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(Received for publication, March 5, 1996; and in revised form, March 18, 1996) From the
A mutant of Escherichia coli lacking
phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1)
directed against a conformationally sensitive epitope (4B1) of lactose
permease were used to establish a novel role for a phospholipid in the
assembly of a membrane protein. Epitope 4B1 is readily detectable in
spheroplasts and right-side-out membrane vesicles from PE-containing
but not from PE-deficient cells expressing lactose permease. Lactose
permease from PE-containing membranes, but not from PE-deficient
membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis and Western blot analysis is also recognized by mAb
4B1. If total E. coli phospholipids or PE (but not
phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted
on nitrocellulose sheets (Eastern blot) prior to transfer of proteins
from SDS-polyacrylamide gels, the permease from PE-deficient cells
regains its recognition by mAb 4B1. Therefore, PE is required during
assembly to form epitope 4B1, but, once formed, sufficient
``conformational memory'' is retained in the permease to
either retain or reform this epitope in the absence of PE. Lactose
permease lacking epitope 4B1 can be induced to form the epitope if
partially denatured and then renatured in the presence of PE
specifically. These results establish for the first time a role for PE
as a molecular chaperone in the assembly of the lactose permease.
Only a limited number of reports address the role of the native
phospholipid environment in the function and assembly of membrane
proteins. Clearly, the amphipathic environment of the membrane is an
important determinant in the folding and maintenance of membrane
protein structure. However, what has not been widely considered is a
role for individual phospholipids in determining the folding pathway
for membrane proteins independent from the maintenance of the final
structure, i.e. to act in the capacity of a non-protein
molecular chaperone. Mutants of Escherichia coli are
available in which membrane phospholipid composition can be varied in a
way that is difficult to achieve in vitro (DeChavigny et
al., 1991; Dowhan, 1992). Such ``phospholipid mutants''
were used to study the in vivo role of PE ( How
phospholipids affect membrane protein assembly and function is still
largely unknown. Do the properties of the target membrane phospholipids
affect assembly and function? Do individual phospholipids act as
conformational determinants? In the present work, we address the
possibility that membrane phospholipid composition, and specifically
PE, determines the conformation of the lactose permease. The
experiments described here combine structural and functional
information on the lactose permease with the use of a conformationally
sensitive monoclonal antibody (Sun et al., 1996) directed
against an epitope in the periplasmic loop [L(VII/VIII)] of
the permease between helices VII and VIII (Fig. 1). Monoclonal
antibodies have been used effectively to detect folding intermediates
of soluble proteins in solution (Fedorov et al., 1992), but
have not been used extensively to follow folding of membrane proteins
as described in this report. The proper assembly of lactose permease,
as assessed by the presence of the conformational epitope, into the
membranes of an E. coli mutant lacking PE (DeChavigny et
al., 1991) was compared to assembly in wild type cells to
establish a requirement for PE. We also introduce a novel adaption of
the Western blot procedure termed an ``Eastern-Western'' in
which phospholipids are applied to the surface of nitrocellulose sheets
(Taki et al., 1994) prior to transfer of proteins from
SDS-polyacrylamide gels by electroblotting. This combination of protein
and ligand on a solid support allows the monitoring by a
conformation-specific monoclonal antibody of the involvement of a
specific phospholipid in the refolding of a denatured protein. The
information gathered from these approaches indicate that PE plays an
important role in facilitating the proper assembly of the lactose
permease into the membrane and thereby demonstrates for the first time
that a phospholipid can act as a molecular chaperone.
Figure 1:
Model of the secondary structure of the
lactose permease (Kaback et al., 1994). Single-letter code is used to denote the amino acids. Boxed areas indicate
the putative hydrophobic transmembrane helical domains numbered
sequentially from the N to C terminus in Roman numerals. The
location of L(VII/VIII) between helices VII and VIII is indicated.
Amino acids in bold including their position in the sequence
are those discussed in the text.
In the Eastern-Western blot procedure, bands of
phospholipids (0.05-0.10 mg) on silica gel thin layer plates were
developed using chloroform/methanol/water/30% ammonium hydroxide
(120:75:6:2, v/v); for total E. coli phospholipids, no
chromatography was performed. Plates were dipped in isopropyl
alcohol/0.2% aqueous CaCl The binding of mAb 4B1 to RSO membrane vesicles and
spheroplasts was utilized to determine whether structural differences
exist between lactose permease assembled in either PE-deficient or
PE-containing membranes. As shown in Fig. 2A, binding
of mAb 4B1 to spheroplasts and RSO membrane vesicles from PE-deficient
cells is reduced to the level observed for binding to control membranes
lacking the permease. High level binding to permease in membranes from
PE-containing cells was observed as reported previously (Carrasco et al., 1982, 1984; Herzlinger et al., 1984; Sun et al., 1996). The amount of permease in each membrane
preparation was established using Western blot analysis with a
site-directed pAb against the C terminus of the permease using either
the ECL chemiluminescence-based method (Fig. 2B) or
radiolabeled protein A (Fig. 2C) for qualitative or
quantitative measurements, respectively. The specific content of
permease in spheroplasts (500 and 600 cpm/mg of total membrane protein)
and RSO membrane vesicles (6000 and 7000 cpm/mg of membrane protein)
was comparable in samples prepared from PE-containing and PE-deficient
cells, respectively. Therefore, the marked difference in mAb 4B1
binding to membranes is due specifically to differences in recognition
of the epitope. The results are consistent with the hypothesis that
membrane phospholipid composition is a determinant for the conformation
of lactose permease.
Figure 2:
Binding of mAb 4B1 to spheroplasts and RSO
membrane vesicles. Aliquots of spheroblasts or membrane vesicles were
treated with mAb 4B1 as described under ``Experimental
Procedures.'' A, radioactivity from bound protein A
associated with equivalent volumes of the final suspensions of
spheroplasts from PE-containing (SPH + PE) and
PE-deficient (SPH - PE) cells, and RSO membrane vesicles
from PE-containing (RSO + PE) and PE-deficient (RSO
- PE) cells was measured by liquid scintillation counting
and plotted as the total counts/min per suspension. Control
spheroplasts or RSO membrane vesicles (SPH/RSO uninduced) made
from PE-containing and PE-deficient cells lacking plasmid
pT7-5/lacY and grown in the absence of IPTG exhibited
individually little binding of antibody. No conclusive results were
obtained from ISO membrane vesicles because of high binding of mAb 4B1
to membranes from cells both induced and uninduced for permease
synthesis. The results are representative of three independent
experiments. B, aliquots of the above final suspensions of
membranes were subjected to SDS-PAGE and transferred to nitrocellulose
sheets and treated with pAb and the ECL detection system as described
under ``Experimental Procedures.'' Only the permease region
of the gel (M
Membrane preparations from PE-containing and
PE-deficient cells were then analyzed by Western blot analysis,
comparing the sensitivity of SDS-denatured permease to detection with
mAb 4B1 and pAb (Fig. 3A). As reported previously
(Carrasco et al., 1982), mAb 4B1 binds to permease from
PE-containing cells after being transferred to a solid support.
Permease from PE-deficient cells exhibits greatly reduced binding of
mAb 4B1, although probing with pAb verified the presence of ample
amounts of permease (Fig. 3B). Therefore, permease
assembled in PE-containing membranes retains sufficient
``conformational memory'' to reform the native structure of
the epitope after removal of SDS. The results confirm the absence of
the conformational epitope in PE-deficient membranes and also suggest
that once the information is imparted to form the epitope in
PE-containing cells, PE is no longer required to maintain the epitope.
Figure 3:
Western blot analysis of lactose permease
with mAb 4B1 and pAb. ISO membrane vesicles containing protein amounts
of 6 µg (lanes 1 and 4), 12 µg (lanes 2 and 5), and 24 µg (lanes 3 and 6)
from PE-containing (lanes 1, 2, and 3) and
PE-deficient (lanes 4, 5, and 6) cells were
subjected to SDS-PAGE and Western blot analysis and detected as
follows. A, detection was with mAb 4B1 (5 mg/ml) at a final
dilution of 1:10
To further investigate the role of phospholipids in the formation of
this conformational epitope, the Eastern-Western blot procedure was
developed. By combining these two procedures (Fig. 4), denatured
proteins are exposed to hydrated phospholipids on the surface of
nitrocellulose sheets as they exit the polyacrylamide gel and begin to
refold in the absence of SDS. The surprising finding is that lactose
permease from PE-deficient membranes subjected to Eastern-Western
blotting with either PE or E. coli phospholipids regains the
ability to bind mAb 4B1. In marked contrast, neither the E. coli anionic phospholipids PG and CL nor PC, which is also zwitterionic
but not present in E. coli, promote the formation of the
epitope. This specificity for PE is consistent with the earlier results
of Chen and Wilson(1984) who showed that only PE and not the other
three phospholipids listed above could restore active transport to
lactose permease reconstituted in proteoliposomes. Therefore,
restoration of mAb 4B1 binding specifically by PE indicates that PE
plays a positive role as a structural determinant, rather than acting
to exclude a negative determinant in the form of anionic phospholipids.
Figure 4:
Antibody detection of permease after
transferring proteins to nitrocellulose overlaid with phospholipids
(Eastern-Western). Phospholipids were transferred to nitrocellulose
sheets prior to transfer by Western blot of lactose permease from
PE-deficient (-PE) or PE-containing (+PE)
cells. The gel was positioned so the proteins in the M
Subjecting membrane preparations to SDS-PAGE in the presence of 5 M urea eliminates detection by mAb 4B1 in both Western and
Eastern-Western blots with permease from both PE-containing and
PE-deficient cells (data not shown). Lactose permease retains about 70%
of its helical structure in SDS as estimated from circular dichroism
measurements, ( The epitope recognized by mAb 4B1 (see Fig. 1) consists
of Phe-247, Phe-250, and Gly-254 on one face of a short What are the special properties of PE which may
be related to its specific role in determining protein conformation?
PE, unlike PC, has a strong tendency to form nonbilayer structures
(Thurmond et al., 1991), and, with its small head group, PE
may interact at the interface between phospholipid and protein domains
or intercalate within the packed helical structure of membrane
proteins; helix VIII contains a region of low information content that
has been postulated to be in contact with phospholipids (Hinkle et
al., 1990). PE (as well as PC) also exhibits a dipole moment over
the ionic head group which can respond to membrane potential (Seelig,
1993). Positive charges have been postulated to interaction with In conclusion, the use of monoclonal antibodies in an
Eastern-Western blot analysis of other membrane proteins may reveal
additional examples of phospholipid-dependent organization of protein
structure, especially for those proteins that require the addition of
lipids to regain function. Direct assay of enzyme activity after
transferring proteins to a solid support in an Eastern-Western blot may
prove to be a useful screening technique to survey the role of specific
lipids in protein function. The method described here could have broad
application for the study of renaturation of proteins from SDS in the
presence of either surface-bound ligands or ligands introduced into the
electroblotting buffers. Certainly the realization that specific
phospholipids can determine the folding pathway of a protein should be
taken into account in designing experiments aimed at understanding the
mechanism for assembling proteins into membranes.
Volume 271,
Number 20,
Issue of May 17, 1996 pp. 11615-11618
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)in
lactose permease (lacY gene product) function (Bogdanov and
Dowhan, 1995). The lactose permease of E. coli is an
extensively studied prototype of most secondary transport systems found
in both prokaryotic and eukaryotic organisms (Kaback et al.,
1994). PE is not required for energy-independent downhill translocation
of substrate mediated by lactose permease, but appears to be essential
for H
-coupled active lactose accumulation in vivo (Bogdanov and Dowhan, 1995). These results parallel the earlier
observation (Chen and Wilson, 1984; Page et al., 1988) with
purified permease reconstituted into proteoliposomes where PE was also
found to be required specifically for active transport.
Strains and Growth Conditions
E. coli strain AD93 (pss93::kan, PE-deficient) cannot
make either PE or phosphatidylserine except when carrying plasmid pDD72 (pss, PE-containing) (DeChavigny et
al., 1991). All strains (mutant and wild type for PE formation)
were grown at 30 °C in LB medium containing 50 mM MgCl
and, except as noted, carried multiple copies of
the lacY gene on plasmid pT7-5/lacY (Bibi and Kaback,
1990). Ampicillin (100 µg/ml) and IPTG (1 mM) were
included in the growth medium to maintain plasmid pT7-5/lacY and induce lactose permease synthesis, respectively.Preparation of Spheroplasts and Membranes
A
modification of the protocol of van Veen et al.(1993) was used
to make spheroplasts. Midexponential phase cells were suspended in 10
mM potassium PIPES (pH 7.0), 0.75 M sucrose, 10
mM MgSO
, 2.5% (w/v) LiCl, and 50 mg/ml
chloramphenicol. After addition of either 1 mg/ml or 0.1 mg/ml lysozyme
to pss or pss
cells, respectively, cell suspensions were chilled to 2 °C,
warmed to room temperature, and subsequently incubated with gentle
shaking at 30 °C for 30 min or 5 min for pss cells or pss cells, respectively.
Intact spheroplasts were collected by centrifugation (3,000 
g
for 10 min) at room temperature and resuspended
at 10 mg/ml total protein in the above buffer without LiCl. RSO
membrane vesicles were made by diluting a pellet of spheroplasts into a
50-fold volume of 10 mM potassium PIPES buffer (pH 7.0). The
solution was adjusted to 10 mM MgCl, 1 mM DTT, and 10 mg/ml deoxyribonuclease and incubated for 10 min on
ice. Intact cells and cell debris were removed by centrifugation at
4000 g for 5 min, and the supernatant was
centrifuged at 30,000 g for 30 min at 4
°C. Resulting RSO membrane vesicles were suspended to a final
concentration of 2 mg of membrane protein/ml in 50 mM potassium PIPES (pH 7.0) containing 10 mM MgCl. ISO membrane vesicles were prepared as described
previously (Bogdanov and Dowhan, 1995) and were resuspended at 10 mg/ml
in ice-cold 100 mM potassium HEPES buffer (pH 8.0) containing
125 mM sucrose, 10 mM MgCl, and 1 mM DTT. Protein concentration was measured using the BCA method as
suggested by the supplier (Pierce).Binding of mAb 4B1 to Membranes
Aliquots (0.05 ml)
of stock spheroplasts, RSO membrane vesicles, or ISO membrane vesicles
were diluted 10-fold into suspension buffer (100 mM potassium
HEPES (pH 8.0), 125 mM sucrose, 10 mM MgCl) containing 5% bovine serum albumin followed by
the addition of 5 µl of affinity-purified mAb 4B1 (5 mg/ml). After
incubation at room temperature for 45 min, mixtures were centrifuged
for 10 min at 10,000 g for spheroplasts
or 30 min at 30,000 g for RSO and ISO
membrane vesicles. Pellets were washed twice with suspension buffer
containing 5% bovine serum albumin and resuspended in a final volume of
0.5 ml of suspension buffer containing 5% bovine serum albumin. 
S-Protein A (2.5 µl, Amersham) was added and
incubation was continued for 45 min. Samples were centrifuged as above,
washed sequentially with suspension buffer first containing 5% bovine
serum albumin and second without bovine serum albumin, and finally
resuspended in suspension buffer without bovine serum albumin to the
final protein concentration of 10.5 mg/ml for spheroplasts and 2.3
mg/ml for RSO or ISO membrane vesicles.SDS-PAGE and Western Blot Analysis
Aliquots of
membrane suspensions were adjusted to the concentration of SDS gel
loading buffer (2.8% SDS, 10% glycerol, 100 mM DTT) and heated
at 37 °C for 15 min prior to being subjected to PAGE in 12.5% gels
containing 0.1% SDS. Transfer of proteins from gels to nitrocellulose
sheets (Schleicher & Schuell, pore size of 0.45 µm) using a
semidry electroblotting system (Milliblot-SDE electroblotting
apparatus) and detection by antibody were carried out as described
previously (Bogdanov and Dowhan, 1995) except as follows. The gel was
incubated for 10 min at room temperature in cathode buffer 1
(electroblotting system) prior to transfer. Bound antibody (mAb 4B1 or
pAb) was visualized by either the ECL detection system (Amersham with
supplied peroxidase-labeled anti-mouse or anti-rabbit antibody,
respectively) and exposure to x-ray film or S-protein A
and analysis with a Betagene Imager as described previously (Bogdanov
and Dowhan, 1995)./methanol (40:20:7, v/v) for 20 s.
A nitrocellulose sheet was placed on top of the silica gel surface
followed by a glass microfiber sheet (Whatman, United Kingdom).
Transfer of phospholipid was accomplished by pressing on the stack with
a metallic heating block at 130 °C for 10 s (modification of Taki et al.(1994)). Western blot transfer (phospholipid side facing
the acrylamide gel) of proteins was accomplished as described above.
= 31,000-33,000) is
shown representing the signal from the following: 10 µg (lanes
1 and 3) and 20 µg (lanes 2 and 4)
of spheroplast protein from PE-containing (lanes 1 and 2) and PE-deficient (lanes 3 and 4) cells; 5
µg (lanes 5 and 7) and 10 µg (lanes 6 and 8) of RSO membrane vesicle protein from PE-containing (lanes 5 and 6) and PE-deficient (lanes 7 and 8) cells. C, samples were prepared and
processed the same as in B above except detection of bound pAb
was via incubation with S-protein A as described under
``Experimental Procedures.'' A Betagene imager was used to
detect and quantify the radiolabel associated with the permease (M = 31,000-33,000 region of the gel)
as reported in the text. The source and amount of each sample in each
respective lane are the same as in B above.
and peroxidase-labeled anti-mouse antibody
supplied with the ECL kit. Use of a semidry rather than an immersion
tank-type electroblotting system was critical to detection by mAb 4B1
in all experiments. B, the nitrocellulose blot from A was treated with 2% SDS in the presence of 100 mM 
-mercaptoethanol and 62.5 mM Tris-HCl (pH 6.7) for
20 min at 50 °C to strip off all antibodies. The sheet was then
treated with pAb followed by the ECL kit to visualize the relative
amounts of lactose permease.
= 31,000-33,000 region of the gel would transfer to
areas of nitrocellulose sheets previously blotted with the following
phospholipids: PC (lanes 1 and 2), PE (lanes 3 and 4), none (lanes 5 and 6), equal
amounts of PG and CL (lanes 7 and 8), and total E. coli phospholipid (lanes 9 and 10).
Sheets were then treated with mAb 4B1 followed by detection using the
ECL method. Identical results were obtained using polyvinylidene
difluoride sheets (Immobilon-P, Millipore Corp.) and when phospholipids
were spotted directly on solid supports rather than being transferred
from silica gel plates by heating.
)and recent evidence indicates that
denaturants such as SDS induce alterations in helical packing within
the permease with little or no change in secondary structure (Jung et al., 1994). However, urea disrupts the secondary structure
of proteins by stabilizing the random coil state (Schein, 1990). In the
case of the permease, urea appears to eliminate structural elements
that either carry conformational memory after assembly in the presence
of PE in vivo or have potential to form the 4B1 epitope. The
possibility that residual PE is bound to permease from PE-containing
cells was eliminated by labeling membrane phospholipids by growth in
the presence of 
P
, followed by SDS-PAGE.
Autoradiography of the gel revealed no difference in the labeling
pattern between samples from PE-containing cells with or without
lactose permease. However, bands in other regions of the gel exhibited
significant radioactivity, indicating that the P was of sufficiently high specific activity to detect low levels
of protein-associated radiolabel such as tightly bound PE. Therefore,
the 4B1 epitope once formed is maintained even in the absence of PE.
Finally, when PE-deficient membranes are solubilized in the presence of
PE-containing liposomes (12.5 mg of total membrane protein plus 50 mg
of either PE or wild type E. coli phospholipid), no
restoration of mAb 4B1 binding is observed (data not shown), indicating
that formation of the epitope is dependent on removal of SDS in the
presence of PE rather than simply exposure of the denatured protein to
PE.
-helical
segment in the periplasmic loop between helices VII and VIII (Sun et al., 1996). Treatment of RSO membrane vesicles with mAb 4B1
inhibits all reactions catalyzed by the permease that involve net
H translocation with no effect on ligand binding and
either equilibrium exchange or counterflow of substrate (Carrasco et al., 1982, 1984). The ``uncoupling'' effect of
mAb 4B1 is remarkably similar to the effects of site-directed mutations
in Glu-325 (helix X) (Carrasco et al., 1986, 1989), as well as
the functional alterations observed in vivo with mutants
devoid of PE (Bogdanov and Dowhan, 1995) and in reconstituted
proteoliposomes lacking PE (Page et al., 1988; Seto-Young et al., 1985). In this respect, it is particularly interesting
that helix VIII contains Glu-269, a residue that is essential for
transport and interacts with another essential residue, His-322 (helix
X), while helix VII contains Asp-237 and Asp-240 which interact with
Lys-358 (helix XI) and Lys-319 (helix X), respectively (Kaback et
al., 1994). Since helices VII and VIII interact with helix X which
contains Glu-325 and mAb 4B1 mimics the effect of mutations at this
position, torsional effects on either of these helices may alter the
pK
of one or more residues in helices VII and/or
VIII in such a manner as to increase the pK of
Glu-325, thereby limiting deprotonation of the permease at the surface
of the membrane (Carrasco et al., 1986, 1989; Sun et
al., 1996). Bogdanov and Dowhan(1995) postulated that lactose
permease in PE-deficient membranes is unable to release a H on the inner surface of the membrane. Binding of mAb 4B1 also
markedly alters the reactivity of a site-specific Cys replacement of
Val-331 (helix X), as well as the fluorescence of this mutant after
labeling the Cys with 2-(4`-maleimidylanilino)naphthalene-6-sulfonic
acid (Sun et al., 1996). Taken together, the data are
consistent with a role for PE in either the folding of helices VII and
VIII or their packing within the bilayer. The observation that
sufficient conformational memory remains in the permease after removal
of PE and renaturation on a solid support to reform the 4B1 epitope
indicates that PE does not act per se to maintain the
conformation within L(VII/VIII). Rather, PE plays a role in the folding
of either the epitope itself or helices VII and VIII which may
determine the conformation of the epitope. Further support for this
conclusion comes from the ability of PE to direct the formation of the
epitope during renaturation from SDS of permease originally assembled
in the absence of PE. Therefore, PE functions as do protein molecular
chaperones in guiding the folding pathway which is a new and novel role
for a phospholipid.
electrons of aromatic side chains of amino acids (Dougherty, 1996). Not
only are two Phe residues part of the determinant recognized by mAb
4B1, but the sequence in the region of L(VII/VIII), which is conserved
among several other secondary transporters (Frillingos et al.,
1994), contains 6 Phe residues. Therefore, head group size and
electrical properties of PE coupled with the ability to assume
nonbilayer structures may combine to influence the final helix
structure and packing of the permease and ultimately its function.
)-D-thiogalactoside; RSO, right-side-out;
ISO, inside-out; DTT, dithiothreitol; mAb, monoclonal antibody; pAb,
polyclonal antibody; PAGE, polyacrylamide gel electrophoresis; PG,
phosphatidylglycerol; CL, cardiolipin; PC, phosphatidylcholine; PIPES,
1,4-piperazinediethanesulfonic acid.)
We thank Drs. Finn Wold and John DeMoss for their
thoughtful comments. Phil Heacock was also very helpful by first
finding references that described methods for applying lipids to
nitrocellulose which was instrumental in developing the Eastern-Western
blot procedure.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 X.-G. Wang, J. P. Scagliotti, and L. T. Hu Phospholipid synthesis in Borrelia burgdorferi: BB0249 and BB0721 encode functional phosphatidylcholine synthase and phosphatidylglycerolphosphate synthase proteins Microbiology, February 1, 2004; 150(2): 391 - 397. [Abstract] [Full Text] [PDF]
|
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W. Zhang, M. Bogdanov, J. Pi, A. J. Pittard, and W. Dowhan Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition J. Biol. Chem., December 12, 2003; 278(50): 50128 - 50135. [Abstract] [Full Text] [PDF]
|
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 |
|
 J. Prades, S. S. Funari, P. V. Escriba, and F. Barcelo Effects of unsaturated fatty acids and triacylglycerols on phosphatidylethanolamine membrane structure J. Lipid Res., September 1, 2003; 44(9): 1720 - 1727. [Abstract] [Full Text] [PDF]
|
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|
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|
S. S. Funari, F. Barcelo, and P. V. Escriba Effects of oleic acid and its congeners, elaidic and stearic acids, on the structural properties of phosphatidylethanolamine membranes J. Lipid Res., March 1, 2003; 44(3): 567 - 575. [Abstract] [Full Text] [PDF]
|
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T. Karnezis, H. C. Fisher, G. M. Neumann, B. A. Stone, and V. A. Stanisich Cloning and Characterization of the Phosphatidylserine Synthase Gene of Agrobacterium sp. Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1->3)-{beta}-D-Glucan (Curdlan) J. Bacteriol., August 1, 2002; 184(15): 4114 - 4123. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 T. Osterbye, K. H. Jorgensen, P. Fredman, J. Tranum-Jensen, A. Kaas, J. Brange, J. L. Whittingham, and K. Buschard Sulfatide promotes the folding of proinsulin, preserves insulin crystals, and mediates its monomerization Glycobiology, June 1, 2001; 11(6): 473 - 479. [Abstract] [Full Text] [PDF]
|
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C. van der Does, J. Swaving, W. van Klompenburg, and A. J. M. Driessen Non-bilayer Lipids Stimulate the Activity of the Reconstituted Bacterial Protein Translocase J. Biol. Chem., January 28, 2000; 275(4): 2472 - 2478. [Abstract] [Full Text] [PDF]
|
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M. Bogdanov and W. Dowhan Lipid-assisted Protein Folding J. Biol. Chem., December 24, 1999; 274(52): 36827 - 36830. [Full Text] [PDF]
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J. Bhattacharyya and K. P. Das Molecular Chaperone-like Properties of an Unfolded Protein, alpha s-Casein J. Biol. Chem., May 28, 1999; 274(22): 15505 - 15509. [Abstract] [Full Text] [PDF]
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M. Bogdanov, M. Umeda, and W. Dowhan Phospholipid-assisted Refolding of an Integral Membrane Protein. MINIMUM STRUCTURAL FEATURES FOR PHOSPHATIDYLETHANOLAMINE TO ACT AS A MOLECULAR CHAPERONE J. Biol. Chem., April 30, 1999; 274(18): 12339 - 12345. [Abstract] [Full Text] [PDF]
|
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|
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|
 J. M. Wood Osmosensing by Bacteria: Signals and Membrane-Based Sensors Microbiol. Mol. Biol. Rev., March 1, 1999; 63(1): 230 - 262. [Abstract] [Full Text] [PDF]
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H. de Cock, K. Brandenburg, A. Wiese, O. Holst, and U. Seydel Non-lamellar Structure and Negative Charges of Lipopolysaccharides Required for Efficient Folding of Outer Membrane Protein PhoE of Escherichia coli J. Biol. Chem., February 19, 1999; 274(8): 5114 - 5119. [Abstract] [Full Text] [PDF]
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R. P. H. Huijbregts, A. I. P. M. de Kroon, and B. de Kruijff Rapid Transmembrane Movement of Newly Synthesized Phosphatidylethanolamine across the Inner Membrane of Escherichia coli J. Biol. Chem., July 24, 1998; 273(30): 18936 - 18942. [Abstract] [Full Text] [PDF]
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H. Duan, C.-Y. Lin, and T. Mazzone Degradation of Macrophage ApoE in a Nonlysosomal Compartment. REGULATION BY STEROLS J. Biol. Chem., December 5, 1997; 272(49): 31156 - 31162. [Abstract] [Full Text] [PDF]
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L. Li, O. P. Karlsson, and A. Wieslander Activating Amphiphiles Cause a Conformational Change of the 1,2-Diacylglycerol 3-Glucosyltransferase from Acholeplasma laidlawii Membranes According to Proteolytic Digestion J. Biol. Chem., November 21, 1997; 272(47): 29602 - 29606. [Abstract] [Full Text] [PDF]
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 J. le Coutre, L. R. Narasimhan, C. K. N. Patel, and H. R. Kaback The lipid bilayer determines helical tilt angle and function in lactose permease of Escherichia coli PNAS, September 16, 1997; 94(19): 10167 - 10171. [Abstract] [Full Text] [PDF]
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