Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7457-7461, August 1992 Biophysics
Refolding and oriented insertion of a membrane protein into a lipid bilayer (unfoldlng/renaturatIon/reconstition/OmpA)
THOMAS SURREY AND FRITZ JAHNIG Max-Planck-Institut for Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, D-7400 Tibingen, Federal Republic of Germany
Communicated by Harden M. McConnell, April 27, 1992
We have studied the refolding and membrane ABSTRACT insertion of the outer membrane protein OmpA of Escherichia coli. The protein was extracted from its native membrane by sonication in the presence ofurea and dissolved in the urea/water miture in unfolded form. In this form it was purified. Upon addition of preformed lipid vesicles, the protein spontaneously refolded and inserted into the vesicle membranes. The vesicles had to be small and the lipids had to be in the fluid state. The insertion occurred in an oriented manner.
OUTER SPACE
LPS PL
In contrast to soluble proteins, membrane proteins are stable only in the presence of an amphipathic environment such as a lipid membrane. Therefore, folding of a membrane protein after its synthesis within the cell can occur only in combination with membrane insertion if no other factors provide the amphipathic environment. Up to now, folding and insertion ofmembrane proteins have usually been studied in vitro as separate processes. The proteins were first unfolded in appropriate solvents and refolded in detergent micelles providing the amphipathic environment, and in a second step they were inserted into membranes (1-3). For the second step, essentially two different ways exist, and they are well known from the reconstitution of membrane proteins without unfolding. In conventional reconstitution, detergent-solubilized lipid is added to the detergentsolubilized protein and the detergent is removed, leading to the formation of lipid bilayers with incorporated protein (4). An alternative method is spontaneous insertion of detergentsolubilized protein into preformed lipid bilayers (5, 6). Combined refolding and insertion might have occurred when a porin in a "water-soluble" form, which presumably is unfolded, spontaneously inserted into vesicle membranes (7). However, detergent had to be added together with the lipid vesicles to induce insertion, and the detergent might have induced refolding before insertion. In this case, the two processes would have again occurred separately. A similar situation was found with a porin secreted by spheroplasts (8). We have studied the combined refolding and insertion of a protein into preformed vesicle membranes without the involvement of any detergent. As a prerequisite, the protein must be solubilized in the unfolded form. This is possible for a class of membrane proteins composed of (-strands and presumably forming (-barrels (9, 10). The amphipathic nature of the /3-strands results in a weak average hydrophobicity, often weaker than that of soluble proteins (10). Such membrane proteins occur, for instance, in the outer membrane of Escherichia coli. By contrast, the proteins of the inner membrane are composed of membrane-spanning a-helices and are strongly hydrophobic, hence exhibiting a strong tendency for aggregation. One of the outer membrane proteins is OmpA, which we have chosen for our studies.
N
PERI PLASM
OmpA
C
FIG. 1. Model of OmpA, showing the membrane topology and sites of cleavage by trypsin and Glu-C endoproteinase. LPS, lipopolysaccharide; PL, phospholipid.
OmpA has been used extensively to investigate protein export across the bacterial inner membrane (11-13). However, little is known about its mode of insertion into the outer membrane, which is true for P-structured membrane proteins in general. When OmpA is unfolded by boiling in SDS (3) or by dissolving in a urea/water mixture (14), it can be refolded in detergent micelles (3, 14). The protein consists of 325 amino acid residues and in its native state is a monomer. The N-terminal half constitutes the membrane part, while the C-terminal half resides in the periplasm. The membrane part has a high percentage of antiparallel p-structure and presumably forms a p-barrel of eight antiparallel p-strands (10) with long loops protruding into the extracellular space (Fig. 1). Together with LPS, these loops mediate OmpA's phage receptor activity (15). A catalytic activity of OmpA is not known. As experimental techniques for our studies, we used SDS/ gel electrophoresis, protease digestion, circular dichroism (CD), and tryptophan fluorescence.
MATERIALS AND METHODS Purification and Unfolding. OmpA was overexpressed in E. coli strain UH203 carrying pRD87-P1 (16). Cells were first grown in 5 liters of NBA (1% Bactotryptone/0.5% yeast extract/1% NaCl) containing 0.5% glucose and 50 Mg of ampicillin per ml at 370C. To remove glucose, they were washed at OD550 = 1.3 with NBA and were then induced at
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Abbreviations: DMPC, dimyristoyl phosphatidylcholine; LPS, lipopolysaccharide; OG, n-octyl f3D-glucopyranoside. 7457
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370C in 20 liters of NBA containing 1 mM isopropyl f-Dthiogalactoside and 50 ,g of ampicillin per ml. Because OmpA overproduction is toxic, these cells were harvested at OD550 = 0.9. Cell membranes were prepared as previously described (17). For preextraction, cell envelopes from a 20-liter culture were resuspended in 120 ml of 4 M urea/20 mM Tris HCl, pH 9/0.05% 2-mercaptoethanol, incubated at 500C for 30 min, and centrifuged at 150,000 x g for 2 hr (Fig. 2). The pelleted membranes were resuspended in 45 ml of 4 M urea/20 mM Tris HCl, pH 9/0.1% 2-mercaptoethanol, and sonicated for 30 min with a Branson sonifier (macrotip, 10°C) to unfold and solubilize OmpA in urea. The sonication time may be reduced by working at 8 M urea. OmpA then was separated from lipid vesicles by centrifugation (4 hr, 150,000 x g). The OmpA-containing supernatant was further purified by anion-exchange chromatography (Q Sepharose Fast Flow, Pharmacia) in 4 M urea/20 mM Tris HCl, pH 9/0.05% 2-mercaptoethanol, using a linear NaCl gradient (0-200 mM) for elution of OmpA. The purity was about 98% as determined by SDS/gel electrophoresis and subsequent scanning of the gel. Purified OmpA was analyzed for LPS contamination according to ref. 18, and no detectable amounts of LPS were found. Protein concentrations were determined by using Pierce BCA protein assay reagents (2-mercaptoethanol had to be removed prior to the reaction). The yield was approximately 15 mg of OmpA, starting from 25 g of wet cells. The purified OmpA was concentrated in 4 M urea/20 mM potassium phosphate (KPO), pH 7.3, by ultrafiltration (Amicon, PM-10) and stored at -20°C. This stock solution was 5 mg/ml in OmpA. For refolding experiments, it was diluted to 100 j&g/ml, if not stated otherwise. The pH was always kept at 7.3 by 20 mM KP1. The 24-kDa tryptic fragment of OmpA was purified as described above, except that cell envelopes were digested with trypsin at 50 ug/ml at room temperature for 90 min before preextraction. Conventional Reconstitution. For refolding in detergent, unfolded OmpA (5 mg/ml) was diluted at least 50-fold into n-octyl 3-Di-glucopyranoside (OG; Sigma) at 10 mg/ml in 20 mM KP1, pH 7.3, resulting in a molar ratio of OmpA to OG of about 1:12,500. Reconstitution of OmpA into vesicles of dimyristoyl phosphatidylcholine (DMPC) (Avanti Polar Lipids) was achieved by dialyzing 3 ml containing OmpA at 100 j&g/ml, OG at 20 mg/ml, and DMPC at 1 mg/ml in 20 mM KPj, pH 7.3, against
FIG. 2. Purification strategy.
Proc. Nad. Acad Sci. USA 89 (1992) 20 mM KPi, pH 7.3/0.02% NaN3 at 370C for 24 hr. The molar ratio of OmpA to DMPC was about 1:500. For reconstitution of OmpA into mixed vesicles composed of LPS and E. coli lipid, 3 mg of E. coli lipid (gift of H. Kiefer from this institute) and 30 mg of OG were dissolved in hexane. After the solvent had been evaporated under a stream of nitrogen and the resulting film had been dried in vacuum for 1 hr, 3 mg of LPS (gift of U. Henning from this institute) dissolved in OG at 10 mg/ml and unfolded OmpA were added. This suspension was brought to 3 ml and made 100 .ug/ml OmpA, 20 mg/ml OG, 20 mM KP, at pH 7.3,5 mM MgCl2. It was sonicated in a water bath for 1 min. Dialysis of the clear solution against 20 mM KP,, pH 7.3/5 mM MgCI2/ 0.02% NaN3 at 30TC for 24 hr yielded OmpA reconstituted in mixed vesicles of LPS/E. coli lipid. Oriented Membrane Insertion. For preparation of lipid vesicles, 10 mg of DMPC was dissolved in 0.2 ml of CHC13. After the solvent had been evaporated and the film had been dried in vacuum for 1 hr, 2 ml of 20 mM KP1, pH 7.3, was added. Sonication of the suspension at 30TC for 30 mm (Branson sonifier, standard tip) yielded small unulamellar vesicles which were kept at 30TC overnight. Prior to insertion experiments, the vesicles were diluted to give DMPC at 1 mg/ml. This product will be called "small preformed vesicles" in the following. Their size has been characterized by quasielastic light scattering and electron microscopy; the diameter is 30-50 nm. HPLC showed 0.5% degradation of DMPC to myristate and lysophosphatidylcholine. Membrane insertion was started by adding a small volume of unfolded OmpA to preformed vesicles at 30TC, thus diluting OmpA to 100 pug/ml (or 25 pg/ml for fluorescence experiments), corresponding to a molar ratio of OmpA to DMPC of about 1:500 (or 1:2000). Samples were analyzed after 40 min. SDS/Gel Electrophoresis and Protease Digestion. SDS/gel electrophoresis was performed according to ref. 19, using 12.5% polyacrylamide gels. Routinely, the samples were not boiled. Typically, 2 .ug ofprotein was applied to one lane. The gels were stained with Coomassie blue. On our gels, unfolded OmpA migrated with an apparent molecular mass of 35 kDa, in contrast to 30 kDa for folded OmpA. In the cited literature, these values vary from 33 to 36 kDa and from 28 to 31 kDa, respectively. Trypsin digestion was employed to determine the extent of refolding and membrane incorporation of OmpA, since unfolded urea-dissolved OmpA is known to be completely digested by trypsin, and refolded membrane-incorporated OmpA is digested only down to a membrane-protected fragment (14). Samples containing OmpA at 100 pg/ml were incubated with trypsin at 10 pg/ml at 150C for 10 hr. Digestion was stopped by addition of 0.1 mM No-p-tosyl-L-lysine chloromethyl ketone. The samples were analyzed by SDS/ gel electrophoresis with subsequent scanning of the gels with an Ultroscan XL (LKB). This permitted us to determine the ratio of membrane-protected fragment of OmpA to OmpA added initially and, thus, the extent of refolding and membrane incorporation. In this calculation the reduced staining intensity of the membrane-protected fragment due to its smaller size had to be taken into account. When the purified tryptic fragment of OmpA was subjected to the same analysis, the samples were incubated with Glu-C endoproteinase (Boehringer Mannheim; EC 3.4.21.19) at 20 pg/ml at 300C for 2 hr. Spectroscopy. OmpA contains five Trp residues, which are all buried in the membrane according to the P-barrel model. Tryptophan fluorescence spectra were recorded with a Perkin-Elmer MPF3 fluorimeter. The excitation wavelength was 296 nm. Circular dichroism spectra were recorded on a Jasco IS00A. All spectra were corrected for light scattering by
FL - _ -35kfOG
Biophysics: Surrey and Rihnig
Proc. Natl. Acad. Sci. USA 89 (1992)
r--_ _ * A-~~~~~~35 k Da imam so
1
2
3
5
4
-3
I ,a X
45
/
^2 6 -\
7
6
'
_
-30 klo
FIG. 3. SDS/gel electrophoresis of preextracted cell membranes during sonication in 8 M urea. Samples were taken before (lane 1) and 30 sec, 90 sec, 3 min, 5 min, 10 min, and 20 miin after (lanes 2-7) start of sonication.
subtracting spectra of samples lacking OmpA. A correction for temperature effects was performed when necessary. Phage Inactivation. OmpA reconstituted in mixed vesicles of LPS/E. coli lipid was tested for biological activity by a phage inactivation test (14, 20), using OmpA-specific bacteriophage K3 and E. coli JM109 (21). Samples (200 gl) of vesicles containing OmpA at 100 pg/ml as well as a control sample without OmpA were incubated with 1000 plaqueforming units of K3 at 370C for 30 min. Subsequently, the suspension was plated out with E. coli JM109, and after incubation for 6 hr at 370C the plaques were counted. RESULTS Unfolding and Conventional Reconstitution. As shown previously, OmpA is soluble in urea (14). Membraneincorporated OmpA, however, could not be solubilized by mere addition of urea. This permitted the preextraction of cell envelopes during purification, whereby mainly peripheral membrane proteins were removed (Fig. 2). When, however, the membrane fraction was sonicated vigorously in the presence of 4 M urea, OmpA and other outer membrane proteins were released from the membrane and dissolved in the urea/water mixture. In this form, OmpA was purified by anion-exchange chromatography. As expected, solubilized OmpA was unfolded. This was demonstrated by a shift in the apparent molecular mass in
7459
A
~//
-7~~~~~~
-1 ? -3 -5
--
200
210
220 230 WAVELENGTH (nm)
240
250
FIG. 5. CD spectra of OmpA (A) and of the 24-kDa fragment of OmpA (B) under various conditions. ., OmpA or fragment unfolded in 8 M urea; -, OmpA or fragment unfolded in 4 M urea;-, OmpA or fragment aggregated in buffer; --, OmpA or fragment refolded in OG at 10 mg/ml; -, OmpA or fragment refolded and inserted spontaneously into small preformed vesicles at 30TC or adsorbed to small preformed DMPC vesicles at 150C.
SDS/gels from 30 kDa, typical for native OmpA, to 35 kDa (Fig. 3 and Fig. 4, lane 1) (3, 14). In addition, unfolded OmpA was completely degraded by trypsin (Fig. 4, lane 6); its CD spectrum indicated a high degree of random-coil structure (Fig. 5A); and its tryptophan fluorescence was relatively weak, with a maximum at 348 nm (Fig. 6), characteristic for Trp residues in a polar environment. When the urea concentration was increased to 8 M, the fluorescence remained unchanged, while the CD spectrum was slightly altered indicating a completely random structure. When the urea concentration was decreased to 80 mM by dilution, OmpA aggregated as judged by ultracentrifugation. Aggregated OmpA could be pelleted within 90 min, in contrast
cF 10o ..
..
..........-...: ..N: .
9-
..
35 kDa -30 kDa
-
M>
24 k Da D CI-
1
2
3
4.
5
6
7
8
9
5
10 11
FIG. 4. SDS/gel electrophoresis of OmpA under various conditions. Lane 1, unfolded OmpA in 4 M urea; lane 2, OmpA refolded in OG; lane 3, OmpA refolded in OG and reconstituted in DMPC vesicles by dialysis at 300C; lane 4, OmpA refolded and inserted spontaneously into small preformed DMPC vesicles at 300C; lane 5, OmpA partially refolded and adsorbed to small preformed DMPC vesicles at 15'C; lane 6, OmpA treated as in lane 1 and digested by trypsin; lane 7, OmpA treated as in lane 4 and digested by trypsin, yielding the 24-kDa fragment; lane 8, OmpA treated as in lane 5 and digested by trypsin; lane 9, trypsin; lane 10, the 24-kDa fragment of OmpA refolded and inserted spontaneously into small preformed DMPC vesicles at 30TC and digested by Glu-C endoproteinase; lane 11, Glu-C endoproteinase.
U,
en C-
0 =>
300
340 360 320 WAVELENGTH (nm)
380
FIG. 6. Tryptophan fluorescence spectra of OmpA under different conditions. - -, OmpA unfolded in 4 M urea; - .. -, OmpA
refolded in OG; -, OmpA refolded and inserted spontaneously into small preformed DMPC vesicles at 30'C; -, OmpA partially refolded and adsorbed to small DMPC vesicles at 150C.
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to solubilized unfolded OmpA. The apparent molecular mass of aggregated OmpA was still 35 kDa. The CD spectrum showed a mixture of a-helix, 3-strand, and random-coil structure (Fig. 5A), and the fluorescence spectrum was slightly blue-shifted compared with unfolded OmpA (data not shown). When the urea concentration was decreased to 80 mM by dilution into a solution of the detergent OG, OmpA refolded, as indicated by the appearance of the apparent molecular mass of 30 kDa characteristic for native OmpA (Fig. 4, lane 2). Refolding, however, was not complete, since 20% of OmpA retained the apparent molecular mass of 35 kDa. This is in agreement with trypsin digestion experiments: 80o of the OmpA molecules were digested down to a fragment of apparent molecular mass of 24 kDa, characteristic for the membrane part of OmpA. The remaining 20%o of OmpA molecules were degraded down to much smaller fragments. The CD spectrum showed a high degree of (-structure (Fig. SA), and the tryptophan fluorescence was relatively strong with a maximum at 330 nm, indicating a hydrophobic environment (Fig. 6). When detergent-solubilized OmpA was reconstituted into vesicle membranes of DMPC by detergent dialysis, the apparent molecular mass of 30 kDa was preserved (Fig. 4, lane 3), hence, OmpA remained folded. As shown previously by Raman and fluorescence spectroscopy, OmpA in DMPC membranes has a high (-strand content of about 60%o and the Trp residues are in a strongly hydrophobic environment (3, 10). To demonstrate that reconstituted OmpA has refolded into its native structure, we tested its function as a phage receptor (14). For this purpose, reconstitution into vesicle membranes containing LPS is necessary, since the phages require LPS for attachment. Indeed, the LPS-containing vesicles did function as phage receptors, as shown by the reduction of the number of plaques in the phage inactivation test from 1080 (vesicles without OmpA) to 45. This ratio is of the expected order of magnitude (20). As shown previously, trypsin digestion of OmpA reconstituted in lipid vesicles yields a 24-kDa fragment representing the membrane part of OmpA (14). However, only about half of the reconstituted OmpA molecules are digested, indicating random orientation of the protein molecules in the vesicle membranes (data not shown) (3). Oriented Insertion Into Preformed Vesicles. When the urea concentration was decreased to 80 mM by dilution of unfolded OmpA into a dispersion of small preformed DMPC vesicles at 30TC, OmpA refolded and inserted into the vesicle membranes. Refolding was demonstrated by the reduction of the apparent molecular mass to 30 kDa (Fig. 4, lane 4). Not all OmpA molecules folded back; a fraction retained the apparent molecular mass of 35 kDa, even after prolonged incubation, and another fraction appeared as oligomers. The CD spectrum indicated a high degree of (3-structure (Fig. 5A), and the tryptophan fluorescence was relatively strong with a maximum at 330 nm (Fig. 6). The time course of the fluorescence at 330 nm revealed a half-time for insertion of about 10 min. Trypsin digestion of spontaneously inserted OmpA yielded the 24-kDa membrane fragment (Fig. 4, lane 7). This indicates insertion of OmpA into the vesicle membranes. Quantitative analysis of the gels by scanning reveals that 40-50%o of the OmpA molecules inserted into the membrane. The trypsin digestion experiment also provides information on the orientation of OmpA within the vesicle membranes. Because all OmpA molecules were degraded, either down to the membrane fragment or down to much smaller fragments, all inserted OmpA molecules were oriented with their peripheral part outside the vesicles. Analyzed quantitatively, more than 99%o of the inserted OmpA molecules were oriented vectorially. This contrasts with the result of trypsin digestion of conventionally reconstituted OmpA (see
above).
Proc. Nad. Acad. Sci. USA 89 (1992)
No membrane insertion of OmpA, but aggregation, was observed, when large preformed vesicles produced by several freeze-thaw cycles were used for insertion experiments (data not shown). Preparation of large vesicles with OG added below the critical micelle concentration again permitted OmpA to spontaneously refold and insert. On the other hand, aggregated OmpA was not able to refold and insert into small preformed vesicles. When unfolded OmpA was diluted into a dispersion of small preformed vesicles of DMPC at 150C-i.e., below the lipid phase transition at 240C-a different behavior was observed. CD spectroscopy indicated the same content of (3-structure as for membrane-incorporated OmpA (Fig. SA), and fluorescence spectroscopy revealed again a hydrophobic environment of the Trp residues (Fig. 6). However, the apparent molecular mass remained at 35 kDa, and OmpA was degraded completely by trypsin (Fig. 4, lanes 5 and 8). Furthermore, it could be unfolded again by increasing the urea concentration to 4 M, in contrast to the case ofinsertion into fluid membranes at 300C (data not shown). These results indicate that below the lipid phase transition, OmpA is in a state with high (3-strand content, adsorbed on the membrane surface but not incorporated in the ordered lipid bilayer. When the temperature was slowly raised to 300C, OmpA refolded and inserted into the membrane as if added at 30"C. Refolding and Membrane Inerto of the Trypic Fragent of OmpA. The 24-kDa fragment of OmpA was prepared by trypsin digestion of cell membranes, followed by the same purification procedure as for complete OmpA. The fragment also dissolved and unfolded in the urea/water mixture. For 8 M urea, the CD spectrum (Fig. SB) and the fluorescence spectrum (data not shown) coincided with the spectra of the complete OmpA molecule. For 4 M urea, the spectra of the fragment remained unaltered, while complete OmpA adopted some secondary structure (see above). This partial refolding can now be attributed to the peripheral part of OmpA. Like complete OmpA, the fragment spontaneously refolded and inserted into small preformed vesicle membranes. The refolding efficiency was 50%6. Unidirectional orientation after insertion was demonstrated by using Glu-C endoproteinase, which removed the small remainder of the periplasmic part left after trypsin digestion and reduced the apparent molecular mass of all fragment molecules from 24 to 21 kDa (Fig. 4, lane 9). This demonstrated that the periplasmic part is not required for oriented insertion of OmpA into vesicle membranes.
DISCUSSION The integral membrane protein OmpA can exist in at least five different conformations, depending on the environment. They are summarized in Fig. 7, together with the observed transitions between them. OmpA could be extracted from its native membrane by sonication in the presence of urea. In the urea/water mixture, it unfolds and thus becomes soluble. This is possible due to the low hydrophobicity of OmpA, which is typical for /3-structured membrane proteins (10). Strongly hydrophobic proteins such as the a-helical membrane proteins will presumably not become soluble under the same conditions. When urea is removed from the solution, OmpA aggregates. The conformation of aggregated OmpA is a mixture of a-helix, (-strand, and random-coil structure. Upon removal of urea in the presence of detergent, unfolded OmpA spontaneously refolds, forming mixed micelles. In a second step, it can be reconstituted into lipid vesicle membranes by removing the detergent. Similar results have been obtained upon refolding SDS-denatured OmpA (3), SDS- and guanidnium hydrochloride-denatured porin (2), and acid-denatured bacteriorhodopsin (1). These results indicate that the complete information for the threedimensional structure of a membrane protein is encoded in its
Biophysics: Surrey and Jdhnig
FIG. 7. Summary of the observed conformations of OmpA and of the transitions between them.
amino acid sequence, as generally accepted for soluble proteins (22, 23). However, while water is required for a soluble protein to fold into the native structure, an amphiphilic environment is required for a membrane protein to fold properly. Our main interest was not detergent-mediated refolding and reconstitution of a membrane protein, but rather the combined refolding and membrane insertion in the absence of any detergent. Unfolded OmpA indeed refolds and inserts spontaneously into lipid bilayers when vesicles of DMPC are offered and urea is removed. However, several conditions have to be fulfilled. (i) The lipid membrane must be in its fluid state. Below the lipid phase transition temperature, OmpA still adopts a high degree of (-strand structure, but it adsorbs onto the membrane without inserting into it. When subsequently the temperature is raised above the phase transition, OmpA inserts into the membrane in the correct structure. (ii) The lipid bilayer has to be highly curved, as in the case of small vesicles. There is no adsorption onto or insertion into large vesicles. As well known, the lipid packing in small vesicles is not optimal, and this kind of defect obviously facilitates the adsorption and insertion of proteins. For large vesicles, defects have to be introduced-e.g., by adding small amounts of detergent. The importance of defects for insertion of detergent-solubilized proteins into lipid vesicles has been emphasized previously (5). (iii) Aggregation of OmpA must be suppressed. Upon removal of urea, OmpA either may refold and insert into vesicles or may aggregate. The relative weight of the two possibilities depends on several factors, among others the concentrations of OmpA and lipid. Strongly hydrophobic membrane proteins such as the a-helical proteins will presumably not insert into membranes as easily as OmpA, because of their stronger tendency for aggregation. The insertion of OmpA into membranes occurred in an oriented manner, with the peripheral part remaining outside the vesicles. This oriented insertion is a difference between our method and conventional reconstitution, which usually leads to random orientation (4). The same oriented insertion was also found for the membrane part of OmpA alone, demonstrating that the peripheral part plays no role in the insertion process. Probably the shape of the membrane part of OmpA also is not responsible for the oriented insertion, since conventional reconstitution leads to a random orientation. Thus, the reason for the oriented insertion seems to be
Proc. Natl. Acad. Sci. USA 89 (1992)
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intimately related to the mechanism by which OmpA inserts into the membrane. One possibility for this mechanism is offered by the partially refolded and adsorbed state of OmpA that occurs below the lipid phase transition. It may represent a folding and insertion intermediate of OmpA (Fig. 7). This state may be viewed as a planar, amphiphilic (3-sheet lying flat on the membrane surface with its hydrophobic side contacting the hydrophobic core of the membrane. In a second step, the (-sheet would roll in and the putative (3-barrel would be formed and insert into the membrane. This mechanism of combined refolding and insertion would differ strongly from the mechanism proposed for the insertion of a-helical membrane proteins, which is based on helical hairpins (24, 25). What can be deduced from these results for the mechanism of membrane insertion of proteins in vivo? As demonstrated, a membrane protein on its own can under certain conditions insert into a lipid bilayer in a definite orientation. Cell membranes are fluid, but not highly curved. Thus, local defects facilitating the contact between the protein and the hydrophobic core of the membrane may be required for insertion into cell membranes. These defects might be induced by other proteins, giving them the opportunity to regulate insertion. Finally, proteins must be shielded from aggregation prior to insertion, which is especially important for exported proteins such as OmpA. This might be the task of chaperones (26). We thank U. Henning and H. Kiefer (this institute) for the gifts of LPS and E. coli lipid. The cooperations with M. Lu (this institute) on the phage test and with H. Rau (University of Stuttgart, Hohenheim) on the CD measurements are gratefully acknowledged. We also thank H.-J. Eibl and I. Kotting (Max-Planck-Institute for Biophysical Chemistry, Gottingen) for the test of lipid purity by HPLC. 1. Huang, K.-S., Bayley, H., Liao, M. J., London, E. & Khorana, H. G. (1981) J. Biol. Chem. 256, 3802-3809. 2. Eisele, J.-L. & Rosenbusch, J. P. (1990) J. Biol. Chem. 265, 10217-10220. 3. Dornmair, K., Kiefer, H. & Jahnig, F. (1990) J. Biol. Chem. 265, 18907-18911. 4. Silvius, J. R. & Allen, T. A. (1989) Biophys. J. 55, 207-208. 5. Scotto, A. W. & Zakim, D. (1988) J. Biol. Chem. 263,18500-18506. 6. Scotto, A. W. & Zakim, D. (1990) Biochemistry 29, 7244-7251. 7. Pfaller, R., Freitag, H., Harmey, A., Benz, R. & Neupert, W. (1985) J. Biol. Chem. 260, 8188-8193. 8. Sen, K. & Nikaido, H. (1990) Proc. Nati. Acad. Sci. USA 87, 743-747. 9. Weiss, M. S., Kreusch, A., Schiltz, E., Nestel, U., Welte, W., Weckesser, J. & Schulz, G. E. (1991) FEBS Lett. 280, 379-382. 10. Vogel, H. & Jahnig, F. (1986) J. Mol. Biol. 190, 191-199. 11. Freudl, R., Schwarz, H., Stierhof, Y.-D., Gamon, K., Hindenach, I. & Henning, U. (1986) J. Biol. Chem. 261, 11355-11361. 12. Tani, K., Shiozuka, K., Tokuda, H. & Mizushima, S. (1989) J. Biol. Chem. 264, 18582-18588. 13. Driessen, A. J. M. & Wickner, W. (1991) Proc. NatI. Acad. Sci. USA 88, 2471-2475. 14. Schweizer, M., Hindenach, I., Garten, W. & Henning, U. (1978) Eur. J. Biochem. 82, 211-217. 15. Morona, R., Klose, M. & Henning, U. (1984) J. Bacteriol. 159, 570-578. 16. Freudl, R., MacIntyre, S., Degen, M. & Henning, U. (1988) J. Biol. Chem. 263, 344-349. 17. Teather, R. M., Bramhall, J., Riede, I., Wright, J. K., Furst, M., Aichele, G., Wilhelm, U. & Overath, P. (1980) Eur. J. Biochem. 108, 224-231. 18. Tsai, C.-M. & Frasch, C. E. (1982) Anal. Biochem. 119, 115-119. 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 20. Van Alphen, L., Havekes, L. & Lugtenberg, B. (1977) FEBS Lett. 75, 285-290. 21. Messing, J., Crea, R. & Seeburg, P. H. (1981) Nucleic Acids Res. 9, 309-321. 22. Dill, K. (1990) Biochemistry 29, 7133-7155. 23. Jaenicke, R. (1991) Biochemistry 30, 3147-3161. 24. Engelman, D. M. & Steitz, T. A. (1981) Cell 23, 411-422. 25. Popot, J.-L. & Engelman, D. M. (1990) Biochemistry 29, 4031-4037. 26. Lecker, S. H., Driessen, A. J. M. & Wickner, W. (1990) EMBO J. 9, 2309-2314.
Refolding and oriented insertion of a membrane protein into a lipid bilayer (unfoldlng/renaturatIon/reconstition/OmpA)
THOMAS SURREY AND FRITZ JAHNIG Max-Planck-Institut for Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, D-7400 Tibingen, Federal Republic of Germany
Communicated by Harden M. McConnell, April 27, 1992
We have studied the refolding and membrane ABSTRACT insertion of the outer membrane protein OmpA of Escherichia coli. The protein was extracted from its native membrane by sonication in the presence ofurea and dissolved in the urea/water miture in unfolded form. In this form it was purified. Upon addition of preformed lipid vesicles, the protein spontaneously refolded and inserted into the vesicle membranes. The vesicles had to be small and the lipids had to be in the fluid state. The insertion occurred in an oriented manner.
OUTER SPACE
LPS PL
In contrast to soluble proteins, membrane proteins are stable only in the presence of an amphipathic environment such as a lipid membrane. Therefore, folding of a membrane protein after its synthesis within the cell can occur only in combination with membrane insertion if no other factors provide the amphipathic environment. Up to now, folding and insertion ofmembrane proteins have usually been studied in vitro as separate processes. The proteins were first unfolded in appropriate solvents and refolded in detergent micelles providing the amphipathic environment, and in a second step they were inserted into membranes (1-3). For the second step, essentially two different ways exist, and they are well known from the reconstitution of membrane proteins without unfolding. In conventional reconstitution, detergent-solubilized lipid is added to the detergentsolubilized protein and the detergent is removed, leading to the formation of lipid bilayers with incorporated protein (4). An alternative method is spontaneous insertion of detergentsolubilized protein into preformed lipid bilayers (5, 6). Combined refolding and insertion might have occurred when a porin in a "water-soluble" form, which presumably is unfolded, spontaneously inserted into vesicle membranes (7). However, detergent had to be added together with the lipid vesicles to induce insertion, and the detergent might have induced refolding before insertion. In this case, the two processes would have again occurred separately. A similar situation was found with a porin secreted by spheroplasts (8). We have studied the combined refolding and insertion of a protein into preformed vesicle membranes without the involvement of any detergent. As a prerequisite, the protein must be solubilized in the unfolded form. This is possible for a class of membrane proteins composed of (-strands and presumably forming (-barrels (9, 10). The amphipathic nature of the /3-strands results in a weak average hydrophobicity, often weaker than that of soluble proteins (10). Such membrane proteins occur, for instance, in the outer membrane of Escherichia coli. By contrast, the proteins of the inner membrane are composed of membrane-spanning a-helices and are strongly hydrophobic, hence exhibiting a strong tendency for aggregation. One of the outer membrane proteins is OmpA, which we have chosen for our studies.
N
PERI PLASM
OmpA
C
FIG. 1. Model of OmpA, showing the membrane topology and sites of cleavage by trypsin and Glu-C endoproteinase. LPS, lipopolysaccharide; PL, phospholipid.
OmpA has been used extensively to investigate protein export across the bacterial inner membrane (11-13). However, little is known about its mode of insertion into the outer membrane, which is true for P-structured membrane proteins in general. When OmpA is unfolded by boiling in SDS (3) or by dissolving in a urea/water mixture (14), it can be refolded in detergent micelles (3, 14). The protein consists of 325 amino acid residues and in its native state is a monomer. The N-terminal half constitutes the membrane part, while the C-terminal half resides in the periplasm. The membrane part has a high percentage of antiparallel p-structure and presumably forms a p-barrel of eight antiparallel p-strands (10) with long loops protruding into the extracellular space (Fig. 1). Together with LPS, these loops mediate OmpA's phage receptor activity (15). A catalytic activity of OmpA is not known. As experimental techniques for our studies, we used SDS/ gel electrophoresis, protease digestion, circular dichroism (CD), and tryptophan fluorescence.
MATERIALS AND METHODS Purification and Unfolding. OmpA was overexpressed in E. coli strain UH203 carrying pRD87-P1 (16). Cells were first grown in 5 liters of NBA (1% Bactotryptone/0.5% yeast extract/1% NaCl) containing 0.5% glucose and 50 Mg of ampicillin per ml at 370C. To remove glucose, they were washed at OD550 = 1.3 with NBA and were then induced at
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Abbreviations: DMPC, dimyristoyl phosphatidylcholine; LPS, lipopolysaccharide; OG, n-octyl f3D-glucopyranoside. 7457
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370C in 20 liters of NBA containing 1 mM isopropyl f-Dthiogalactoside and 50 ,g of ampicillin per ml. Because OmpA overproduction is toxic, these cells were harvested at OD550 = 0.9. Cell membranes were prepared as previously described (17). For preextraction, cell envelopes from a 20-liter culture were resuspended in 120 ml of 4 M urea/20 mM Tris HCl, pH 9/0.05% 2-mercaptoethanol, incubated at 500C for 30 min, and centrifuged at 150,000 x g for 2 hr (Fig. 2). The pelleted membranes were resuspended in 45 ml of 4 M urea/20 mM Tris HCl, pH 9/0.1% 2-mercaptoethanol, and sonicated for 30 min with a Branson sonifier (macrotip, 10°C) to unfold and solubilize OmpA in urea. The sonication time may be reduced by working at 8 M urea. OmpA then was separated from lipid vesicles by centrifugation (4 hr, 150,000 x g). The OmpA-containing supernatant was further purified by anion-exchange chromatography (Q Sepharose Fast Flow, Pharmacia) in 4 M urea/20 mM Tris HCl, pH 9/0.05% 2-mercaptoethanol, using a linear NaCl gradient (0-200 mM) for elution of OmpA. The purity was about 98% as determined by SDS/gel electrophoresis and subsequent scanning of the gel. Purified OmpA was analyzed for LPS contamination according to ref. 18, and no detectable amounts of LPS were found. Protein concentrations were determined by using Pierce BCA protein assay reagents (2-mercaptoethanol had to be removed prior to the reaction). The yield was approximately 15 mg of OmpA, starting from 25 g of wet cells. The purified OmpA was concentrated in 4 M urea/20 mM potassium phosphate (KPO), pH 7.3, by ultrafiltration (Amicon, PM-10) and stored at -20°C. This stock solution was 5 mg/ml in OmpA. For refolding experiments, it was diluted to 100 j&g/ml, if not stated otherwise. The pH was always kept at 7.3 by 20 mM KP1. The 24-kDa tryptic fragment of OmpA was purified as described above, except that cell envelopes were digested with trypsin at 50 ug/ml at room temperature for 90 min before preextraction. Conventional Reconstitution. For refolding in detergent, unfolded OmpA (5 mg/ml) was diluted at least 50-fold into n-octyl 3-Di-glucopyranoside (OG; Sigma) at 10 mg/ml in 20 mM KP1, pH 7.3, resulting in a molar ratio of OmpA to OG of about 1:12,500. Reconstitution of OmpA into vesicles of dimyristoyl phosphatidylcholine (DMPC) (Avanti Polar Lipids) was achieved by dialyzing 3 ml containing OmpA at 100 j&g/ml, OG at 20 mg/ml, and DMPC at 1 mg/ml in 20 mM KPj, pH 7.3, against
FIG. 2. Purification strategy.
Proc. Nad. Acad Sci. USA 89 (1992) 20 mM KPi, pH 7.3/0.02% NaN3 at 370C for 24 hr. The molar ratio of OmpA to DMPC was about 1:500. For reconstitution of OmpA into mixed vesicles composed of LPS and E. coli lipid, 3 mg of E. coli lipid (gift of H. Kiefer from this institute) and 30 mg of OG were dissolved in hexane. After the solvent had been evaporated under a stream of nitrogen and the resulting film had been dried in vacuum for 1 hr, 3 mg of LPS (gift of U. Henning from this institute) dissolved in OG at 10 mg/ml and unfolded OmpA were added. This suspension was brought to 3 ml and made 100 .ug/ml OmpA, 20 mg/ml OG, 20 mM KP, at pH 7.3,5 mM MgCl2. It was sonicated in a water bath for 1 min. Dialysis of the clear solution against 20 mM KP,, pH 7.3/5 mM MgCI2/ 0.02% NaN3 at 30TC for 24 hr yielded OmpA reconstituted in mixed vesicles of LPS/E. coli lipid. Oriented Membrane Insertion. For preparation of lipid vesicles, 10 mg of DMPC was dissolved in 0.2 ml of CHC13. After the solvent had been evaporated and the film had been dried in vacuum for 1 hr, 2 ml of 20 mM KP1, pH 7.3, was added. Sonication of the suspension at 30TC for 30 mm (Branson sonifier, standard tip) yielded small unulamellar vesicles which were kept at 30TC overnight. Prior to insertion experiments, the vesicles were diluted to give DMPC at 1 mg/ml. This product will be called "small preformed vesicles" in the following. Their size has been characterized by quasielastic light scattering and electron microscopy; the diameter is 30-50 nm. HPLC showed 0.5% degradation of DMPC to myristate and lysophosphatidylcholine. Membrane insertion was started by adding a small volume of unfolded OmpA to preformed vesicles at 30TC, thus diluting OmpA to 100 pug/ml (or 25 pg/ml for fluorescence experiments), corresponding to a molar ratio of OmpA to DMPC of about 1:500 (or 1:2000). Samples were analyzed after 40 min. SDS/Gel Electrophoresis and Protease Digestion. SDS/gel electrophoresis was performed according to ref. 19, using 12.5% polyacrylamide gels. Routinely, the samples were not boiled. Typically, 2 .ug ofprotein was applied to one lane. The gels were stained with Coomassie blue. On our gels, unfolded OmpA migrated with an apparent molecular mass of 35 kDa, in contrast to 30 kDa for folded OmpA. In the cited literature, these values vary from 33 to 36 kDa and from 28 to 31 kDa, respectively. Trypsin digestion was employed to determine the extent of refolding and membrane incorporation of OmpA, since unfolded urea-dissolved OmpA is known to be completely digested by trypsin, and refolded membrane-incorporated OmpA is digested only down to a membrane-protected fragment (14). Samples containing OmpA at 100 pg/ml were incubated with trypsin at 10 pg/ml at 150C for 10 hr. Digestion was stopped by addition of 0.1 mM No-p-tosyl-L-lysine chloromethyl ketone. The samples were analyzed by SDS/ gel electrophoresis with subsequent scanning of the gels with an Ultroscan XL (LKB). This permitted us to determine the ratio of membrane-protected fragment of OmpA to OmpA added initially and, thus, the extent of refolding and membrane incorporation. In this calculation the reduced staining intensity of the membrane-protected fragment due to its smaller size had to be taken into account. When the purified tryptic fragment of OmpA was subjected to the same analysis, the samples were incubated with Glu-C endoproteinase (Boehringer Mannheim; EC 3.4.21.19) at 20 pg/ml at 300C for 2 hr. Spectroscopy. OmpA contains five Trp residues, which are all buried in the membrane according to the P-barrel model. Tryptophan fluorescence spectra were recorded with a Perkin-Elmer MPF3 fluorimeter. The excitation wavelength was 296 nm. Circular dichroism spectra were recorded on a Jasco IS00A. All spectra were corrected for light scattering by
FL - _ -35kfOG
Biophysics: Surrey and Rihnig
Proc. Natl. Acad. Sci. USA 89 (1992)
r--_ _ * A-~~~~~~35 k Da imam so
1
2
3
5
4
-3
I ,a X
45
/
^2 6 -\
7
6
'
_
-30 klo
FIG. 3. SDS/gel electrophoresis of preextracted cell membranes during sonication in 8 M urea. Samples were taken before (lane 1) and 30 sec, 90 sec, 3 min, 5 min, 10 min, and 20 miin after (lanes 2-7) start of sonication.
subtracting spectra of samples lacking OmpA. A correction for temperature effects was performed when necessary. Phage Inactivation. OmpA reconstituted in mixed vesicles of LPS/E. coli lipid was tested for biological activity by a phage inactivation test (14, 20), using OmpA-specific bacteriophage K3 and E. coli JM109 (21). Samples (200 gl) of vesicles containing OmpA at 100 pg/ml as well as a control sample without OmpA were incubated with 1000 plaqueforming units of K3 at 370C for 30 min. Subsequently, the suspension was plated out with E. coli JM109, and after incubation for 6 hr at 370C the plaques were counted. RESULTS Unfolding and Conventional Reconstitution. As shown previously, OmpA is soluble in urea (14). Membraneincorporated OmpA, however, could not be solubilized by mere addition of urea. This permitted the preextraction of cell envelopes during purification, whereby mainly peripheral membrane proteins were removed (Fig. 2). When, however, the membrane fraction was sonicated vigorously in the presence of 4 M urea, OmpA and other outer membrane proteins were released from the membrane and dissolved in the urea/water mixture. In this form, OmpA was purified by anion-exchange chromatography. As expected, solubilized OmpA was unfolded. This was demonstrated by a shift in the apparent molecular mass in
7459
A
~//
-7~~~~~~
-1 ? -3 -5
--
200
210
220 230 WAVELENGTH (nm)
240
250
FIG. 5. CD spectra of OmpA (A) and of the 24-kDa fragment of OmpA (B) under various conditions. ., OmpA or fragment unfolded in 8 M urea; -, OmpA or fragment unfolded in 4 M urea;-, OmpA or fragment aggregated in buffer; --, OmpA or fragment refolded in OG at 10 mg/ml; -, OmpA or fragment refolded and inserted spontaneously into small preformed vesicles at 30TC or adsorbed to small preformed DMPC vesicles at 150C.
SDS/gels from 30 kDa, typical for native OmpA, to 35 kDa (Fig. 3 and Fig. 4, lane 1) (3, 14). In addition, unfolded OmpA was completely degraded by trypsin (Fig. 4, lane 6); its CD spectrum indicated a high degree of random-coil structure (Fig. 5A); and its tryptophan fluorescence was relatively weak, with a maximum at 348 nm (Fig. 6), characteristic for Trp residues in a polar environment. When the urea concentration was increased to 8 M, the fluorescence remained unchanged, while the CD spectrum was slightly altered indicating a completely random structure. When the urea concentration was decreased to 80 mM by dilution, OmpA aggregated as judged by ultracentrifugation. Aggregated OmpA could be pelleted within 90 min, in contrast
cF 10o ..
..
..........-...: ..N: .
9-
..
35 kDa -30 kDa
-
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24 k Da D CI-
1
2
3
4.
5
6
7
8
9
5
10 11
FIG. 4. SDS/gel electrophoresis of OmpA under various conditions. Lane 1, unfolded OmpA in 4 M urea; lane 2, OmpA refolded in OG; lane 3, OmpA refolded in OG and reconstituted in DMPC vesicles by dialysis at 300C; lane 4, OmpA refolded and inserted spontaneously into small preformed DMPC vesicles at 300C; lane 5, OmpA partially refolded and adsorbed to small preformed DMPC vesicles at 15'C; lane 6, OmpA treated as in lane 1 and digested by trypsin; lane 7, OmpA treated as in lane 4 and digested by trypsin, yielding the 24-kDa fragment; lane 8, OmpA treated as in lane 5 and digested by trypsin; lane 9, trypsin; lane 10, the 24-kDa fragment of OmpA refolded and inserted spontaneously into small preformed DMPC vesicles at 30TC and digested by Glu-C endoproteinase; lane 11, Glu-C endoproteinase.
U,
en C-
0 =>
300
340 360 320 WAVELENGTH (nm)
380
FIG. 6. Tryptophan fluorescence spectra of OmpA under different conditions. - -, OmpA unfolded in 4 M urea; - .. -, OmpA
refolded in OG; -, OmpA refolded and inserted spontaneously into small preformed DMPC vesicles at 30'C; -, OmpA partially refolded and adsorbed to small DMPC vesicles at 150C.
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to solubilized unfolded OmpA. The apparent molecular mass of aggregated OmpA was still 35 kDa. The CD spectrum showed a mixture of a-helix, 3-strand, and random-coil structure (Fig. 5A), and the fluorescence spectrum was slightly blue-shifted compared with unfolded OmpA (data not shown). When the urea concentration was decreased to 80 mM by dilution into a solution of the detergent OG, OmpA refolded, as indicated by the appearance of the apparent molecular mass of 30 kDa characteristic for native OmpA (Fig. 4, lane 2). Refolding, however, was not complete, since 20% of OmpA retained the apparent molecular mass of 35 kDa. This is in agreement with trypsin digestion experiments: 80o of the OmpA molecules were digested down to a fragment of apparent molecular mass of 24 kDa, characteristic for the membrane part of OmpA. The remaining 20%o of OmpA molecules were degraded down to much smaller fragments. The CD spectrum showed a high degree of (-structure (Fig. SA), and the tryptophan fluorescence was relatively strong with a maximum at 330 nm, indicating a hydrophobic environment (Fig. 6). When detergent-solubilized OmpA was reconstituted into vesicle membranes of DMPC by detergent dialysis, the apparent molecular mass of 30 kDa was preserved (Fig. 4, lane 3), hence, OmpA remained folded. As shown previously by Raman and fluorescence spectroscopy, OmpA in DMPC membranes has a high (-strand content of about 60%o and the Trp residues are in a strongly hydrophobic environment (3, 10). To demonstrate that reconstituted OmpA has refolded into its native structure, we tested its function as a phage receptor (14). For this purpose, reconstitution into vesicle membranes containing LPS is necessary, since the phages require LPS for attachment. Indeed, the LPS-containing vesicles did function as phage receptors, as shown by the reduction of the number of plaques in the phage inactivation test from 1080 (vesicles without OmpA) to 45. This ratio is of the expected order of magnitude (20). As shown previously, trypsin digestion of OmpA reconstituted in lipid vesicles yields a 24-kDa fragment representing the membrane part of OmpA (14). However, only about half of the reconstituted OmpA molecules are digested, indicating random orientation of the protein molecules in the vesicle membranes (data not shown) (3). Oriented Insertion Into Preformed Vesicles. When the urea concentration was decreased to 80 mM by dilution of unfolded OmpA into a dispersion of small preformed DMPC vesicles at 30TC, OmpA refolded and inserted into the vesicle membranes. Refolding was demonstrated by the reduction of the apparent molecular mass to 30 kDa (Fig. 4, lane 4). Not all OmpA molecules folded back; a fraction retained the apparent molecular mass of 35 kDa, even after prolonged incubation, and another fraction appeared as oligomers. The CD spectrum indicated a high degree of (3-structure (Fig. 5A), and the tryptophan fluorescence was relatively strong with a maximum at 330 nm (Fig. 6). The time course of the fluorescence at 330 nm revealed a half-time for insertion of about 10 min. Trypsin digestion of spontaneously inserted OmpA yielded the 24-kDa membrane fragment (Fig. 4, lane 7). This indicates insertion of OmpA into the vesicle membranes. Quantitative analysis of the gels by scanning reveals that 40-50%o of the OmpA molecules inserted into the membrane. The trypsin digestion experiment also provides information on the orientation of OmpA within the vesicle membranes. Because all OmpA molecules were degraded, either down to the membrane fragment or down to much smaller fragments, all inserted OmpA molecules were oriented with their peripheral part outside the vesicles. Analyzed quantitatively, more than 99%o of the inserted OmpA molecules were oriented vectorially. This contrasts with the result of trypsin digestion of conventionally reconstituted OmpA (see
above).
Proc. Nad. Acad. Sci. USA 89 (1992)
No membrane insertion of OmpA, but aggregation, was observed, when large preformed vesicles produced by several freeze-thaw cycles were used for insertion experiments (data not shown). Preparation of large vesicles with OG added below the critical micelle concentration again permitted OmpA to spontaneously refold and insert. On the other hand, aggregated OmpA was not able to refold and insert into small preformed vesicles. When unfolded OmpA was diluted into a dispersion of small preformed vesicles of DMPC at 150C-i.e., below the lipid phase transition at 240C-a different behavior was observed. CD spectroscopy indicated the same content of (3-structure as for membrane-incorporated OmpA (Fig. SA), and fluorescence spectroscopy revealed again a hydrophobic environment of the Trp residues (Fig. 6). However, the apparent molecular mass remained at 35 kDa, and OmpA was degraded completely by trypsin (Fig. 4, lanes 5 and 8). Furthermore, it could be unfolded again by increasing the urea concentration to 4 M, in contrast to the case ofinsertion into fluid membranes at 300C (data not shown). These results indicate that below the lipid phase transition, OmpA is in a state with high (3-strand content, adsorbed on the membrane surface but not incorporated in the ordered lipid bilayer. When the temperature was slowly raised to 300C, OmpA refolded and inserted into the membrane as if added at 30"C. Refolding and Membrane Inerto of the Trypic Fragent of OmpA. The 24-kDa fragment of OmpA was prepared by trypsin digestion of cell membranes, followed by the same purification procedure as for complete OmpA. The fragment also dissolved and unfolded in the urea/water mixture. For 8 M urea, the CD spectrum (Fig. SB) and the fluorescence spectrum (data not shown) coincided with the spectra of the complete OmpA molecule. For 4 M urea, the spectra of the fragment remained unaltered, while complete OmpA adopted some secondary structure (see above). This partial refolding can now be attributed to the peripheral part of OmpA. Like complete OmpA, the fragment spontaneously refolded and inserted into small preformed vesicle membranes. The refolding efficiency was 50%6. Unidirectional orientation after insertion was demonstrated by using Glu-C endoproteinase, which removed the small remainder of the periplasmic part left after trypsin digestion and reduced the apparent molecular mass of all fragment molecules from 24 to 21 kDa (Fig. 4, lane 9). This demonstrated that the periplasmic part is not required for oriented insertion of OmpA into vesicle membranes.
DISCUSSION The integral membrane protein OmpA can exist in at least five different conformations, depending on the environment. They are summarized in Fig. 7, together with the observed transitions between them. OmpA could be extracted from its native membrane by sonication in the presence of urea. In the urea/water mixture, it unfolds and thus becomes soluble. This is possible due to the low hydrophobicity of OmpA, which is typical for /3-structured membrane proteins (10). Strongly hydrophobic proteins such as the a-helical membrane proteins will presumably not become soluble under the same conditions. When urea is removed from the solution, OmpA aggregates. The conformation of aggregated OmpA is a mixture of a-helix, (-strand, and random-coil structure. Upon removal of urea in the presence of detergent, unfolded OmpA spontaneously refolds, forming mixed micelles. In a second step, it can be reconstituted into lipid vesicle membranes by removing the detergent. Similar results have been obtained upon refolding SDS-denatured OmpA (3), SDS- and guanidnium hydrochloride-denatured porin (2), and acid-denatured bacteriorhodopsin (1). These results indicate that the complete information for the threedimensional structure of a membrane protein is encoded in its
Biophysics: Surrey and Jdhnig
FIG. 7. Summary of the observed conformations of OmpA and of the transitions between them.
amino acid sequence, as generally accepted for soluble proteins (22, 23). However, while water is required for a soluble protein to fold into the native structure, an amphiphilic environment is required for a membrane protein to fold properly. Our main interest was not detergent-mediated refolding and reconstitution of a membrane protein, but rather the combined refolding and membrane insertion in the absence of any detergent. Unfolded OmpA indeed refolds and inserts spontaneously into lipid bilayers when vesicles of DMPC are offered and urea is removed. However, several conditions have to be fulfilled. (i) The lipid membrane must be in its fluid state. Below the lipid phase transition temperature, OmpA still adopts a high degree of (-strand structure, but it adsorbs onto the membrane without inserting into it. When subsequently the temperature is raised above the phase transition, OmpA inserts into the membrane in the correct structure. (ii) The lipid bilayer has to be highly curved, as in the case of small vesicles. There is no adsorption onto or insertion into large vesicles. As well known, the lipid packing in small vesicles is not optimal, and this kind of defect obviously facilitates the adsorption and insertion of proteins. For large vesicles, defects have to be introduced-e.g., by adding small amounts of detergent. The importance of defects for insertion of detergent-solubilized proteins into lipid vesicles has been emphasized previously (5). (iii) Aggregation of OmpA must be suppressed. Upon removal of urea, OmpA either may refold and insert into vesicles or may aggregate. The relative weight of the two possibilities depends on several factors, among others the concentrations of OmpA and lipid. Strongly hydrophobic membrane proteins such as the a-helical proteins will presumably not insert into membranes as easily as OmpA, because of their stronger tendency for aggregation. The insertion of OmpA into membranes occurred in an oriented manner, with the peripheral part remaining outside the vesicles. This oriented insertion is a difference between our method and conventional reconstitution, which usually leads to random orientation (4). The same oriented insertion was also found for the membrane part of OmpA alone, demonstrating that the peripheral part plays no role in the insertion process. Probably the shape of the membrane part of OmpA also is not responsible for the oriented insertion, since conventional reconstitution leads to a random orientation. Thus, the reason for the oriented insertion seems to be
Proc. Natl. Acad. Sci. USA 89 (1992)
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intimately related to the mechanism by which OmpA inserts into the membrane. One possibility for this mechanism is offered by the partially refolded and adsorbed state of OmpA that occurs below the lipid phase transition. It may represent a folding and insertion intermediate of OmpA (Fig. 7). This state may be viewed as a planar, amphiphilic (3-sheet lying flat on the membrane surface with its hydrophobic side contacting the hydrophobic core of the membrane. In a second step, the (-sheet would roll in and the putative (3-barrel would be formed and insert into the membrane. This mechanism of combined refolding and insertion would differ strongly from the mechanism proposed for the insertion of a-helical membrane proteins, which is based on helical hairpins (24, 25). What can be deduced from these results for the mechanism of membrane insertion of proteins in vivo? As demonstrated, a membrane protein on its own can under certain conditions insert into a lipid bilayer in a definite orientation. Cell membranes are fluid, but not highly curved. Thus, local defects facilitating the contact between the protein and the hydrophobic core of the membrane may be required for insertion into cell membranes. These defects might be induced by other proteins, giving them the opportunity to regulate insertion. Finally, proteins must be shielded from aggregation prior to insertion, which is especially important for exported proteins such as OmpA. This might be the task of chaperones (26). We thank U. Henning and H. Kiefer (this institute) for the gifts of LPS and E. coli lipid. The cooperations with M. Lu (this institute) on the phage test and with H. Rau (University of Stuttgart, Hohenheim) on the CD measurements are gratefully acknowledged. We also thank H.-J. Eibl and I. Kotting (Max-Planck-Institute for Biophysical Chemistry, Gottingen) for the test of lipid purity by HPLC. 1. Huang, K.-S., Bayley, H., Liao, M. J., London, E. & Khorana, H. G. (1981) J. Biol. Chem. 256, 3802-3809. 2. Eisele, J.-L. & Rosenbusch, J. P. (1990) J. Biol. Chem. 265, 10217-10220. 3. Dornmair, K., Kiefer, H. & Jahnig, F. (1990) J. Biol. Chem. 265, 18907-18911. 4. Silvius, J. R. & Allen, T. A. (1989) Biophys. J. 55, 207-208. 5. Scotto, A. W. & Zakim, D. (1988) J. Biol. Chem. 263,18500-18506. 6. Scotto, A. W. & Zakim, D. (1990) Biochemistry 29, 7244-7251. 7. Pfaller, R., Freitag, H., Harmey, A., Benz, R. & Neupert, W. (1985) J. Biol. Chem. 260, 8188-8193. 8. Sen, K. & Nikaido, H. (1990) Proc. Nati. Acad. Sci. USA 87, 743-747. 9. Weiss, M. S., Kreusch, A., Schiltz, E., Nestel, U., Welte, W., Weckesser, J. & Schulz, G. E. (1991) FEBS Lett. 280, 379-382. 10. Vogel, H. & Jahnig, F. (1986) J. Mol. Biol. 190, 191-199. 11. Freudl, R., Schwarz, H., Stierhof, Y.-D., Gamon, K., Hindenach, I. & Henning, U. (1986) J. Biol. Chem. 261, 11355-11361. 12. Tani, K., Shiozuka, K., Tokuda, H. & Mizushima, S. (1989) J. Biol. Chem. 264, 18582-18588. 13. Driessen, A. J. M. & Wickner, W. (1991) Proc. NatI. Acad. Sci. USA 88, 2471-2475. 14. Schweizer, M., Hindenach, I., Garten, W. & Henning, U. (1978) Eur. J. Biochem. 82, 211-217. 15. Morona, R., Klose, M. & Henning, U. (1984) J. Bacteriol. 159, 570-578. 16. Freudl, R., MacIntyre, S., Degen, M. & Henning, U. (1988) J. Biol. Chem. 263, 344-349. 17. Teather, R. M., Bramhall, J., Riede, I., Wright, J. K., Furst, M., Aichele, G., Wilhelm, U. & Overath, P. (1980) Eur. J. Biochem. 108, 224-231. 18. Tsai, C.-M. & Frasch, C. E. (1982) Anal. Biochem. 119, 115-119. 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 20. Van Alphen, L., Havekes, L. & Lugtenberg, B. (1977) FEBS Lett. 75, 285-290. 21. Messing, J., Crea, R. & Seeburg, P. H. (1981) Nucleic Acids Res. 9, 309-321. 22. Dill, K. (1990) Biochemistry 29, 7133-7155. 23. Jaenicke, R. (1991) Biochemistry 30, 3147-3161. 24. Engelman, D. M. & Steitz, T. A. (1981) Cell 23, 411-422. 25. Popot, J.-L. & Engelman, D. M. (1990) Biochemistry 29, 4031-4037. 26. Lecker, S. H., Driessen, A. J. M. & Wickner, W. (1990) EMBO J. 9, 2309-2314.
