(Received for publication, June 15, 1995; and in revised form, September 14, 1995)
From the
We have studied the kinetics of folding and membrane insertion
of the outer membrane protein OmpA of Escherichia coli. In the
native structure, its membrane-inserted domain forms a -barrel.
The protein was unfolded in solubilized form in water/urea, and
refolding was induced by dilution of urea and simultaneous addition of
lipid vesicles. Three transitions along the folding pathway could be
distinguished. Their characteristic times lie below a second, in the
range of minutes, and in the range of an hour. The fast process
corresponds to the transition from the unfolded state in water/urea to
a misfolded state in water, the moderately slow process to a transition
from the misfolded state to a partially folded state in the membrane,
and the slow process to the transition from the partially folded to the
native state. The partially folded state in the membrane is interpreted
as the analogue of the molten globule state of soluble proteins.
Folding of soluble proteins in vitro has been studied
extensively in the past, and much insight into the details of folding
has been gained. Folding proceeds along a pathway with a number of more
or less stable intermediates (for a review, see (1) ). By
contrast, the study of folding of membrane proteins is still in its
infancy. Only a few integral membrane proteins have been unfolded and
refolded in vitro: the -helical proteins
bacteriorhodopsin (2) and the photosynthetic antenna
complex(3) , as well as the
-barrel proteins OmpA (4) and the porin OmpF(5) . The kinetics of refolding
were studied only for bacteriorhodopsin (6) and
OmpF(5) . In all these cases, refolding took place into mixed
micelles of detergent and lipid, which are considered as a good model
system for a lipid membrane. This is certainly true as far as protein
structure is concerned, but does not necessarily hold for the kinetics
of folding which may be much slower for insertion into a membrane than
into a micelle. This problem may be solved by studying refolding into
lipid membranes(7) .
The main problem with membrane proteins
lies in their insolubility in water which makes it difficult to keep
them solubilized and unfolded before starting the refolding process. If
water is replaced by another solvent such as trifluoroacetic acid, they
may become soluble in the unfolded form as shown for
bacteriorhodopsin(2) , but such a model system bears less
resemblance to the in vivo situation given by a membrane in
water. In another approach, the proteins bacteriorhodopsin and OmpF
were made soluble by attaching polyethylene glycol to them(8) ,
but again this represents a drastic change compared to the in vivo situation. An exceptionally favorable case is provided by members
of the -barrel class of membrane proteins which can be kept
soluble in water in the unfolded form(4, 5) . This is
a consequence of their low hydrophobicity which itself is a
characteristic feature of their
-barrel structure and
distinguishes them from the
-helical membrane
proteins(9) . We have shown previously that OmpA, a protein of
the outer membrane of Escherichia coli, refolds from its
unfolded state and inserts directly into lipid vesicle
membranes(7) .
In the present paper, the kinetics of this
process are investigated. This is the first kinetic study of refolding
and direct membrane insertion of an integral membrane protein. As
experimental techniques, we used CD spectroscopy, Trp fluorescence, ANS ()fluorescence, and protease digestion with ensuing SDS-gel
electrophoresis.
Folding and insertion of OmpA into vesicle membranes was started by adding a small volume of the stock solution of unfolded OmpA to preformed vesicles at 30 °C (only in the experiment of Fig. 3A, unfolded OmpA was diluted into water prior to addition of vesicles). The experiments were routinely performed with highly sonicated lipid vesicles consisting of DMPC or the mixture DMPC/DMPG at a molar ratio of 95/5. The vesicles were prepared essentially as described previously(7) . After sonication, the lipid concentrations and the pH values of the vesicle dispersions were adjusted to their final values, followed by storage of the vesicles overnight at 30 °C. The size of the vesicles was characterized by quasielastic light scattering (Coulter N4/SD) and electron microscopy (Philips 201), the diameter was 30-40 nm. For one experiment, unsonicated vesicles were prepared by extrusion of lipid dispersions through filters (Nuclepore). The diameter of these vesicles (again after 1 night at 30 °C) was about 200 nm. The end concentrations of the folding and insertion experiments were 2.4 µM OmpA, 1.5 mM (or 0.75 mM) DMPC/DMPG, 100 mM NaCl, 16 mM urea, 20 mM buffer when parallel measurements of CD, Trp fluorescence, and protease digestion were performed, and 0.6 µM OmpA, 1.5 mM (or 4.5 mM) DMPC, 4 mM urea, 20 mM buffer when only Trp fluorescence was measured. Spectra of inserted OmpA were recorded 3 h after the start of insertion. Since the kinetics of folding and insertion depend on the vesicle size, it was important to use the same preparation of vesicles in performing the parallel kinetic measurements.
Figure 3: Yield of folding and membrane insertion of 2.4 µM OmpA. A, after different incubation times of partially folded OmpA in water at pH 7.3 and pH 10 prior to addition of 7.5 mM DMPC; B, at different concentrations of DMPC at pH 7.3 and zero incubation time; and C, at different pH and zero incubation time in 7.5 mM DMPC. The yield of insertion has been determined by quantitating the membrane-protected fragment on SDS/gels after insertion and subsequent trypsin digestion (lane 3 of the inset). Inset, SDS-gel of OmpA unfolded in urea (lane 1), inserted into DMPC vesicles at pH10 without digestion (lane 2), and after digestion by 0.1 µM trypsin (lane 3) (Samples of lane 1 and 2 were not boiled before being loaded on the gel.)
Peptidyl-prolyl-isomerase from pig kidney was a gift of F.-X. Schmid (10) and was added in one experiment at an equimolar ratio of isomerase/OmpA.
Figure 1:
Circular
dichroism (A) of OmpA (2.4 µM), Trp fluorescence (B) of OmpA (0.6 µM), and ANS fluorescence (C) in the absence and presence of OmpA (2.4 and 4.8
µM) under different conditions. -
-, unfolded OmpA in 8 M urea at pH 7.3; - -
-, partially folded OmpA in water at pH 7.3; --, partially
folded OmpA in water at pH 10.0; -, folded OmpA in 0.75
mM DMPC/DMPG (A) or 1.5 mM DMPC (B)
at pH 7.3. In C, the ANS fluorescence before the addition of
OmpA is included in water at pH 7.3 (-
-) and in the
presence of 8 M urea
(
).
Unfolded OmpA in 8 M urea is characterized by a CD spectrum indicating a completely random structure. Its Trp fluorescence is relatively weak, with a maximum at 350 nm indicating a polar environment. The ANS fluorescence remains unchanged by the addition of OmpA, i.e. OmpA does not bind the hydrophobic probe ANS.
When the urea concentration is decreased to
20 mM by dilution of unfolded OmpA into water, the CD spectrum
indicates that OmpA adopts secondary structure, which has been
described previously as a mixture of ,
, and random
structure(7) . The Trp fluorescence is still weak, but the
emission maximum is already shifted to 343 nm. The hydrophobic probe
ANS (12) binds to this partially folded OmpA as indicated by an
increase of the intensity of the ANS fluorescence and a shift of the
emission maximum from 520 nm to 480 nm. This means that in contrast to
unfolded OmpA, partially folded OmpA has hydrophobic regions which are
accessible to ANS. Increase of the pH from 7.3 to 10.0 leads to a loss
of secondary structure, as indicated by CD, and a reduced Trp
fluorescence. The increase of ANS fluorescence at pH 10 is less
pronounced than at pH 7.3 (data not shown).
After dilution of
urea-unfolded OmpA into a dispersion of small DMPC vesicles, the CD
spectra indicate an increased content of -structure. The intensity
of the Trp fluorescence is high and the emission maximum is at 325 nm.
The CD and fluorescence spectra agree with those of conventionally
reconstituted OmpA(7) . ANS binding is not suitable to test
accessible hydrophobic regions of membrane-inserted OmpA, because in
this case ANS fluorescence is always high due to its partitioning into
the lipid membranes.
Figure 2: Kinetics of partial folding of 2.4 µM OmpA into water at pH 7.3 as observed by CD at 220 nm, Trp fluorescence at 330 nm, and ANS fluorescence at 480 nm.
In contrast to ellipticity, the intensity of the Trp fluorescence at 330 nm was slowly increasing within 30 min, but did not reach the value of membrane-inserted OmpA. This rise in Trp fluorescence is indicative of aggregation of OmpA, as will be demonstrated below.
Both processes, i.e. fast folding and slow aggregation in water, can be visualized by ANS fluorescence. Within the mixing time, the ANS fluorescence increased rapidly due to binding of ANS to partially folded OmpA, followed by a slow increase of ANS fluorescence due to aggregation of OmpA. The latter increase parallels the slow increase of the Trp fluorescence.
The effect of incubation time on the yield is
shown in Fig. 3A. At pH 7.3, the yield decreases from
68% for zero incubation time to 10% for 27-h incubation time. This
indicates that aggregation of the partially folded protein in water
competed with membrane insertion. By centrifugation, aggregated OmpA
could indeed be precipitated. ()At pH 10, refolding and
membrane insertion was almost quantitative, and the yield decreased
only slightly with increasing incubation time. This implies a reduced
tendency to aggregate which presumably is caused by an increased charge
of the protein at this pH.
The effect of lipid concentration on the yield is shown in Fig. 3B. The yield increased with lipid concentration and saturated above about 4 mM DMPC, corresponding to a molar lipid/protein ratio of roughly 1000 or a molar vesicle/protein ratio of about 1/10.
In Fig. 3C, the
pH dependence of the yield is shown for the range from pH 2.5 to 11.5.
At pH 10, the yield was optimal with 98%. At higher pH, the yield
decreased, presumably due to the drastically increased charge of the
protein which interferes with folding. Decreasing the pH from 10 to
5.7, the theoretical isoelectric point of OmpA, led to a reduced yield,
probably caused by a stronger tendency to aggregate. Below
the isoelectric point, virtually no membrane insertion was observed.
Fig. 4A shows the mean residue ellipticity at 206 nm over
a time range of 3 h after the start of folding and insertion. At least
three phases can be distinguished. The initial value (-7.5
10
degrees cm
dmol
) is already characteristic for partially
folded OmpA in water (Fig. 2); hence, the fast transition from
the unfolded state in urea to the partially folded state in water
occurred within the mixing time of the experiment (about 1 s). The
subsequent process of folding from the partially folded state in water
to the completely folded and membrane-inserted state is slow and at
least biphasic, the half-times being about 5 min for the moderately
slow step and about 40 min for the slow step, with amplitudes of 75%
and 25%, respectively.
Figure 4: Kinetics of folding and membrane insertion of 2.4 µM OmpA into vesicles of 0.75 mM (A, B) or 1.5 mM (C) DMPC/DMPG at pH 7.3 as observed by CD at 206 nm (A), Trp fluorescence at 330 nm (B), and protease digestion (C). In B, a logarithmic plot of the Trp fluorescence is shown as an inset. In C, Trp fluorescence at the higher lipid concentration is included for comparison and the SDS-gel is shown as an inset.
The time course of the Trp fluorescence shown in Fig. 4B is also at least biphasic with half-times of 4 min and 35 min and amplitudes of 73% and 27%, respectively. This demonstrates that CD at 206 nm and Trp fluorescence detect the same transitions.
The time course of the yield of insertion as determined by protease digestion is shown in Fig. 4C. Because the lipid concentration in this experiment was twice as high as in the CD and fluorescence experiments (Fig. 4, A and B), the time course of fluorescence at this lipid concentration is included. Both time courses are biphasic with roughly equal half-times of 2 min and 30 min, but different amplitudes. The amplitude of the fast phase is only 25% for the yield of insertion, compared to 80% for the fluorescence intensity. This would indicate that upon contact with membranes, in a first step all OmpA molecules adopt secondary structure and a small fraction inserts into the membrane, while in the second step the major part inserts and all fold into the final structure. However, it is also possible that in the first step actually no molecules insert, but a fraction of them inserts during cooling through the lipid phase transition (which is done to stop insertion).
The slowest step observed can probably not be attributed to proline isomerization of part of the OmpA molecules, because it was not accelerated by a peptidyl-prolyl-isomerase added in equimolar amounts (data not shown). Likewise, any effect of the periplasmic part of OmpA on folding and membrane insertion is negligible, because the purified tryptic fragment exhibited the same folding behavior (data not shown). Finally, the possibility that insertion is slow due to a coupling of protein insertion to vesicle fusion must be rejected, because fusion was shown to proceed still slower (data not shown)(14) .
The velocity of insertion increased with lipid concentration as shown in Fig. 5A, the half-times of the fluorescence increase being inversely proportional to the lipid concentration. Increasing the pH from 7.3 to 10.0 decreased the velocity of insertion, as shown also in Fig. 5A. At pH 10, the half-times are 5 times larger than at pH 7.3. The effect of membrane curvature is demonstrated in Fig. 5B. Insertion into unsonicated vesicles of low curvature was extremely slow compared to insertion into sonicated lipid vesicles of high curvature. The fastest kinetics were observed for folding into detergent micelles of dodecyl maltoside. Essentially the same behavior was obtained with micelles of octyl glucoside (data not shown). The moderately slow step of insertion is accelerated compared to insertion into vesicle membranes, its half-time decreasing from 5 min to 20 s, but the slow step proceeds with roughly the same kinetics as for insertion into vesicle membranes. The observed increase in fluorescence is not caused simply by binding of detergent molecules to the partially folded state of OmpA, but reflects structural changes of the protein as indicated by CD measurements(7) . The faster folding and insertion of OmpA into micelles results either from the higher curvature of the micelles or from the formation of a new micelle around the folding protein.
Figure 5:
Influence of different parameters on the
kinetics of folding and membrane insertion of 0.6 µM OmpA
as observed by Trp fluorescence. A, insertion into vesicles of
DMPC at a lipid concentration of 1.5 or 4.5 mM and pH 7.3
(-- and ) or pH 10.0 (-); B, insertion at pH 10.0 into sonicated or not sonicated
vesicles of DMPC at 4.5 mM and into micelles of dodecyl
maltoside at 1 mM. As control, the constant fluorescence of
partially folded OmpA in water at pH 10.0 is included, indicating that
OmpA does not aggregate at this pH.
Folding and membrane insertion of OmpA was found to proceed
in at least three steps. In water, OmpA undergoes a transition from the
unfolded state U into an intermediate state I
with a characteristic time below a second. Upon association with
membranes, two slower transitions take place with characteristic times
of about 5 min and h leading to the folded and inserted state
F
. These results will now be interpreted within the
framework of a kinetic model for folding and membrane insertion of OmpA (Fig. 6).
Figure 6:
Scheme of the proposed folding pathway of
OmpA. U, unfolded in water/urea; I
, misfolded in water; A,
aggregated in water; I
, partially folded
and inserted in membrane; F
, fully folded
and inserted in membrane.
The process of partial folding in water is
reminiscent of the first step in folding of soluble proteins. They
often adopt a partially folded state within ms. The state is called
molten globule and believed to arise from a hydrophobic collapse in
which the hydrophobic core of soluble proteins forms, but not their
detailed structure(15) . Postulating such a hydrophobic
collapse to take place in the case of OmpA, it should be fast as indeed
observed, but the state I would not be a partially
correctly folded state, but a completely misfolded state, something
like an inside-out version of native OmpA.
Guided again by the
analogy to soluble proteins(16) , from state I two
pathways seem possible. The protein may associate with membranes or it
may aggregate, and both processes are indeed observed. Both are driven
by the hydrophobic effect. Aggregation may be suppressed by increasing
the repulsion between OmpA molecules (e.g. due to charges
and/or loss of structure with exposed hydrophobic patches) as well as
by increasing the interaction with membranes (e.g. by
increasing the lipid concentration or the curvature of the vesicle
membranes). Under optimal conditions, aggregation is negligible, and
the yield of folding and insertion is virtually quantitative.
The
two transitions which occur upon association with membranes are
relatively slow. Which structural changes might they reflect or, in
other words, what is the nature of the intermediate I?
Folding of soluble proteins is sometimes rate-limited by proline
isomerization (1) or by pairing of domains (17) . Both
possibilities could be excluded for OmpA, as was the case with fusion
of vesicles. We therefore propose a model for folding of OmpA which
closely resembles the standard model for folding of soluble proteins.
Upon contact with a membrane, OmpA starts to insert and to fold into a
state I
whose structure is globally correct, but in detail
incorrect, i.e. a barrel-like structure is formed, but the
details remain wrong. By this structural characterization, I
would be the analogue of the molten globule state of soluble
proteins (and not the misfolded state I
in water).
Furthermore, I
would resemble the intermediate state in
folding of
-helical membrane proteins proposed within the
framework of a two-state model(18) . A vaguely related
molten-globule state has been postulated for the pore-forming domain of
colicin(19) . Because the membrane can be considered as a
reactant, the rate of the transition from I
to
I
should be proportional to the lipid concentration, as
observed for an amphipathic helix(20) . The final transition
from I
to F
would require fine-tuning of the
protein structure and should be the slowest step.
Comparing the
predictions of the model with the experimental data, the rate of the
moderately slow transition from I to I
was
indeed proportional to the lipid concentration and was different for
insertion into vesicle membranes and into micelles. As expected,
insertion into micelles was faster. By contrast, the slow transition
from I
to F
proceeded with the same kinetics
for membranes and micelles. This again would be expected, because
fine-tuning of the structure should be roughly independent of the
environment. To which extent OmpA is inserted in the membrane in state
I
may be estimated from the data on protease digestion.
Unfortunately, these data are not completely conclusive, but indicate
that at least the major part of OmpA molecules became protected with
the half-time of the slow transition from I
to
F
. Hence, in I
, the major part if not all of
the OmpA molecules are associated with the membrane but not inserted.
They insert upon folding into the final state F
.
Concerning folding in vivo, OmpA faces two kinetic problems after being translocated across the inner bacterial membrane. It is prone to aggregation(13) , and its interaction with the outer bacterial membrane, which has negligible curvature, is extremely slow. Periplasmic chaperones and catalysts for sorting to and insertion into the outer membrane may help to overcome these problems. Our approach should be useful to elucidate in vitro the effect of periplasmic chaperones and catalysts on folding and sorting of OmpA.