above the CMC, but OMPs would not fold in the presence of monomers (7). Also,
the prefolding of OMPs into LPS micelles and subsequent fusion of the
micelles with the outer membrane can lead to two different orientations
of OMPs in the lipid bilayer, but only one direction of OMPs is
observed in cells. The long
-strand-connecting loops are facing the
extracellular space.
In this study, we sought to explore possible assisted folding pathways
of OmpA. We have addressed the following questions. 1) How do Skp and
LPS (either separately or in combination) affect unfolded OmpA in
solution? 2) Are there effects of Skp (or LPS) on the folding of OmpA
into lipid bilayers? 3) At which stoichiometries and how strong does
Skp (or LPS) bind to OmpA? 4) Does Skp (or LPS) lead to secondary
structure formation of OmpA in solution?
 |
EXPERIMENTAL PROCEDURES |
Purification of Skp--
The Skp protein was purified from the
periplasmic fraction of E. coli CAG16037 (35) harboring the
plasmids pSkp (pQE60 from Qiagen carrying the skp gene) and
pPLT13 (mini-F carrying lacIq, see Ref. 36). Cells were
grown in LB medium at 37 °C and were induced at an
A600 of 0.3 with 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3-4 h. The
cells were harvested at an A600 of ~1.5,
resuspended in sucrose buffer (100 mM Tris-HCl, pH 8.0, 20% sucrose) and equilibrated on ice for 10 min. After centrifugation,
cells were resuspended in sucrose buffer with 10 mM EDTA
and incubated with lysozyme (10 µg/ml) for 30 min on ice.
Spheroplasts were sedimented at low speed after the addition of
MgSO4 (20 mM). The resulting periplasmic fraction was dialyzed overnight at 4 °C against buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl), run through
a Poros® DEAE column and then loaded onto a Poros CM column.
The DEAE column was removed, and the CM column was washed with 3 volumes of buffer A. Skp was eluted from the column by a NaCl gradient
(100-750 mM NaCl in buffer A), dialyzed overnight at
4 °C against buffer A, and concentrated in Centriprep YM-3
concentrator units (Amicon). The Skp concentration was determined
photometrically at 280 nm with a molar absorption coefficient of 1280 M
1 cm
1 (37) and using the
method of Lowry et al. (38) with bovine serum albumin as a standard.
Purification of OmpA--
OmpA was purified from E. coli as described previously (16). OmpA concentrations of stock
solutions were determined using the method of Lowry et al.
(38).
Purification of R-LPS--
E. coli rough mutant
F576 was cultivated as described previously (39), and its LPS
(R2 core type, M
3900 g/mol) was isolated as reported
previously (40).
Preparation of Lipid
Bilayers--
1,2-Dioleoyl-sn-glycero-3-phosphocholine
(DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG)
were from Avanti Polar Lipids (Alabaster, AL). Lipids were dissolved in
chloroform and mixed to a molar ratio of 50% DOPC, 30% DOPE, and 20%
DOPG. The lipid mixtures were first dried under a stream of nitrogen
and then in the desiccator under high vacuum for at least 3 h.
Lipids were then hydrated in 10 mM HEPES buffer, pH 7.0, with 2 mM EDTA and dispersed by vortexing. Small
unilamellar vesicles (SUV) were prepared by sonicating the lipid
dispersions with a Branson W450D ultrasonicator for 40 min with a 50%
pulse cycle.
Kinetics of OmpA Insertion and Folding into Membranes Detected by
Electrophoresis--
When isolated from bacteria using the method by
Surrey and Jähnig (16), OmpA is completely unfolded in 8 M urea. The folding of OmpA was initiated by rapidly mixing
5 µl of denatured OmpA first with 46 µl of HEPES buffer (10 mM, pH 7.0, with 2 mM EDTA) containing Skp
followed by the immediate addition of 10 µl of LPS and then by the
addition of 35 µl of preformed lipid bilayers. The final
concentrations were 7.1 µM OmpA, 28.4 µM
Skp, 1.4 mM lipid, and from 7.1 to 280 µM
LPS. When experiments were performed in the absence of Skp, LPS, or
lipid vesicles, the corresponding solutions were replaced by the same
volume of buffer. Unless indicated otherwise, all of the mixing steps
were performed in this sequence very rapidly.
Samples of the reaction mixture were taken at different times after the
initiation of folding, and an equal volume of 0.125 M Tris
buffer, pH 6.8, containing 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol was added. SDS binds to both folded and unfolded OmpA
and inhibits further folding (10, 11). SDS-PAGE was performed as
described previously (41, 42) but without heat denaturation of the
samples. The different electrophoretic mobilities of folded (apparent
molecular mass = 30 kDa) and unfolded OmpA (apparent molecular
mass = 35 kDa) were used to determine the fraction of folded OmpA by
densitometry as described previously (10). Time courses of folding were
monitored over 180 min. Experimental errors were estimated from
three to six experiments.
Folding Monitored by CD Spectroscopy--
5.3 µl of a stock
solution of OmpA (3 mg/ml) were diluted first into buffer. The buffer
volume was calculated for each experiment to obtain a final volume of
150 µl. In experiments with Skp, LPS, or lipid vesicles, 5.9 µl of
a stock solution of Skp (1.8 mg/ml), 16.6 µl of a stock solution of
LPS (0.5 mg/ml), or 23.5 µl of a stock solution of lipid vesicles
(3.9 mM) were added in this sequence. The mixing of all of
the reactants was done rapidly in an Eppendorf vial. Samples were
incubated for at least 3 h and then transferred into a CD cuvette.
Far-UV CD spectra of 3.0 µM OmpA were recorded at
20 °C on a Jasco 715 CD spectrometer using a 0.5-mm thermostated
cuvette. Five scans were accumulated from 190 to 250 nm (205-250 nm in
presence of 8 M urea) with a response time of 16 s, a
bandwidth of 1 nm, and a scan speed of 10 nm/min. Background spectra
without OmpA and Skp were subtracted. The recorded CD spectra were
normalized to obtain the mean residue molar ellipticity [
](
) as
shown in Equation 1,
|
(Eq. 1)
|
where l is the path length of the thermostated
cuvette and
(
) is the recorded ellipticity at wavelength
.
c1 and c2 are the
concentrations, and n1 and
n2 are the numbers of the amino acid residues of
OmpA and Skp, respectively.
Skp and LPS Binding to OmpA Monitored by Fluorescence
Spectroscopy--
LPS (M = 3900 g/mol) binding to unfolded OmpA
was determined by first recording background spectra of LPS at
different concentrations in 490 µl of glycine buffer (10 mM, pH 8.5). The LPS concentration ranged from 0 to
0.13 mg/ml. After the addition of 10 µl of urea-denatured OmpA (1 mg/ml), the fluorescence spectra of OmpA were recorded at each LPS
concentration. The background spectra exhibited only very weak
intensity because of light scattering and were subtracted. Skp binding
to OmpA was determined analogously in 500 µl of glycine buffer at Skp
concentrations ranging from 0 to 0.08 mg/ml and at an OmpA
concentration of 0.02 mg/ml. Background spectra of Skp were subtracted.
Skp does not contain tryptophan and is only weakly fluorescent when
excited at 290 nm.
Fluorescence spectra were recorded as described previously (10) on a
Spex Fluorolog-3 spectrofluorometer with double monochromators in the
excitation and emission pathways. The excitation wavelength was 290 nm,
and the bandwidths of the excitation monochromators were 3 nm. The
bandwidths of the emission monochromators were 3.5 nm. The integration
time was 0.1 s, and an increment of 0.5 nm was used to scan
spectra in the range of 310-370 nm.
The association constant K for the binding of the ligand L
(i.e. LPS or Skp) to a protein binding site Px
(Px + L
PxL) as defined by the mass action
law, i.e. K = [PxL]/([Px][L]) was determined by fitting
the fluorescence intensities as a function of molar ratio of ligand
added to the protein. It was assumed that all of the binding sites on
the protein are equivalent for all of the molecules of the selected
ligand (either LPS or Skp).
 |
RESULTS |
Skp and LPS Affect the Folding of OmpA--
To investigate how Skp
and LPS affect the folding kinetics of OmpA, we used an electrophoretic
mobility assay as described previously (6, 10, 11, 16). In SDS-PAGE,
folded OmpA migrates at 30 kDa, whereas unfolded OmpA migrates at 35 kDa if not heat-denatured prior to electrophoresis (43). The 30-kDa form has been shown by Raman, Fourier Transform Infrared, and CD
spectroscopy (7, 11, 16, 44-46) by phage inactivation assays (43) and
by single channel conductivity measurements (47) to correspond to
completely folded and active OmpA. In the experiments shown in Fig.
1, the folding of OmpA upon dilution of
the denaturant urea was monitored at pH 7.0 over a time course of 180 min. In presence of Skp in aqueous solution, OmpA did not fold (Fig.
1A, gel 1). When OmpA was incubated with a 5-fold
molar excess of LPS (Fig. 1A, gel 2),
approximately 45% OmpA folded as analyzed by densitometry (Fig.
1B,
). OmpA folding quickly leveled off within 1 h
after initiation of folding. However, folding into LPS was strongly
inhibited when OmpA was first reacted with Skp prior to the addition of
LPS (Fig. 1, A, gel 3, and B,
).

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 1.
Membrane insertion and folding of OmpA is
affected by LPS and Skp. A, SDS-polyacrylamide gels
showing the time courses of OmpA folding at 30 °C. The
arrows indicate the migration of unfolded (U) and
folded (F) OmpA. The folding of OmpA was monitored after
dilution of the denaturant in the presence of a 4-fold molar excess of
Skp (gel 1), a 5-fold molar excess of LPS (gel
2), a 4-fold molar excess of Skp followed by a 5-fold molar excess
of LPS (gel 3), a 200-fold molar excess of phospholipids
(gel 4), and a 4-fold molar excess of Skp followed by a
5-fold molar excess of LPS followed by the immediate addition of a
200-fold molar excess of phospholipids (gel 5). For
comparison, the migration of native OmpA (F) at 30 kDa is
shown in the first lane of gel 1. Where present,
the migration of Skp (17 kDa) is also indicated. The experiments 2-5
were done in parallel. In all of the five experiments, OmpA, LPS, Skp,
and lipid vesicles were from the same stock solutions. B,
densitometric analysis of the fraction of folded OmpA as determined
from SDS gels as shown panel A: Skp/OmpA = 4:1 mol/mol
( ); LPS/OmpA = 5:1 mol/mol ( ); Skp/LPS/OmpA = 4/5/1
mol/mol/mol ( ); phospholipid bilayers ( ); and Skp/LPS/OmpA = 4/5/1 mol/mol/mol in presence of phospholipid bilayers ( ).
|
|
Consistent with previous reports (11), OmpA refolded to 75% when
reacted with preformed phospholipid bilayers (Fig. 1A, gel 4, and B,
). When OmpA was preincubated
first with Skp and then with LPS before vesicles were added, up to 90%
folding of OmpA into phospholipid bilayers was observed (Fig.
1A, gel 5). Also, the SDS gels and their
densitometric analysis indicated much faster folding kinetics (Fig.
1B,
) in comparison with OmpA refolding into lipid
bilayers in the absence of Skp and LPS. Before preincubation with Skp
and LPS, the denaturant urea was diluted 10-fold (see "Experimental
Procedures"), i.e. to a concentration lower then necessary
to keep OmpA unfolded. Because the same vesicle preparations were used
and the reactions analyzed in gels 4 and 5 were performed in parallel,
we concluded that the interaction with Skp and LPS prevented a
hydrophobic collapse of OmpA after urea dilution.
Skp and LPS Are Jointly Required for Efficient Membrane Insertion
and Folding of OmpA--
The improved folding kinetics of OmpA into
phospholipid bilayers after preincubation with Skp and LPS raised the
question of whether these periplasmic components were mutually required to assist the folding of OmpA into lipid bilayers or whether one component, either Skp or LPS, would suffice. Fig.
2 shows the time dependence of OmpA
folding into lipid bilayers as analyzed by SDS-PAGE and subsequent
densitometry. In panel A, preformed lipid bilayers were
added to OmpA immediately after urea dilution in the presence of Skp
(
), in the presence of LPS (
), in the presence of Skp and LPS
(
), and in the absence of Skp and LPS (
). The four experiments
were performed in parallel with the same stock solutions. Folding
kinetics into lipid bilayers were fastest and folding was most complete
when Skp and LPS were both present. When Skp and LPS were both absent,
the folding into lipid bilayers was less efficient. However, when only
one of the periplasmic components, either Skp or LPS, was added, the
efficiency of OmpA folding into lipid bilayers was further reduced.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Efficient folding of OmpA into lipid bilayers
requires the simultaneous presence of Skp and LPS. The
OmpA-folding kinetics were determined by electrophoresis and
densitometry. OmpA, Skp, LPS, and lipid were reacted as described under
"Experimental Procedures" at 30 °C. The Skp/OmpA ratio was 4 mol/mol. The LPS/OmpA ratio was 5 mol/mol, and the lipid/OmpA ratio was
200 mol/mol. A, folding was initiated after urea dilution by
immediate addition of lipid bilayers in absence of Skp and LPS ( ),
in presence of Skp ( ), in presence of LPS ( ), and in presence of
both Skp and LPS ( ). B, the corresponding experiments
were performed but with a 30-min delay between urea dilution in
presence or absence of Skp and the addition of lipid or addition of
first LPS and then lipid. In an additional experiment, only LPS added
was to OmpA 30 min before lipid addition ( ).
|
|
In a second set of experiments (Fig. 2, panel B), LPS and
lipid were added 30 min after dilution of the denaturant with buffer in
the presence or absence of Skp (panel B). The introduction of this delay led to much stronger dependencies of the folding kinetics
and yields of folded OmpA in the presence of Skp and LPS. When LPS was
added to Skp·OmpA complexes prior to lipid addition (
), the
largest folding yields were obtained (>90%). When only Skp was
present (
), OmpA folding into lipid bilayers was slowest and the
lowest folding yields were observed. Slow folding and low yields were
also observed when LPS and lipid bilayers were added 30 min after
dilution of urea by addition of Skp-free buffer to OmpA (
). When
OmpA was incubated in the presence of LPS but in the absence of Skp for
30 min prior to the addition of preformed lipid bilayers (
), OmpA
partially folded as observed previously (Fig. 1,
). In this
experiment, the folding yields were lower than observed for OmpA
folding into lipid bilayers in the absence of LPS (
).
With one exception, even slower folding kinetics and lower yields of
folded OmpA were observed when the delay time was increased to 1 h
for all of the folding reactions (data not shown). Still, up to 90%
OmpA folded after 180 min when OmpA was preincubated with Skp for
1 h before LPS and lipid were added. In contrast, a 1-h
preincubation of OmpA in buffer in the absence of LPS and Skp led to
<60% folding after the addition of lipid bilayers.
Based on these results, we concluded the following. 1) Skp alone
prevents the aggregation or misfolding of OmpA in solution, because the
addition of LPS and preformed lipid bilayers after >30 min still leads
to efficient folding. 2) LPS is required for efficient folding of OmpA
into lipid bilayers from Skp·OmpA complexes. In the absence of LPS,
Skp strongly inhibits insertion and folding of OmpA.
Skp and LPS Bind to OmpA in Solution with Different Stoichiometries
and Affinities--
To determine the stoichiometry and the strength of
the binding of Skp and LPS to unfolded OmpA in solution, we used
intrinsic fluorescence spectroscopy (Fig.
3).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
Binding of LPS and Skp to unfolded OmpA in
solution. The intrinsic fluorescence intensity of OmpA at 330 nm
is shown as a function of the molar LPS/OmpA ratio (A) and
the molar Skp/OmpA ratio (B) at an OmpA concentration of 0.6 µM. Each data point is the average of three separate
measurements at the same ligand/OmpA ratio. The binding data were
fitted (solid lines) to the corresponding mass action laws
of ligand binding assuming the equivalent binding sites. Background
intensities of LPS or Skp in absence of OmpA were subtracted. All of
the experiments were performed at 20 °C.
|
|
First, LPS binding to unfolded OmpA in aqueous solution was
investigated. We observed an increasing Trp fluorescence of OmpA at 330 nm with increasing LPS concentration until a saturation was reached
(Fig. 3A). When these fluorescence intensities were fitted
as a function of the LPS concentration to a simple mass action law for
LPS binding (assuming equivalent LPS binding sites), a stoichiometry of
25 ± 2 LPS molecules bound to unfolded OmpA was obtained. The
association constant for LPS·OmpA complex formation was
KLPS = 1.2 ± 0.7 µM
1, corresponding to a free energy of
G
=
34.7 ± 1.5 kJ/mol for
LPS binding to OmpA.
We then investigated the binding of Skp to unfolded OmpA. Small
fluorescence contributions of Skp were measured at each Skp concentration and subtracted from the OmpA fluorescence intensity after
mixing. The fluorescence intensity of OmpA increased strongly as a
function of the molar Skp/OmpA ratio (Fig. 3B). We fitted our data to the mass action law for Skp binding and found a binding stoichiometry of 2.8 ± 0.1
3 Skp molecules bound per
OmpA molecule. The binding curve sharply levels off at this
stoichiometry, indicating strong binding of Skp to unfolded OmpA. From
the fit to the mass action law of Skp binding, we estimated
KSkp = 46 ± 30 µM
1, corresponding to a free energy of
G
=
43 ± 2 kJ/mol. This
binding constant is between 8- and 150-fold higher than the binding
constant of LPS and demonstrates a preferred binding of Skp to OmpA
over LPS.
OmpA Folding into Lipid Bilayers Is Optimal at Specific
Concentrations of LPS and Skp--
To examine how different LPS/OmpA
ratios affect the folding of OmpA into a 200-fold molar excess of
lipid, we performed folding experiments in the absence (Fig.
4A) and presence (Fig.
4B) of a 4-fold molar excess of Skp at molar LPS/OmpA ratios
ranging from 0 to 40. Experiments with and without Skp at the same
LPS/OmpA ratio were performed in parallel using the same lipid vesicle preparation (Fig. 4).

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 4.
Dependence of OmpA folding into lipid
bilayers on the concentration of LPS. Insertion and folding
of 7.1 µM OmpA into a 200-fold molar excess of lipid at
LPS/OmpA ratios of 0, 1, 5, 7, and 40 mol/mol (from top to
bottom) as a function of time after initiation of folding.
A, folding kinetics monitored in absence of Skp.
B, folding kinetics monitored in presence of 4 mol/mol
Skp/OmpA. Skp migrated at 17 kDa in the gels shown in B, but
only the bands of unfolded (U) and folded (F) OmpA are
shown. C and D, densitometric analysis of OmpA
folding as a function of the LPS concentration obtained from SDS gels
including those shown in panels A and B. Folding
experiments were performed either in the absence (C) or
presence (D) of a 4-fold molar excess of Skp over OmpA. The
LPS to OmpA ratios were 0 ( ), 1 ( ), 2 ( ), 5 ( ), 7 ( ), 30 ( ), and 40 ( ).
|
|
Representative gels shown in Fig. 4A demonstrate the time
courses of OmpA folding into lipid bilayers at molar LPS/OmpA ratios of
0, 1, 5, 7, and 40 in absence of Skp. The corresponding mole fractions
of folded OmpA are shown in Fig. 4C. In absence of LPS, ~70% OmpA folded within 180 min (as observed in our previous
experiment) (Fig. 1A, gel 4, and B,
). When LPS was present, the rates of folding and the yields of
folded OmpA decreased with increasing LPS/OmpA ratios. At a 40-fold
molar excess of LPS (Fig. 4, A and C), only
~30% OmpA folded within 180 min, indicating that insertion and
folding of OmpA into phospholipid bilayers are inhibited by LPS in the
absence of Skp.
In presence of Skp, LPS increased the folding rates and yields of
folded OmpA as indicated by the gels (Fig. 4B) and the
densitometric analysis (Fig. 4D). When LPS was added to
complexes of Skp and OmpA (at 4 Skp/OmpA) prior to the addition of
preformed phospholipid bilayers, the highest yields of folded OmpA were
obtained at molar LPS/OmpA ratios between 2 and 7 and >90% OmpA
folded. When we further increased the amounts of LPS added to
OmpA·Skp complexes, LPS inhibited OmpA folding into lipid bilayers at
LPS/OmpA molar ratios of >10, similar to the LPS effect on the folding
of OmpA in the absence of Skp (panel A). In comparison with
the set of experiments in the absence of Skp (panel A), 50%
OmpA still folded even at 40 LPS/OmpA. In the absence of LPS, Skp
slightly inhibited the folding of OmpA into lipid bilayers (Fig. 4,
A and B, gels 0) as determined by
densitometry (Fig. 4, C and D,
). This
inhibitory effect of Skp in the absence of LPS was stronger at a higher
Skp/OmpA ratio of 8 mol/mol (gel not shown) or in experiments with
delayed lipid addition (Fig. 2B,
). The yields of OmpA
folding after 180 min are summarized in Fig.
5.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Final yields of folded OmpA, both in the
presence ( ) and absence ( ) of Skp as a function of the LPS/OmpA
molar ratio.
|
|
OmpA Remains in an Unfolded State When Bound to Skp in
Solution--
To obtain more information on the folding of OmpA in the
presence of equimolar amounts of Skp, we recorded circular dichroism spectra (Fig. 6) of OmpA·Skp complexes
(
) in aqueous solution (panel A) in the presence of a
5-fold molar excess of LPS (panel B) and in presence of both
LPS and preformed phospholipid bilayers (panel C).
Furthermore, the spectra of Skp (- -) were recorded in the absence of
OmpA under otherwise identical conditions. At last, we recorded the
spectra of OmpA (·····) in absence of Skp under these
conditions. All of the spectra were normalized to the mean molar
ellipticity per residue (see "Experimental Procedures") and are
described in three separate paragraphs below for panels A-C. The secondary structure of OmpA in OmpA·Skp complexes can be obtained by subtraction of the spectrum of Skp from the spectrum of
Skp·OmpA complexes if the secondary structure of Skp remains largely
unaffected in the presence of folded or unfolded OmpA. The line shapes
and relative amplitudes of the spectra indicated that changes in Skp
secondary structure in presence of OmpA are comparably small.
Therefore, we also calculated the component spectra of OmpA (- · - ·), which are shown in panels A-C for comparison.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Secondary structure of OmpA-Skp complexes by
circular dichroism spectroscopy. CD spectra of Skp (- -), OmpA
(····), and OmpA bound to Skp ( ) in aqueous buffer
(A), in presence of LPS (B), and in presence of
LPS and lipid bilayers (C). The component spectrum of OmpA
in Skp-OmpA complexes (- · - ·) is also shown in panels
A-C. This spectrum was obtained by subtracting the spectrum of
Skp from the spectrum of the complex of OmpA and Skp. The mean residue
molar ellipticity was calculated for all spectra according to Eq. 1.
|
|
In aqueous solution (panel A), the amplitude of the spectrum
of OmpA·Skp complexes was only 45% of the amplitude of the CD spectrum of Skp at a wavelength of 215 nm. Despite the strongly reduced
amplitude, the spectral line shape of the complex was characteristic
for relatively large contents of
-helix secondary structure, similar
to the spectrum of Skp in solution in the absence of OmpA. Therefore,
we concluded that the secondary structures of Skp and OmpA remained
largely unaffected by the binding of unfolded OmpA to Skp. The
subtraction of the spectrum of Skp (- -) from the spectrum of the
Skp·OmpA complex (
) resulted in a spectrum (- · - ·)
indicating the random coil structure of OmpA, because this spectrum and
the spectrum of urea-denatured OmpA (see Refs. 7, 11, and 16) were very
similar. The spectrum of OmpA in aqueous solution in the absence of LPS
and Skp (Fig. 6A, ·····) is shown for comparison.
The relatively large amplitude of this spectrum suggests more secondary
structure in the hydrophobically collapsed state of OmpA than in
complex with Skp. Skp apparently prevents the misfolding of OmpA in solution.
When OmpA·Skp complexes or Skp were incubated in presence of a 5-fold
molar excess of LPS (panel B), the amplitude of the spectrum
of the complex (
) increased to 56% of the amplitude of the spectrum
of Skp (- -). Therefore, minor amounts of secondary structure may
have formed in OmpA. The OmpA spectrum (- · - ·) obtained by
subtraction of the Skp spectrum from the spectrum of Skp·OmpA
complexes (
) was characterized by a minimum located at 208 nm. The
amplitude of this minimum was still relatively small, also suggesting
that only minor amounts of secondary structure are formed in OmpA in
complex with Skp and LPS. In absence of Skp, the incubation of OmpA
with a 5-fold molar excess of LPS led to spectra that were
characterized by a minimum at 215 nm and an amplitude that indicated
larger amounts of
-sheet secondary structure. This partial formation
of
-sheet structure was consistent with our previous observation of
partial folding of OmpA into LPS in the absence of Skp as shown by
SDS-PAGE (see Fig. 1, A, gels 2 and 3,
and B,
and
).
After the addition of lipid bilayers (Fig. 6C), the
amplitude of the circular dichroism spectra of Skp·OmpA complexes
increased further to 78% of the amplitude of Skp. This increase was
stronger than the increase observed upon the addition of LPS, and it
demonstrated that completion of secondary structure formation in OmpA
required the presence of membranes. Because our SDS-PAGE results
indicated efficient OmpA folding in the presence of both Skp and
LPS and bilayers, we calculated the spectrum of OmpA in
Skp·OmpA complexes by subtraction of the spectrum of Skp from the
spectrum of the complexes. This calculated spectrum (- · - ·) had
indeed the same line shape as OmpA that was completely refolded into
lipid bilayers in the absence of Skp and LPS (·····).
 |
DISCUSSION |
Upon investigating the roles of the periplasmic components Skp and
LPS on the insertion and folding of OmpA into phospholipid bilayers in
detail, we found a first assisted folding pathway for the integral
membrane protein OmpA that is described by three major stages. First,
although Skp binding to OmpA alone is sufficient to keep OmpA unfolded
in solution, thus effectively replacing the denaturant urea, it is
inhibiting the membrane insertion and folding of OmpA. Second, the
interaction of Skp·OmpA complexes with LPS does not lead to OmpA
folding but facilitates OmpA insertion and folding into phospholipid
bilayers in the third stage. Membrane insertion and folding of OmpA is
optimal at specific molar ratios of Skp, LPS, and OmpA.
Skp Solubilizes Unfolded OmpA by Forming a Positively Charged
Complex--
Previous studies have described OmpA insertion and
folding into phospholipid bilayers in the absence of any folding
catalysts (6-11, 16). It was previously reported that the rates and
yields of OmpA folding are higher when deprotonated at pH 10, i.e. at a negative net charge of the protein that leads to
increased solubility of OmpA (11). At pH 7.0, the binding of the highly
basic Skp to OmpA at a 3:1 stoichiometry leads to a positively charged
protein complex and, therefore, also increases the solubility of OmpA. To prevent the aggregation of OmpA in solution, Skp binding must shield
the more hydrophobic regions of unfolded OmpA from the aqueous space.
The observed increase in Trp fluorescence upon binding of Skp (Fig.
3B) indicates that in solution, Skp specifically recognizes
and binds to OmpA segments that will later form the transmembrane
region of the
-barrel, because all of Trps of OmpA are located in
the transmembrane
-strands. In vivo, Skp binds to OmpA
close to the periplasmic surface of the inner membrane and is required
for the release of OmpA into the periplasm (26). This is consistent
with our observation that the folding kinetics of Skp-bound OmpA into
phospholipid bilayers were inhibited when compared with the folding
kinetics of urea-unfolded OmpA (Fig. 2, panel B,
and
).
Skp Prevents Partial Folding and Unspecific Interactions of
Unfolded OmpA with LPS--
We observed that OmpA partially folds into
LPS micelles when Skp is absent (we estimated a critical micelle
concentration of CMCLPS
8 µM as
described in Ref. 48). Folding into LPS micelles was incomplete at
LPS/OmpA ratios between 5 and 40 (cf. Fig. 4A),
indicating that OmpA interacts unspecifically with LPS micelles. In our
previous study (7), OmpA folded into micelles of neutral
(i.e. uncharged or zwitterionic) detergents,
independent of the hydrophobic chain length and chemical structure of
the headgroup of the amphiphile, but never into negatively charged SDS
micelles as determined by CD spectroscopy (data not shown) and SDS-PAGE
(6, 7, 10, 11, 16). LPS is negatively charged with two monophosphate
and two diphosphate groups in the inner core region (49). In the
absence of Skp in solution, the different phosphate groups may interact
in different modes with unfolded OmpA partially inhibiting OmpA folding
into LPS micelles. Consistent with unspecific OmpA-LPS interactions,
the folding of OmpA into phospholipid bilayers was also inhibited in
the absence of Skp when LPS was present (Fig. 4, A and
C). When both LPS and Skp were present (Fig. 4, B
and D), an inhibition of folding into lipid bilayers was
only observed at LPS/OmpA ratios of >10 mol/mol. Skp binding to OmpA
was much stronger than the binding of LPS to OmpA, indicating that LPS
cannot easily replace Skp bound to OmpA. Only at very high LPS
concentrations and low Skp concentrations, the thermodynamic binding
equilibria would favor LPS binding over Skp binding. For this reason,
the folding of OmpA into lipid bilayers may be partially inhibited even
in presence of Skp if the LPS concentration is high enough (Fig. 4,
B and D). Therefore, in cells, it may be
important to keep a balance between the concentrations of Skp and LPS
in the periplasm. At balanced LPS/Skp ratios, Skp suppresses a partial
folding of OmpA into LPS-micelles (Fig. 1, gels 2 and
3). Schäfer et al. (26) show that Skp binds
immediately after translocation of OmpA across the inner membrane. Our
present study indicates that this early binding of Skp also prevents a partial folding and unspecific interactions of unfolded OmpA with LPS
in the periplasm (Figs. 1, gel 3, and 6B).
Structure Formation in OmpA Requires the Presence of the
Phospholipid Bilayer--
OmpA neither developed the native tertiary
structure (Fig. 1A, gel 3) nor the large amounts
of
-sheet secondary structure (Fig. 6B) when in complex
with Skp and LPS in solution at Skp/OmpA ratios of
1. The formation
of native secondary (Fig. 6C) and tertiary (Fig.
1A, gel 5) structure required the insertion of OmpA from the complex with Skp and LPS into preformed lipid bilayers. This finding is consistent with our previous reports (6, 8, 9) that
OmpA folds and inserts from a urea-unfolded form into phospholipid
bilayers by a concerted mechanism in which the formation of secondary
and tertiary structures are strictly coupled and synchronized to
insertion into the bilayer.
Insertion of OmpA into Lipid Bilayers from the Complexes with Skp
and LPS--
OmpA folding into phospholipid bilayers was most
efficient in the presence of 4 mol of Skp and 2-7 mol of LPS/mol OmpA.
Because we determined a binding stoichiometry of 25 LPS/unfolded OmpA in the absence of Skp in solution (Fig. 3B), only a small
number of LPS molecules (10-25%) apparently bind to Skp·OmpA
complexes and are required for efficient OmpA folding. From the crystal structures of LPS (49) and from the structure of OmpA (50, 51), we
found that ~5 LPS molecules can form a first shell around folded OmpA
in the outer leaflet of the outer membrane, which is consistent with
the stoichiometry of 2-7 mol/mol LPS/OmpA found for the fastest
folding kinetics of OmpA in presence of Skp. To the best of our
knowledge, there are no enzymes in the outer membrane that actively
generate a trans-bilayer asymmetry. We propose that this asymmetry may
be a direct result of specific binding of Skp and LPS to OMPs prior to
OMP insertion and folding into the phospholipid bilayer. Interestingly,
such interactions were observed in the crystal structure of LPS bound
to another outer membrane protein, FhuA, of E. coli
(49).
A First Pathway for OmpA Membrane Insertion and Folding into Lipid
Membranes--
The results of our present in vitro study
are evidence for a first pathway of assisted OmpA folding and membrane
insertion that is summarized in Fig. 7.
In an unfolded form that has been trans-located across the inner
membrane, OmpA binds up to 3 molecules of Skp and forms a soluble
complex, USkp3, in which OmpA is kept largely unfolded as
indicated by circular dichroism spectroscopy. This complex then binds a
small number (n = 2-7) of LPS per OmpA in solution to
form a folding and insertion-competent form of OmpA bound to Skp and
LPS (FCSkp3LPSn). In this complex, OmpA develops minor amounts of secondary structure (Fig. 6B).
When this insertion-competent intermediate of OmpA is reacted with preformed phospholipid bilayers, OmpA rapidly inserts and folds to its
native state N as indicated by the formation of native secondary and
tertiary structures (see Figs. 1, gel 5, and 6C). The functions of Skp and LPS as "co-chaperones" may be directly reflected in the close neighborhood of the genes skp and
firA in the 4-min region on the chromosome, in particular,
as LPS and Skp are required at specific molar ratios of ~0.5-1.7 mol
LPS/mol Skp for optimal folding rates and yields of membrane-inserted OmpA. This co-assisted folding of OmpA by Skp and LPS very likely is a
general folding pathway of outer membrane proteins because Skp was
found to bind several OMPs (23), because deletion of the skp
gene resulted in reduced expression levels of several OMPs in the outer
membrane (23), and because specific LPS binding motifs were reported
for FhuA (49) and, more recently, also for OmpT (52).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 7.
Scheme for an assisted folding pathway of a
bacterial outer membrane protein. OmpA is translocated through the
cytoplasma membrane in an unfolded form (U) and binds to a
small number of molecules of the periplasmic chaperone Skp, which
solubilizes OmpA in the unfolded state (USkp3). The
complex of unfolded OmpA and Skp interacts with a small number of LPS
molecules to form a folding competent intermediate of OmpA
(FCSkp3LPSn). In the final step,
folding competent OmpA inserts and folds into the lipid bilayer.
|
|
The Skp-LPS-assisted folding pathway that we found in this study may
not be the only mechanism by which OMPs like OmpA insert and fold into
the outer membrane, because the deletion of the skp gene
only decreases the concentration of OMPs in the outer membrane but does
not entirely eliminate their presence. However, our experiments
demonstrate for the first time the successful in vitro
folding of a membrane protein into lipid bilayers from an unfolded
soluble state that only involves components from the cell and that does
not require urea as a denaturant. The strategy that we used here to
investigate the Skp/LPS-assisted folding pathway will also prove useful
to discover other parallel pathways in future studies.