Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel -strands, forming an amphiphilic connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of -strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed -barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

Export of altered forms of an Escherichia coli K-12 outer membrane protein (OmpA) can inhibit synthesis of unrelated outer membrane proteins

Expression of mutant ompA genes, encoding the 325 residue Escherichia coli outer membrane protein OmpA, caused an inhibition of synthesis of the structurally unrelated outer membrane porins OmpC and OmpF and of wild-type OmpA, but not of the periplasmic beta-lactamase. There was no accumulation of precursors of the target proteins and the inhibitory mechanism operated at the level of translation. So far only alterations around residue 45 of OmpA have been found to affect this phenomenon. Linkers were inserted between the codons for residues 45 and 46. A correlation between size and sequence of the resulting proteins and presence or absence of the inhibitory effect was not found, indicating that the added residues acted indirectly by altering the conformation of other parts of the mutant OmpA. To be effective, the altered polypeptides had to be channelled into the export pathway. Internal deletions in effector proteins, preventing incorporation into the membrane, abolished effector activity. The results suggest the existence of a periplasmic component that binds to OmpA prior to membrane assembly; impaired release of this factor from mutant OmpA proteins may trigger inhibition of translation. The factor could be a See B-type protein, keeping outer membrane proteins in a form compatible with membrane assembly.     

In vivo membrane assembly of split variants of the E.coli outer membrane protein OmpA.

The two-domain, 325 residue outer membrane protein OmpA of Escherichia coli is a well-established model for the study of membrane assembly. The N-terminal domain, consisting of approximately 170 amino acid residues, is embedded in the membrane, presumably in the form of a beta-barrel consisting of eight antiparallel transmembrane beta-strands. A set of 16 gene variants carrying deletions in the membrane-embedded domain of OmpA was constructed. When pairs of these mutant genes were co-expressed in E.coli, it was found that a functional OmpA protein could be assembled efficiently from two complementary protein fragments. Assembly was found when the polypeptide chain was split at the second or third periplasmic turn. All four protein termini were located in the periplasmic space. Interestingly, duplication of transmembrane strands five and six led to a variant with an unusual topology: the N-terminus of one fragment and the C-terminus of the other fragment were exposed at the cell surface. This is the first demonstration of correct membrane assembly of split beta-structured membrane proteins. These findings are important for a better understanding of their folding/assembly pathway and may have implications for the development of artificial outer membrane proteins and for the cell surface display of heterologous peptides or proteins.     

OmpA can form folded and unfolded oligomers

The monomeric outer membrane protein OmpA from Escherichia coli has long served as a model protein for studying the folding and membrane insertion of β-barrel membrane proteins. Here we report that when OmpA is refolded in limiting amounts of surfactant (close to the cmc), it has a high propensity to form folded and unfolded oligomers. The oligomers exist both in a folded and (partially) unfolded form which both dissociate under denaturing conditions. Oligomerization does not require the involvement of the periplasmic domain and is not strongly affected by ionic strength. The folded dimers can be isolated and show native-like secondary structure; they are resistant to proteolytic attack and do not dissociate in high surfactant concentrations, indicating high kinetic stability once formed. Remarkably, OmpA also forms significant amounts of higher order structures when refolding in the presence of lipid vesicles. We suggest that oligomerization occurs by domain swapping favored by the high local concentration of OmpA molecules congregating on the same micelle or vesicle. In this model, the unfolded oligomer is stabilized by a small number of intermolecular β-strand contacts and subsequently folds to a more stable state where these intermolecular contacts are consolidated in a native-like fashion by contacts between complementary β-strands from different molecules. Our model is supported by the ability of complementary fragments to associate with each other in vitro. Oligomerization is probably avoided in the cell by the presence of cellular chaperones which maintain the protein in a monomeric state.     

A Naturally Occurring Novel Allele of Escherichia coli Outer Membrane Protein A Reduces Sensitivity to Bacteriophage

A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.     

Association of neighboring β-strands of outer membrane protein A in lipid bilayers revealed by site-directed fluorescence quenching

We present a detailed study on the formation of neighboring β-strands during the folding of a monomeric integral membrane protein of the β-barrel type. β-Strand and β-barrel formations were investigated for the eight-stranded transmembrane domain of outer membrane protein A (OmpA) with single-tryptophan (W), single-cysteine (C) OmpA mutants. Based on the OmpA structure, W and C were introduced in two neighboring β-strands oriented toward the hydrocarbon core of the membrane. Replaced residue pairs were closer to either the periplasmic turns (named cis-side) or the outer loops (named trans-side) of the strand. W_nC_m OmpA mutants containing W at position n and C at position m along the polypeptide chain were labeled at the C by a nitroxyl spin label, which is a short-range fluorescence quencher. To monitor the association of neighboring β-strands, we determined the proximity between fluorescent W and labeled C in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native β-strand contacts in folding experiments required the lipid membrane. Residues in the trans-side of strands β₁, β₂, and β₃, represented by mutants W₁₅C₃₅ (β₁β₂, trans) and W₅₇C₃₅ (β₃β₂, trans), reached close proximity prior to residues in the N(β₁)- and C(β₈)-terminal strands as examined for mutants W₁₅C₁₆₂ (β₁β₈, trans) and W₇C₁₇₀ (β₁β₈, cis). Tryptophan and cysteine converged slightly faster in W₁₅C₁₆₂ (β₁β₈, trans) than in W₇C₁₇₀ (β₁β₈, cis). The last folding step was observed for residues at the cis-ends of strands β₁ and β₂ for the mutant W₇C₄₃ (β₁β₂, cis). The data also demonstrate that the neighboring β-strands associate upon insertion into the hydrophobic core of the lipid bilayer.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Outer Membrane Protein A (OmpA): A New Player in Shigella flexneri Protrusion Formation and Inter-Cellular Spreading

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.     

Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.     

The influence of amino substitutions within the mature part of an Escherichia coli outer membrane protein (OmpA) on assembly of the polypeptide into its membrane

The membrane part of the 325-residue outer membrane protein OmpA of Escherichia coli encompasses residues 1-177. This part is thought to cross the membrane eight times in antiparallel beta-strands, forming four loops of an amphipathic beta-barrel. With the aim of gaining some insight into the mechanism of sorting, i.e. the way the protein recognizes and assembles into its membrane, a set of point mutants in the ompA gene has been generated. Selection for toxicity of ompA expression following mutagenesis with sodium bisulfite yielded genes with multiple base pair substitutions, the majority of which resulted in amino acid substitutions in the membrane moiety of the protein. None of the altered proteins was blocked in membrane incorporation. A proline residue exists at or near each of the presumed turns at the inner side of the outer membrane. Using oligonucleotide-directed mutagenesis, each of them was replaced by a leucine residue which is thought to be a turn blocking residue. None of these proteins had lost the ability to be incorporated into the membrane. Apparently, leucine residues are tolerated at turns in this protein. To interfere with the formation of antiparallel beta-strands, four double mutants were prepared: ompA-ON3 (Ala11----Pro, Leu13----Pro), -ON4 (Ala11----Asp, Leu13----Pro), -ON5 (Gly160----Val, Leu162----Arg), and -ON6 (Leu164----Pro, Val166----Asp). The former three proteins and even quadruple mutants consisting of a combination of ompA-ON2 or -ON4 with -ON5 were not defective in membrane assembly. In contrast, the OmpA-ON6 protein was translocated across the plasma membrane but could not be incorporated into the outer membrane. It is concluded that at least one rather small area of the polypeptide is of crucial importance for the assembly of OmpA into the outer membrane.     

SurA assists the folding of Escherichia coli outer membrane proteins.

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1–171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180–325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA^180–325). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA^180–325 with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA^1–325) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the periplasmic domain of OmpA above physiological temperatures, which may induce dimerization and play a role in triggering the previously reported larger pore formation.     

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12

Summary The gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO₂H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature, mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.     

The soluble, periplasmic domain of OmpA folds as an independent unit and displays chaperone activity by reducing the self-association propensity of the unfolded OmpA transmembrane β-barrel

OmpA is one of only a few transmembrane proteins whose folding and stability have been investigated in detail. However, only half of the OmpA mass encodes its transmembrane β-barrel; the remaining sequence is a soluble domain that is localized to the periplasmic side of the outer membrane. To understand how the OmpA periplasmic domain contributes to the stability and folding of the full-length OmpA protein, we cloned, expressed, purified and studied the OmpA periplasmic domain independently of the OmpA transmembrane β-barrel region. Our experiments showed that the OmpA periplasmic domain exists as an independent folding unit with a free energy of folding equal to − 6.2 (± 0.1) kcal mol^-1 at 25 °C. Using circular dichroism, we determined that the OmpA periplasmic domain adopts a mixed alpha/beta secondary structure, a conformation that has previously been used to describe the partially folded non-native state of the full-length OmpA. We further discovered that the OmpA periplasmic domain reduces the self-association propensity of the unfolded barrel domain, but only when covalently attached (in cis). In vitro folding experiments showed that self-association competes with β-barrel folding when allowed to occur before the addition of membranes, and the periplasmic domain enhances the folding efficiency of the full-length protein by reducing its self-association. These results identify a novel chaperone function for the periplasmic domain of OmpA that may be relevant for folding in vivo. We have also extensively investigated the properties of the self-association reaction of unfolded OmpA and found that the transmembrane region must form a critical nucleus comprised of three molecules before undergoing further oligomerization to form large molecular weight species. Finally, we studied the conformation of the unfolded OmpA monomer and found that the folding-competent form of the transmembrane region adopts an expanded conformation, which is in contrast to previous studies that have suggested a collapsed unfolded state.     

Structural and Functional Roles of the Surface-Exposed Loops of the β-Barrel Membrane Protein OmpA from Escherichia coli

The N-terminal domain of the OmpA protein from Escherichia coli, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel beta-barrel whose eight transmembrane beta-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest beta-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.     

Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins

β-Barrel proteins are frequently found in the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. In Escherichia coli, these proteins are inserted in the outer membrane by the Bam (β-barrel assembly machinery) complex, a multiprotein machinery formed by the β-barrel protein BamA and the four peripheral membrane proteins BamB, BamC, BamD and BamE. The periplasmic part of BamA binds prefolded β-barrel proteins by a β-augmentation mechanism, thereby stabilizing the precursors prior to their membrane insertion. However, the role of the associated proteins within the Bam complex remains unknown. Here, we describe the crystal structure of BamB, a nonessential component of the Bam complex. The structure shows a typical eight-bladed β-propeller fold. Two sequence stretches of BamB were previously identified to be important for interaction with BamA. In our structure, both motifs are located in close proximity to each other and contribute to a conserved region forming a narrow groove on the top of the propeller. Moreover, crystal contacts reveal two interaction modes of how BamB might bind unfolded β-barrel proteins. In the crystal lattice, BamB binds to exposed β-strands by β-augmentation, whereas peptide stretches rich in aromatic residues can be accommodated in hydrophobic pockets located at the bottom of the propeller. Thus, BamB could simultaneously bind to BamA and prefolded β-barrel proteins, thereby enhancing the folding and membrane insertion capability of the Bam complex.     

Export and sorting of theEscherichia coli outer membrane protein OmpA

Results of studies, mostly using the outer membrane, 325 residue protein OmpA, are reviewed which concern its translocation across the plasma membrane and incorporation into the outer membrane ofEscherichia coli. For translocation, neither a unique export signal, acting in a positive fashion within the mature part of the precursor, nor a unique conformation of the precursor is required. Rather, the mature part of a secretory protein has to be export-compatible. Export-incompatibility can be caused by a stretch of 16 (but not 8 or 12) hydrophobic residues, too low a size of the polypeptide (smaller than 75 residue precursors), net positive charge at the N-terminus, or lack of a turn potential at the same site. It is not yet clear whether binding sites for chaperonins (SecB, trigger factor, GroEL) within OmpA are importantin vivo. The mechanism of sorting of outer membrane proteins is not yet understood. The membrane part of OmpA, encompassing residues 1 to about 170, it thought to traverse the membrane eight times in antiparallel -sheet conformation. At least the structure of the last -strand (residues 160–170) is of crucial importance for membrane assembly. It must be amphiphilic or hydrophobic, these properties must extend over at least nine residues, and it must not contain a proline residue at or near its center. Membrane incorporation of OmpA involves a conformational change of the protein and it could be that the last -strand initiates folding and assembly in the outer membrane.     

Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins.

Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA). Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane. In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins. Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane. Deltaskp mutant cells are viable and show only slight changes in the physiology of their outer membranes. In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37 degrees C in rich medium. We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm. Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.