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INTRODUCTION |
Bacterial outer membrane proteins are synthesized as precursor
proteins in the cytoplasm. After their translocation across the inner
membrane via the Sec machinery (1) and processing to mature protein,
they are assembled into the outer membrane (OM).1 Pore proteins, such as
the PhoE protein, are assembled as a trimer in the OM. How membrane
proteins fold and assemble into a specific membrane is largely unknown.
Kinetic and equilibrium folding studies of mostly soluble, hydrophilic
proteins have indicated the existence of at least three main stages in
protein folding, the formation of secondary structure, the folding
pattern, and detailed tertiary structure (2). In vivo, the
folding process is mediated by molecular chaperones that are proteins
that will act primarily by preventing misfolding and aggregation of
partially folded and unassembled protein subunits (3). In contrast to
soluble proteins, membrane proteins will expose much more hydrophobic
surfaces, exposed to the lipid phase, whereas the hydrophilic areas
will be buried during folding. It can therefore be anticipated that membrane protein folding and assembly in vivo will require a
membrane environment and molecular chaperones at or in this membrane.
Indeed, refolding of bacterial outer membrane proteins can be
accomplished in the presence of detergent and phospholipids (4-7).
However, the kinetics of refolding of these membrane proteins were slow and, in the case of the trimeric porin OmpF, the yield was also low.
Interestingly, phosphatidylethanolamine has recently been demonstrated
to act as a non-protein molecular chaperone in the assembly of a
bacterial cytoplasmic membrane transporter (8). Thus, certain lipids
might also be required as molecular chaperone in the folding and
assembly of bacterial outer membrane proteins.
Various in vivo and in vitro studies have
implicated an important role for LPS in the folding and assembly of
bacterial outer membrane proteins (9-14). LPS are the major
amphiphilic components in the outer leaflet of the bacterial OM (15,
16). After synthesis in the inner membrane, LPS are translocated in an
as yet unknown manner to the OM. Inhibition of fatty acid synthesis
with the antibiotic cerulenin interferes with the assembly of pore
proteins into the outer membrane (12, 17-19). Thus, there appears to
be a direct relationship between de novo synthesis of
lipids, LPS and/or phospholipids, and correct folding and assembly of
pore proteins in vivo. Recently, an early role for LPS in
folding of PhoE protein was demonstrated in vitro (14).
Both, LPS and divalent cations were shown to be involved in the
formation of an early intermediate, i.e. a folded monomer of
PhoE protein. The subsequent assembly of monomers into trimers requires
an additional incubation with outer membranes and Triton X-100 (0.08%
w/v). The kinetics of the folding of PhoE with LPS is much higher (20)
as compared with the kinetics observed in refolding studies with OmpA
and OmpF in the presence of phospholipids (5-7). These in
vitro results implicate a special role for LPS in outer membrane
protein folding.
In order to understand how LPS can support outer membrane protein
folding, it is important to elucidate the molecular mechanism of
LPS-protein interactions. Both hydrophilic and hydrophobic interactions, involving the negatively charged phosphates in the core
and at the C-1 and C-4' positions of the lipid A backbone and the fatty
acids, respectively, might be directly involved in LPS-protein
interactions. In addition, the state of order of the acyl chains
(described by the order parameter S37) and the three-dimensional supramolecular structures of LPS might directly influence the capacity to support protein folding (for an overview see
Refs. 21 and 22). The relationship between the molecular shape of the
lipid molecule and the structural polymorphism of its aggregates is
well documented for phospholipids (23). The same principles also apply
for LPS (21, 22). Thus, the supramolecular structure of LPS aggregates
can either be lamellar (L, M) or non-lamellar (cubic, Q or
HII). Factors like degree of saturation of acyl chains, temperature, head group size and ionization (pH, divalent cation concentration), and water content can influence the type of aggregate structures (lamellar and various non-lamellar phases).
We have used a large set of different LPS molecules, of which the
chemical and biophysical properties were previously characterized, in
the developed in vitro folding system to investigate which properties of LPS are involved in folding of PhoE protein into its
folded monomeric, native-like state.
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MATERIALS AND METHODS |
In Vitro Translation and Folding of PhoE Protein--
Isolation
of S135 cell extract from Escherichia coli strain MC4100
(24) and the in vitro transcription and translation
reactions were performed as described previously (25). Plasmid pJP370 (26) was used to direct the synthesis of the mature form of PhoE
protein that was radioactively labeled due to the incorporation of
[35S]methionine during protein synthesis. Folding of PhoE
protein was initiated by addition of purified LPS and 0.015% Triton
X-100 after inhibition of protein synthesis with puromycin and was
essentially performed as described previously (14). In short, 20 µl
of a mixture of LPS with Triton X-100 (0.0338%, w/v) in buffer L (50 mM triethanolamine acetate, pH 7.5, 250 mM
sucrose, 1 mM dithiothreitol) was mixed with 25 µl of a
translation mixture and incubated for 30 min at 37 °C. Samples were
treated with trypsin (45 µg/ml) for 15 min at 37 °C,
phenylmethylsulfonyl fluoride (1 mM) was added, and the
samples were transferred to ice. Prior to electrophoresis, sample
buffer containing 2% SDS was added to the protein samples which were
divided into two equal portions. One portion was incubated for 10 min
at room temperature (lane a in Figs. 2-4) and the other portion at 100 °C (lane c in Figs. 2-4). In addition, 5 µl of the total translation mixture (lacking LPS, Triton X-100) was
incubated for 10 min at 100 °C in sample buffer (Figs. 2-3,
lanes TL) and was used for determining the total amount of
full-length PhoE present in 25 µl of translation mixture.
SDS-polyacrylamide gels (27) were run at 20 mA in a
temperature-controlled room at 4 °C to prevent denaturation of the
various folded forms of the PhoE protein during electrophoresis. The
folded monomer (Figs. 2-4, lanes a and
designated m*) often runs as a smear originating from a
species with a molecular mass of approximately 31 kDa up to the
position of the denatured PhoE form (m; 38 kDa). Gels were incubated with Amplify, dried, and exposed to film (Fuji) at
70 °C. Data were quantified with a PhosphorImager (Molecular
Dynamics). The amount of trypsin-resistant PhoE is calculated from the
amount of denatured PhoE (m) present in lanes designated
c (Figs. 2-4) as percentage of the total amount of
full-length PhoE present in 25 µl of translation mixture.
LPS Preparation and Chemical Modifications--
Bacteria from
enterobacterial strains as well as the non-enterobacterial species
Paracoccus denitrificans and Rhodobacter capsulatus were cultured as described (28, 29). The
non-enterobacterial strain Chromobacterium violaceum was
obtained from the Institute for Fermentation, Osaka, Japan (IFO number
12614), and was cultivated in polypeptone growth media. LPS was
extracted from phenol-killed bacteria, and rough mutant strains were
extracted according to a modified PCP (PCP I, phenol/chloroform/petrol
ether, 2:5:8 volume %) procedure (30) and for the wild type strain of
Salmonella minnesota by a phenol/water extraction (31). LPS
in the water phase was enzyme-treated with proteinase K, RNase, and
DNase (purchased from Sigma, Deisenhofen, Germany, and Boehringer,
Mannheim, Germany) and further purified by the PCP procedure (PCP II,
phenol/chloroform/petrol ether, 5:5:8 volume %). LPS were lyophilized
and usually used in their natural salt form. In some cases, LPS Re
samples were converted to various defined salt forms (Na+,
Li+, Mg2+, Ba2+, and
Ca2+) by extensive dialysis for 48 h against the
corresponding salts. The preparations of de-O-acylated LPS,
de-O-acylated and de-phosphorylated LPS, de-acylated LPS and
of lipid A from LPS of E. coli and K. pneumoniae
R20 were performed as described (33, 34). The structures of the
modified LPS form of J5 are schematically indicated in Fig.
1B.
The chemical structures of the various lipid A and LPS from S. minnesota strains are given in Ref. 32 (and schematically in Fig.
1A), those of E. coli J-5 in Refs. 33 and 34,
those of Klebsiella pneumoniae in Ref. 35, and those of the
non-enterobacterial LPS and lipid A in Refs. 28, 29, and 36.
Order Parameter S--
The state of order of the hydrocarbon
chains of the endotoxins was determined by Fourier-transform
spectroscopy by evaluating the symmetric stretching vibration
s(CH2) of the methylene groups. The position of
this vibrational band is a sensitive marker of lipid order, and an
estimation of the order parameter S at 37 °C, S37, can
be performed according to a procedure described earlier (37), in which
S can be calculated from the peak position xs of
s (CH2) by S =
941.8 + 0.7217 xs
7.823 10
5
xs2
2.068 10
8
xs3. It should be noted that
S37 defined in this way should be similar but not identical
to the order parameter used in NMR spectroscopy (S = 1 for
perfectly aligned and 0 for isotropic acyl chains).
Endotoxin Aggregate Structure--
The supramolecular structures
of LPS and lipid A aggregates were deduced from x-ray diffraction
patterns, which were obtained by measurements at the European Molecular
Biology Laboratory (EMBL) outstation at the Hamburg synchrotron
radiation facility HASYLAB kindly performed by M. H. J. Koch using
the double-focusing monochromator-mirror camera X33 (38). The
evaluation of the x-ray spectra was achieved according to procedures
described (39) and in previous papers (37, 40, 41) which allow us to
assign the spacing ratios of the main scattering maxima to defined
three-dimensional structures. Here, in particular lamellar (L) and
non-lamellar inverted cubic (Q) structures are of relevance. From this,
the conformation of the individual molecules can be derived, which is
cylindrical in the case of L-structures and conical, the cross-section
of the hydrophobic moiety is larger than that of the hydrophilic moiety, in the case of inverted Q-structures.
For some endotoxin derivatives, de-O-acyl-LPS and
de-O-acyl-dephospho-LPS, for which only limited amounts were
available, the aggregate structures were predicted according to
procedures published earlier (42).
Phospholipid Purification and Phosphor
Determination--
Phospholipids were purified from E. coli
MC4100 (24) grown overnight at 30 °C in L broth (43) as described
(44). The phosphor content was determined by the methods of Rouser
et al. (45).
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RESULTS |
Folding of PhoE Protein with LPS of Wild Type, Mutant Forms, and of
Lipid A Variants--
Folding of PhoE with LPS of a deep rough mutant,
lacking the complete core region up to the
3-deoxy-D-manno-octulopyranosonic acid residues,
was previously shown to be much less efficient as compared with
wild-type LPS (14). In order to identify which region of the core was
important to support protein folding, we made use of a well
characterized series of chemotypes of LPS derived from S. minnesota (Table I). The primary
chemical structures of wild type LPS and of the LPS mutants down to the
deep rough mutant (LPS Re) are largely known (Fig.
1A; 32, 46, and references therein) except for the precise substitution with phosphate groups, in
particular for lipopolysaccharides with longer sugar chains (>LPS Rc).
In addition to differences in total amount of negative charges, the
type of supramolecular structure of the LPS aggregates (Refs. 40 and
41; aggregate structure of the lipid A part) and the state of order of
the acyl chains at 37 °C (Ref. 46; order parameter S37)
are indicated (Table I).
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Table I
Folding of in vitro synthesized PhoE protein with LPS
LPS was purified from the indicated strains. Folding was performed at
37 °C with 0.015% Triton X-100 and 138 nmol/ml LPS (Kdo content).
No folding was observed in the absence of LPS. The total negative
charge, preferred aggregate structure of the lipid A part of LPS, and
the order parameter (S37) of the lipid A acyl chains are
indicated. The amount of trypsin-resistant PhoE (TrypR) as % of the total amount of full-length PhoE synthesized is an average of
two independent experiments. Q, inverted cubic structure. L, lamellar
structure, ND, not determined. NO, not observed.
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Fig. 1.
Schematic chemical structures of LPS S. minnesota and E. coli. For detailed
structural information see Refs. 32-34. A, rough mutant
lipopolysaccharide (R from LPS) from S. minnesota and its
derivatives (Ra to Re). The positions of further non-stoichiometric
substituents like phosphates are not indicated. For example, the Rd1
mutant of strain Rz has two phosphate groups that are absent in LPS of
the R7 strain. B, chemical structure of LPS of an Rc mutant
of E. coli O111 (strain J5). The sites where the chemical
modification occurs for the preparation of lipid A (HAc),
de-O-acylated LPS (hydrazin, Hy), or
dephosphorylated LPS (HF) are indicated. The lipid A part
containing phosphates (P) and fatty acids (14O12,
14O14, and 14OH) and the phosphates in the core region
are schematically indicated. Kdo,
3-deoxy-D-manno-octulopyranosonic acid.
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Folding of in vitro synthesized PhoE protein with LPS of
chemotype S, Ra, Rb1, and Rd1/P+ were more efficient as
compared with the folding efficiencies obtained with LPS of chemotypes
Rc/P
, Rd1/P
, Rd2, and Re (Table I and Fig.
2). The efficiency of folding is very
reproducible and varies usually between 10% of the obtained average
folding efficiency (as indicated in Tables I and II; e.g.
60 ± 6%). The reduction of the folding efficiencies with this
latter group appears to be mainly due to the absence of a negative
charge in the inner core region. The negative charge of the phosphate
group at the first heptopyranose, designated Hep I, appears to be most
critical in this respect, as has been suggested previously (13). A
further reduction in folding efficiency, as observed with LPS of
chemotype Re, should be due to a further reduction of the net negative
charge present. The variations in the folding efficiencies were not due
to changes in the supramolecular structure and changes in the state of
order of the acyl chains since the lipid A moiety of all LPS chemotypes
prefers a nonlamellar cubic phase (Q) and the order parameter
S37 decreased gradually, due to the decrease of
Tc from 37 to 30 °C accompanying the
reduction of the size of the core structure (21, 46).

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Fig. 2.
LPS-dependent protein
folding. Folding of in vitro synthesized PhoE protein
was performed with LPS of various chemotypes from S. minnesota. Samples, containing the folded proteins, were loaded
onto an SDS-polyacrylamide gel after incubation for 10 min in sample
buffer at room temperature (lanes indicated with
a) or 100 °C (lanes indicated with
c). The positions of denatured mature PhoE (m)
and the folded monomer of PhoE (m*) are indicated.
TL, total translation products containing the cytosolic
protein chloramphenicol acetyltransferase (Cat) and mature
PhoE (mPhoE).
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All LPS chemotypes used above were in their natural salt form. The
folding efficiencies did not change significantly when the counterions
in the LPS of chemotype Re were exchanged by dialysis for
Na+ or Li+ (Table I and Fig.
3A). Interestingly, the
folding efficiency of Re LPS in the Mg2+ salt form was
significantly reduced, whereas those of the Ba2+ and
Ca2+ forms had lost the capacity to support protein folding
completely (Table I and Fig. 3A). This reduced capacity to
support folding seems to be correlated with a change in the
supramolecular structure from a cubic, i.e. non-lamellar, to
a lamellar phase and an increase in S37 (41). Parallel to
the change in aggregate structure, there is also a further reduction of
the amount of free negative charges that might influence the capacity
to support folding. The exact charge density of the LPS in the divalent
cation salt forms as indicated in Table I, however, can only be
estimated to be
2 from the measurement of the electrophoretic
mobility of the lipid aggregates (Zeta potential, kindly performed by
B. Lindner, Borstel Research Institute).

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Fig. 3.
LPS-dependent protein
folding. Folding of in vitro synthesized PhoE protein
was performed with LPS of chemotype Re (A) in its natural
salt form ( ) or in the salt form as indicated. B, folding
of PhoE protein with LPS derived from S. minnesota (S. min), C. violaceum (C. viol), R. capsulatus (Rb. caps), and P. denitrificans
(P. den), all of chemotypes S. Protein samples were analyzed
as described in the legend of Fig. 1. The positions of denatured PhoE
(m) and the folded monomer of PhoE (m*) are
indicated. TL, total translation products containing
chloramphenicol acetyltransferase (Cat) and mature PhoE
(mPhoE).
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In order to further substantiate the influence of the supramolecular
structure further, LPS purified from C. violaceum, R. capsulatus, and P. denitrificans were used. The LPS
from these non-enterobacterial species contain a lipid A structure that
is different to the basic chemical structure of enterobacterial lipid A, mainly at the level of fatty acid composition (28, 29, 36, 47). The
aggregate structures of the lipid A moiety of LPS from C. violaceum and P. denitrificans were determined by x-ray
diffraction. The diffraction patterns showed reflections at equidistant
ratios which is typical for the existence of lamellar structures found
also for that of R. capsulatus (37, 48). Interestingly,
these LPS forms were much less capable to support folding as compared
with LPS of chemotype S or Ra from S. minnesota or E. coli (Table I and Fig. 3B). The decreased efficiencies of folding with LPS of the lipid A variants is not due to a decrease in
total negative charges in the core region or due to an increase in the
order parameter S37. Apparently, LPS with a lipid A moiety adopting a lamellar state is much less capable to support folding of
PhoE protein into a native-like conformation. The folding assays were
performed in the presence of 0.015% Triton X-100 (approximately the
critical micelle concentration), whereas the endoxin aggregate structure is determined in the absence of this detergent. However, we
expect that the physical state of the LPS is dominant as Triton X-100
is only a minor component as compared with LPS.
Folding of PhoE Protein with Chemically Modified LPS--
In order
to study the influence of phosphate groups and fatty acids on the
protein folding further, a variety of chemically modified LPS
preparations was used. The E. coli J-5 strain contains a
well characterized LPS of chemotype Rc (Fig. 1B; see Refs.
33 and 34). The lipid A portion, obtained from the LPS after mild acetic acid hydrolysis and centrifugation, showed only low efficiency (Table II and Fig.
4) most likely due to removal of the
negative charges from the core region (phosphates and
3-deoxy-D-manno-octulopyranosonic acid) and
increase in the order parameter. Interestingly, removal of
O-linked acyl chains with hydrazine drastically decreased
the efficiency of folding (Table II and Fig. 4). This seems to be a
direct consequence of the change in the aggregate structure from
non-lamellar to direct micellar. A reduction of negative charges by
removal of the phosphate groups from the de-O-acylated J-5
LPS additionally decreased the folding efficiency (Table II and Fig.
4). However, the de-O-acylated and de-phosphorylated J-5 LPS
is still more efficient as compared with the lipid A of this LPS. These
results suggest that the presence of negatively charged
3-deoxy-D-manno-octulopyranosonic acid residues
are important for LPS-protein interactions as well. Alternatively or in
addition, the increased state of order in the acyl chains of this lipid A could directly affect LPS-protein interactions. Removal of all fatty
acids from LPS of J-5 resulted in complete inactivation of the molecule
with regard to protein folding (Fig. 4).
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Table II
Folding of in vitro synthesized PhoE protein with chemically
modified LPS
Purified LPS of E. coli J-5 and K. pneumoniae R20
were chemically modified and re-purified as described under
"Materials and Methods" (see also Fig. 1B). The amount
of lipid A of J-5 and of R20 used was 291 nmol/ml and 311 µg/ml,
respectively. M, direct micellar structure. For further details, see
Table I.
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Fig. 4.
Folding of PhoE with chemically modified LPS
of E. coli strain J5. Folding was performed with
intact LPS of E. coli strain J5 (WT) and its
derivatives, lipid A, de-O-acylated LPS, de-acylated LPS,
and de-O-acylated and de-phosphorylated LPS. Preparation of
these chemically modified forms was performed as described under
"Materials and Methods." Protein samples were analyzed by
SDS-polyacrylamide gel electrophoresis as described in the legend of
Fig. 1. The positions of mature PhoE (m) and the folded
monomer of PhoE (m*) are indicated. The results in Figs.
2-4 are all derived from one experiment using equal amounts of
full-length PhoE protein of which the amount is determined from
lanes TL in Figs. 2 and 3.
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Similar results were obtained with rough mutant and chemically modified
LPS derived from K. pneumoniae rough strain R20 (Table II).
Interestingly, the free lipid A of this LPS does not form a pure cubic
phase as found for most enterobacterial strains, but x-ray diffraction
patterns indicate a superposition of diffraction maxima typical for
cubic and lamellar structures (Q/L). The chemical structure of this LPS
has recently been determined (35). Removal of O-acyl chains
results in a decreased efficiency of folding due to a transition of the
aggregate state from non-lamellar to lamellar. The removal of the two
phosphates at the lipid A part of de-O-acylated LPS of R20
or the complete removal of the core region (forming pure lipid A from)
does not further affect the capacity to support folding. Interestingly,
this is the only LPS type known so far that lacks phosphate residues in
the core region. Instead, the negative charges in the R20 core are
derived from galacturonic acid residues. Apparently, if negative
charges in the core region are important for LPS-protein interactions,
they do not need to originate from phosphate groups.
Folding of PhoE Protein with a Glycosphingolipid and
Phospholipids--
It is well established that Gram-negative bacteria
contain LPS in the outer leaflet of the OM, and, at least, the presence of a deep rough LPS molecule appears to be essential in E. coli since conditional lethal mutations have been obtained in
genes encoding proteins that are involved in the early steps in lipid A
biosynthesis (49). Interestingly, some exceptions of this rule have
been described. The Gram-negative bacterium Sphingomonas paucimobilis contains a glycosphingolipid in the outer leaflet (50). Furthermore, an lpxA mutant of Neisseria
meningitidis has been described recently which completely lacks
LPS due to an early block in lipid A biosynthesis (51), whereas it
still contains outer membrane proteins. We therefore investigated
whether folding of the E. coli outer membrane protein PhoE
can proceed in an LPS-independent manner. Folding of PhoE with GSL-1
was relatively inefficient (10% trypsin-resistant PhoE with 427 µg/ml GSL-1). This could either be due to its different chemical
structure compared with that of LPS or could be correlated with the
preferred lamellar state of
GSL-1.2 Furthermore, folding
of PhoE into a monomer is hardly achieved with purified phospholipids
from E. coli (2% trypsin-resistant PhoE with 62 nmol/ml of
phospholipid), whereas similar amounts of LPS would support the folding
of nearly all synthesized PhoE protein. Interestingly, a synthetic
non-E. coli phospholipid
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) could
support folding of PhoE albeit inefficiently (11% trypsin-resistant
PhoE with 62 nmol/ml DOPC). Possibly, certain phospholipid species
might take over the role of LPS partially and promote outer membrane
protein folding, e.g. in the Neisseria lpxA
mutant. This process might be less efficient as in the wild type
Neisseria strain which contains LPS, and the observed
reduced growth rate of the lpxA mutant strain might be a
consequence of this.
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DISCUSSION |
Lipids play many different roles in cells. Clearly, their
organization in membranes provides the correct environment for many different enzymes and membrane-associated processes to occur (52). However, it is only recently that lipids have been recognized as
important components that can be involved in the process of folding and
assembly of membrane proteins. In this study we describe for the first
time which properties of LPS are required in order to support
LPS-dependent folding of PhoE protein into its folded monomeric state. We demonstrate that specific negative charges in the
inner core region of LPS and a non-lamellar structure of the lipid
contribute to the efficiency of LPS-dependent protein folding. The results explain the longstanding observation that deep
rough mutants of E. coli and Salmonella contain
reduced amounts of outer membrane proteins (9-11). We propose that
important LPS-protein interactions are mediated by salt bridges formed
between divalent cations and the negative charges in inner core and
certain negative charges in the pore protein. These negative charges
are likely to participate in the strong and tight network of divalent
cation and carboxylate groups within the layer of LPS-core units in
such a way that the interface between these core units and protein would become as tight as the LPS layer itself (53). Furthermore, it may
be expected that besides the negative charges the hydroxy fatty acids
of the lipid A moiety also are of importance for the similar PhoE
binding as has been proposed recently for the binding of LPS to the
OmpF protein (54).
Evidence was obtained that the three-dimensional supramolecular
structure of LPS is important in order to support the folding of PhoE
protein in vitro. LPS with lipid A that prefers the lamellar or even the direct micellar state was much less capable to support folding as compared with LPS with lipid A that preferred a non-lamellar inverted state. LPS in the lamellar state could be less available or
not available for LPS-protein interactions and/or the specific molecular shape of the LPS molecule is not compatible for LPS-protein interaction. Thus, extensive hydrophilic and hydrophobic interactions between LPS and protein, involving the negative charges in the inner
core and lipid A, respectively, seem to be required for efficient
protein folding. The lipid A moiety of LPS in the outer leaflet of the
outer membrane is likely to be in a lamellar state and, thus, might not
be suitable for folding of the porin, although one could argue that
some mechanism exists that allows the exposure of this residential LPS
to the outer membrane protein during its biogenesis. Furthermore, LPS
could adopt a different structure in contact with a porin than in a
porin-free bilayer. Alternatively, de novo synthesized LPS
is required for assembly of pore proteins in vivo as was
previously suggested (12, 17-19). This de novo synthesized
LPS might be preferentially in a monovalent cation form (or at least
not in its full divalent cation form) since biosynthesis of LPS occurs
at the site of the inner membrane that is facing the cytoplasm and that
is known to contain especially free K+ as available cation
to compensate for negative charges in the cytoplasm (55), whereas
Mg2+ and Ca2+ concentrations are very low (56,
57). Thus, de novo synthesized LPS might be transported from
its site of synthesis to the OM in a salt form (K+ salt)
that is optimal to support protein folding. At later stages in the
biogenesis, the LPS are most likely converted to (partial) Mg2+ and/or Ca2+ salt forms. New research into
the molecular mechanism of LPS biogenesis is required to investigate
its possible connection with porin biogenesis, and this research has to
take into account the specific chemical and biophysical properties of LPS.