In vivo membrane assembly of split variants of the E. coli outer membrane protein OmpA (original) (raw)
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Insights into the structure and assembly of Escherichia coli outer membrane protein A
The FEBS journal, 2012
Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA have remained controversial. A review of studies to date supports the hypothesis that native OmpA is a single-domain large pore, while a two-domain narrow-pore structure is a folding intermediate or minor conformer. The in vitro refolding of OmpA to the large-pore conformation requires isolation of the protein from outer membranes with retention of an intact disulfide bond followed by sufficient incubation in lipids at temperatures of ≥ 26 °C to overcome the high energy of activation for refolding. The in vivo maturation of the protein involves covalent modification of serines in the eighth β-barrel of the N-terminal domain by oligo-(R)-3-hydroxybutyrates as the protein is escorted across the cytoplasm by SecB for post-translational secretion across the secretory translocase in the inner membrane. After cleavage of the signal se...
Membrane protein folding on the example of outer membrane protein A of Escherichia coli
Cellular and Molecular Life Sciences (CMLS), 2003
The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded b-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the b-barrel transmembrane domain into the lipid bilayer are highly synchronized, i. e. the formation of large amounts of
Biochimie, 1990
Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coil B and K-12, have been used to study the assembly and the topology of the protein. Thi-~ paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies. A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows u~ io propose a rough model including 2 antigenic sites. The second part is focused on the assembly of the OmpF protein in the outer membrane. Various forms, precursor, unussembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process. The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope. The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly.
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-barrel whose eight transmembrane-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-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-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.
Biochemistry, 1999
Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H., and Tamm, L. K. (1999) Biochemistry 38, 4996-5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in ViVo and in Vitro and, as judged by SDS-PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA. TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30°C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 Å from the bilayer center. In a narrow temperature range between 30 and 35°C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. When monitored at 40°C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 Å from the center after refolding was complete. In contrast Trp-7 approached the 10 Å distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four-hairpins of the eight-stranded-barrel of OmpA were very similar to one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.
Resolving the native conformation of Escherichia coli OmpA
FEBS Journal, 2010
Outer membrane protein A (OmpA), a major outer membrane protein of Escherichia coli, is a highly conserved and multifunctional integral membrane protein that has served as a model system for studies of outer membrane targeting and protein folding [1]. However, despite intense study for several decades, the native structure of the protein has not yet been resolved.
Biogenesis of inner membrane proteins in< i> Escherichia coli
2011
The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis—protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary.
Structural requirements for membrane assembly of proteins spanning the membrane several times
The Journal of Cell Biology, 1989
We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the op-posite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.