Force as a probe of membrane protein structure and function

Force measurement techniques are being used increasingly to explore the mechanical properties of proteins, as well as the structural origins of intermolecular forces. Developments in instrumentation and the increasing availability of engineered and purified membrane proteins have widely expanded the range of biological systems that can be addressed. Within the past year, force measurements have identified novel mechanisms of binding between cell-surface proteins, as well as the mechanical properties of integral membrane proteins and the intramolecular interactions that stabilize their structures.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.     

Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

Abstract Outer membranes of Escherichia coli K-12 were used to isolate hybridoma cell lines that produce monoclonal antibodies against the FhuA (TonA) protein. Two monoclonal antibodies were obtained from independent immunization and fusion experiments. The antibodies belonged to the subclass IgG₁ and κ, and IgG_2b and κ, respectively. The latter antibody was purified by affinity chromatography on protein A-Sepharose. The culture supernatants of the hybridoma cell lines and the isolated antibody inhibited adsorption of the phages T5 and T1 to E. coli cells while binding of phage ø80, which also uses the FhuA protein as a receptor, remained unaffected. The specificity of the antibodies to the FhuA protein was supported by the prevention of killing of cells by colicin M and by the lack of inhibition of colicin B and of phage BG23. Transport of iron(III) as ferrichrome complex was not inhibited by the isolated antibody. However, partial competition with the adsorption of the phages T2, TuIb and T6 was observed which may indicate an organization of certain functional phage receptors into clusters.     

Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli

We describe a lesion, lamB701-708, affecting the hydrophilic portion of the lambda receptor signal sequence. The C to A transversion of the sixth codon of the signal sequence changes a positively charged arginine to a neutral serine. The phenotype conferred by this alteration is unique among previously described signal sequence mutations. The results suggest an essential role for the charged amino acids of the hydrophilic segment in the initial interaction between a nascent secreted protein and a membrane export site. The results further suggest that synthesis of lambda receptor is coupled to its export.     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

Projected structure of the pore-forming OmpC protein from Escherichia coli outer membrane.

A single-projection structure analysis of a bacterial outer membrane protein, OmpC, has been carried out by electron microscopy of frozen hydrated specimens. Two distinct crystal polymorphs have been observed in the frozen-hydrated samples, and projection structures of both forms have been obtained to a resolution of 13.5 A. Preliminary examination of negatively stained samples revealed the expected, trimeric appearance of pores in the OmpC specimens. Electron microscopy of unstained, frozen-hydrated OmpC reveals the trimeric pore structure with equal clarity. In addition, the overall molecular envelope of the protein is readily discerned, and a major lipid-containing domain can also be seen. Because of the small coherent patch size, mosaic disorder, and unpredictable polymorphism of the presently available specimens, three-dimensional reconstruction of frozen-hydrated OmpC has not been carried out.     

Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F

The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin-sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000-molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and which reacted with only one of the two classes of protein F-specific monoclonal antibodies. The 2-kilobase fragment was used in Southern blot hybridizations to construct a restriction map of the cloned and subcloned fragments and to demonstrate with restriction digests of whole P. aeruginosa DNA that only one copy of the protein F gene was present in the P. aeruginosa chromosome. The protein F produced by the original cosmid clone in a porin-deficient E. coli background was purified. To demonstrate retention of porin function after cloning, the protein F from the E. coli clone was incorporated into black lipid bilayer membranes. Two major classes of channels were revealed. The predominant class of channels had an average conductance of 0.36 nS in 1 M KCl, whereas larger channels (4 to 7 nS) were seen at a lower frequency. Similar channel sizes were observed for porin protein F purified by the same method from P. aeruginosa outer membranes.     

Role of a major outer membrane protein in Escherichia coli.

Mutants of Escherichia coli B/r lacking a major outer membrane protein, protein b, were obtained by selecting for resistance to copper. These mutants showed a decreased ability to utilize a variety of metabolites when the metabolites were present at low concentrations. Also, mutants of E. coli K-12 lacking proteins b and c from the outer membrane were shown to have an identical defect in the uptake of various metabolites. These results are discussed with regard to their implications as to the role of these proteins in permeability of the outer membrane,     

Monoclonal antibody, a novel probe for protein folding

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Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.     

Primary structure of the tolC gene that codes for an outer membrane protein of Escherichia coli K12.

We present the nucleotide sequence of the tolC gene of Escherichia coli K12, and the amino acid sequence of the TolC protein (an outer membrane protein) as deduced from it. The mature TolC protein comprises 467 amino acid residues, and, as previously reported (1), a signal sequence of 22 amino acid residues is attached to the N-terminus. The C-terminus of the gene is followed by a stem-loop structure (8 base pair stem, 4 base loop) which may be a rho-independent termination signal. The codon usage of the gene is nonrandom; the major isoaccepting species of tRNA are preferentially utilised, or, among synonomous codons recognized by the same tRNA, those codons are used which can interact better with the anticodon (2,3). In contrast to the codon usage for other outer membrane proteins of E. coli (4) the rare arginine codons AGA and AGG are used once and twice respectively.     

Mutations affecting antigenic determinants of an outer membrane protein of Escherichia coli.

The Escherichia coli LamB protein is located in the outer membrane. It is both a component of the maltose and maltodextrin transport system, and the receptor for phages lambda and K10. It is a trimer composed of three identical polypeptide chains, each containing 421 residues. Six independent mutants have been isolated, in which the LamB protein is altered in its interaction with one or more monoclonal antibodies specific for regions of the protein that are exposed at the cell surface. Some of the mutations also altered the binding site for phage lambda. All of the mutations were clustered in the same region of the lamB gene, corresponding to residues 333-394 in the polypeptide. This and previous results strongly suggest that a rather large segment of the LamB polypeptide, extending from residue 315 to 401, is exposed at the outer face of the outer membrane. This segment would bear the epitopes for the four available anti-LamB monoclonal antibodies that react with the cell surface, and part of the binding site for phage lambda.     

Engineering the Escherichia coli outer membrane protein OmpC for metal bioadsorption

The outer membrane protein, OmpC, from Escherichia coli was used to display metal-binding poly-histidine peptides on the surface of this bacterium. SDS-PAGE analysis of outer membrane protein preparations confirmed the expression of the metal-binding epitopes inserted in position 162 of the mature OmpC protein. Display of these epitopes was confirmed by epifluorescence microscopy of cells bound to Ni²⁺-NTA-agarose beads and metal adsorption experiments. The cells harboring one or two copies of the metal binding epitope were able to adsorb 3 to 6 times more Zn²⁺ (13.8 mol g^–1 cell), Fe³⁺ (35.3 mol g^–1 cell), and Ni²⁺ (9.9 mol g^–1 cell) metallic ions than control cells expressing the wild-type OmpC.     

Apparent bacteriophage-binding region of an Escherichia coli K-12 outer membrane protein.

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli. It serves as the receptor for several T-even-like phages and is required for the action of certain colicins and for the stabilization of mating aggregates in conjugation. We have isolated two mutant alleles of the cloned ompA gene which produce a protein that no longer functions as a phage receptor. Bacteria possessing the mutant proteins were unable to bind the phages, either reversibly or irreversibly. However, both proteins still functioned in conjugation, and one of them conferred colicin L sensitivity. DNA sequence analysis showed that the phage-resistant, colicin-sensitive phenotype exhibited by one mutant was due to the amino acid substitution Gly leads to Arg at position 70. The second mutant, which contained a tandem duplication, encodes a larger product with 8 additional amino acid residues, 7 of which are a repeat of the sequence between residues 57 and 63. In contrast to the wild-type OmpA protein, this derivative was partially digested by pronase when intact cells were treated with the enzyme. The protease removed 64 NH2-terminal residues, thereby indicating that this part of the protein is exposed to the outside. It is argued that the phage receptor site is most likely situated around residues 60 to 70 of the OmpA protein and that the alterations characterized have directly affected this site.     

The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin

Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the blood stream and eventually invasion of the meninges. The latter two aspects have been well characterized at a molecular level in the last decade. Less is known about the early stages of pathogenesis, i.e. adhesion to and invasion of gastrointestinal cells. Here, the characterization of the Hek protein is reported, which is expressed by neonatal meningitic E. coli (NMEC) and is localized to the outer membrane. It is demonstrated that this protein can cause agglutination of red blood cells and can mediate autoaggregation. Escherichia coli expressing this protein can adhere to and invade epithelial cells. So far, this is the first outer membrane protein in NMEC to be directly implicated in epithelial cell invasion.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

Tricarboxylate transport in a Cit+ Escherichia coli: evidence for the role of an outer membrane protein

A strain of Escherichia coli of bovine origin able to use tricarboxylates as single carbon source is described. Tricarboxylate utilization (Cit+) and fluorocitrate sensitivity (FCs) could be transferred conjugatively to E. coli K12 and were not plasmid borne. No evidence was found for tct gene products of Salmonella typhimurium. A citrate-inducible outer membrane protein of 21-22 kilodaltons (kd) was found only in Cit+ strains. A protein (21-22kd) protein was also found in wild-type E. coli K12 and in fluorocitrate-resistant mutants of Cit+ strains, but it was present in a cryptic form no longer inducible by citrate. Fluorocitrate-resistant mutants of Cit+ strains were still able to transport citrate by a fluorocitrate-insensitive system. High levels of the 22-kd protein correlated with reduced growth induction times on citrate and with the ability to effectively transport citrate.