Immunogenicity of in vitro folded outer membrane protein PorA of Neisseria meningitidis

In vitro folded and the denatured form of PorA P1.6 from Neisseria meningitidis strain M990 were used for immunization studies in mice. Previously, the antigen was isolated from cytoplasmic inclusion bodies, folded and purified. Its immunogenicity without adjuvant appeared to be low. The addition of the adjuvant QuilA, but not of galE lipooligosaccharide, considerably enhanced the immunogenicity. Moreover, when immunized with folded PorA P1.6 plus QuilA, a clear switch towards the IgG2a subclass of antibodies and concomitantly, the appearance of serum bactericidal activity, which is believed to be important for protective immunity, was observed. Hence, a tool for preparing vaccines against serogroup B meningococci devoid of endotoxin is available.     

Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.     

A conformational unfolding reaction activates phage fd for the infection of Escherichia coli

Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function.     

In vitro study of the metabolic effects of D-amino acids

While the L -configuration of amino acids predominates in all known living systems, D -enantiomers of amino acids have been detected with highly sensitive chromatographic techniques in human physiological fluids. In the present study, the survival of Chinese hamster ovary cells (CHO) and HeLa cells was inhibited by exposure to high concentrations of some D - or L -amino acids. Inhibition of colony formation, though, was not necessarily observed to be chiral-dependent. Some L -amino acids (L AAS) were found to be toxic while other D -amino acids (D AAS) were innocuous in both cultures. This is contradictory to the previous observations that D AAS were generally considered to be harmful. Frequently it was implied, although not experimentally proven, that the L AAS were not toxic. One of the metabolites produced by oxidative deamination of D - or L AAS is hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS) that is decomposed by catalase. Increased intracellular H₂O₂ can result in peroxidation of lipids. We measured catalase activity and the lipid peroxide levels (LPO) after incubating cells in either D - or L AAS. The amino acids (AAS) that were found to inhibit colony formation were found to be associated with higher levels of catalase activity and LPO. Therefore, we hypothesize that enhanced ROS generation may be, in part, responsible for the observed toxicity of some amino acids. © 1996 Wiley-Liss, Inc.     

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30°C or 37°C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic \-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30°C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A ₆₆₀ at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.     

Structure of -amino acid oxidase: new insights from an old enzyme

-amino acid oxidase is the prototype of flavin-dependent oxidases. The recent resolution of its 3D structure has provided an explanation for several of its properties and has led to a substantial revision of the mechanism of -amino acid dehydrogenation, with significant implications for the general uderstanding of flavin-dependent catalysis     

Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356: evidence for protein-protein interaction of two subdomains

The structure of the crystallisation domain, SAN, of the S(A)-protein of Lactobacillus acidophilus ATCC 4356 was analysed by insertion and deletion mutagenesis, and by proteolytic treatment. Mutant S(A)-protein synthesised in Escherichia coli with 7-13 amino acid insertions near the N terminus or within regions of sequence variation in SAN (amino acid position 7, 45, 114, 125, 193), or in the cell wall-binding domain (position 345) could form crystalline sheets, whereas insertions in conserved regions or in regions with predicted secondary structure elements (positions 30, 67, 88 and 156) destroyed this capacity. FACscan analysis of L.acidophilus synthesising three crystallising and one non-crystallising S(A)-protein c-myc (19 amino acid residues) insertion mutant was performed with c-myc antibodies. Fluorescence was most pronounced for insertions at positions 125 and 156, less for position 45 and severely reduced for position 7. By cytometric flow sorting a transformant harbouring the mutant S(A)-protein gene (position 125) was isolated that showed an increased fluorescense signal. Immunofluorescence microscopy suggested that the transformant synthesized mutant S(A)-protein only. PCR analysis of the transformant grown in the absence of selection pressure indicated that the mutant allele was stably integrated in the chromosome. Proteolytic treatment of S(A)-protein indicated that only sites near the middle of SAN are susceptible, although potential cleavage sites are present through the entire molecule. Expression in E.coli of DNA sequences encoding the two halves of SAN yielded peptides that could oligomerize. Our results indicate that SAN consists of a approximately 12kDa N and a approximately 18kDa C-terminal subdomain linked by a surface exposed loop. The capacity of S(A)-protein of L.acidophilus to present epitopes, up to approximately 19 amino acid residues in length, at the bacterial surface in a genetically stable form, makes the system, in principle, suitable for application as an oral delivery vehicle.     

Mutations affecting beta-tubulin folding and degradation

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.     

The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.     

Amino Acid Analyses of Desert Varnish from the Sonoran and Mojave Deserts

There has long been a debate as to whether desert varnish deposits are microbially mediated or are deposited by inorganic processes. Several researchers have cultured bacteria from the surface of desert varnish suggesting that bacteria are intimately associated with varnish coatings and may play a role in their formation. To test this hypothesis, we have collected scrapings of desert varnish from the Sonoran Desert in Arizona and the Mojave Desert in California and analyzed them for amino acids. Thirteen amino acids were found in desert varnish indicating a biogenic component of these varnishes. Two protein amino acids that were not detected in any of the varnishes are cysteine and tryptophan. Two nonprotein amino acids,β-alanine andγ-amino butyric acid, were found. These are known to be formed by enzymatic decarboxylation, thereby indicating possible organismal activity in varnish. Some D -enantiomers of the amino acids were also found. In addition to small amounts of the D -enantiomer of aspartic acid, which is rapidly formed by racemization and was present in most samples, D -alanine and D -glutamic acid were found. These latter two amino acids are components of the peptidoglycan cell wall material of bacteria. L -lysine was also detected, but not diaminopimelic acid. The combination of L -lysine, D -alanine, and D -glutamic acid is characteristic of the peptidoglycan from Gram-positive bacteria. Although the presence of these biomarkers does not prove that Gram-positive bacteria produce the coatings, finding them is consistent with the hypothesis that they may play a role in desert varnish formation.     

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.     

A testis antigen related to the brain D2 adhesion protein

The localization of an antigen immunochemically crossreactive with the rat brain interneuronal adhesion molecule, D2-protein, has in testis been demonstrated by immunoelectrophoresis and immunocytochemistry. This D2-like antigen is localized to spermatids and residual bodies in testis, but it is absent both from spermatogonia and mature spermatozoa and from Sertoli cells. By immunoperoxidase electronmicroscopy D2-like antigen was observed in the head region posterior to the acrosomal membrane of late spermatids. In the nervous system D2-protein is involved in interneuronal adhesion. It is suggested that D2-like antigen in testis may be involved in a similar adhesion between late spermatids and Sertoli cells during spermiation.     

Comparison of conformation and antimicrobial activity of synthetic analogs of gramicidin S: Stereochemical consideration of the role of D-phenylalanine in the antibiotic.

In order to study the role of D -amino acid residues in keeping the stable β-sheet conformation and in the antimicrobial activity of gramicidin S (GS), the four analogs of GS containing D -Ala, L -Ala, Gly, and Aib (α-aminoisobutyric acid) in place of D -Phe were synthesized. D -Ala-and Gly-containing analogs showed antimicrobial activity, while those containing L -Ala and Aib showed no activity. Conformation of these analogs and their derivatives were studied by comparison of ORD and CD spectra and by slective methylation method. It is concluded that the biologically active analogs have β-sheet conformation while inactive analogs have a much different conformation from that of GS. This indicates that D -Ala-Pro and Gly-Pro sequences favor taking a β-bend form but L -Ala-Pro and Aib-Pro sequences do not because the presence of L -side methyl group on the α-carbon atom of L Ala and Aib residues destabilizes the β-bend form. This would explain why the inactive analogs which take a different conformation from that of the active ones result in the loss of activity.     

Experimental study of the conformation of two stereoregular polymethionines: poly(D-methionyl-L-methionine) and poly(L-methionyl-D-methionyl-L-methionine) (author's transl)]

Infrared spectroscopy, X-ray diffraction, and nuclear magnetic resonance spectroscopy have been used in investigating the conformation of two stereoregular polymethionines, poly(D -methionyl-L -methionine) and poly(L -methionyl-D -methionyl-L -methionine). When dissolved in a helicogenic solvent, such as chloroform and hexafluoroisopropanol, the polytripeptide is in an α-helical conformation. A helix-to-coil transition can then be induced by addition of trifluoroacetic acid. On the other hand, it appears that the most stable conformation of poly(D -Met-L -Met) is a β antiparallel folded structure in which the linear polypeptide segments are near to the planar extension. This structure has been evidenced through X-ray examination of oriented films, casted from solutions in chloroform. It has also been identified in solution in the same solvent, by use of infrared spectroscopy and by measuring the δ_Hα chemical shift which characterizes the H^α proton in the peptide units. This δ_Hα value is found equal to 5.4 ppm and differs significantly from those which are usually attributed to the α-helical conformation (δ_Hα = 4.2 ppm) and to the random coil (δ_Hα = 4.6 ppm). The β folded conformation of the poly(D -Met-L -Met) appears to be comparatively less stable than the α-helical one for the poly(L -Met) macromolecular stereoisomer since hexafluoroisopropanol is a helicogenic solvent for this last solute and a destabilizing one for the poly(D -Met-L -Met) β folded conformer. X-ray examinations carried out with stretched films, casted from a solution of poly(D -Met-L -Met) in chloroform, result in several data concering the cross β structure of this stereoregular polypeptide in the solid state.     

Successive optical resolution by replacing crystallization of DL-threonine

DL -Threonine [DL -Thr; (2RS,3SR)-2-amino-3-hydroxybutanoic acid] was optically resolved by replacing crystallization using L -serine (L -Ser) and 4-hydroxy-L -proline (L -Hyp) as optically active cosolutes. D -Thr was allowed to crystallize preferentially from racemic aqueous solutions in the presence of these L -α-amino acids. The optical resolution of DL -Thr was more successfully achieved by using L -Ser, whose structure is more similar to that of DL -Thr than L -Hyp, and successively gave D - and L -Thr of 87—92% optical purities. The D - and L -Thr obtained were then recrystallized from water to give optically pure D - and L -Thr. © 1994 Wiley-Liss, Inc.     

Entrapping unusual folding characteristics of the beta-Ala residues in a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3.

Crystal structure analysis of a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3 (beta-Ala: 3-amino propionic acid; Aib: alpha-aminoisobutyric acid) revealed distinct conformational preferences for folded [phi approximately 136 degrees, mu approximately -62 degrees, psi approximately 100 degrees] and semifolded [phi approximately 83 degrees, mu approximately -177 degrees, psi approximately -117 degrees] structures of the N-and C-terminus beta-Ala residues, respectively. The overall folded conformation is stabilized by unusual Ni...H-Ni+1 and nonconventional C-H...O intramolecular hydrogen bonding interactions.     

Cloning, purification, and preliminary characterization by circular dichroism and NMR of a carboxyl-terminal domain of the bacteriophage P22 scaffolding protein.

Assembly of double-stranded DNA viruses and bacteriophages involves the polymerization of several hundred molecules of coat protein, directed by an internal scaffolding protein. A 163-amino acid carboxyl-terminal fragment of the 303-amino acid bacteriophage P22 scaffolding protein was cloned, overexpressed, and purified. This fragment is active in procapsid assembly reactions in vitro. The circular dichroism spectrum of the fragment, as well as the 1D-NMR and 15N-1H HSQC spectra of the uniformly-labeled protein, indicate that stable secondary structure elements are present. Determination of the three dimensional packing of these elements into the folded scaffolding protein fragment is underway. Structure-based drug design targeted at structural proteins required for viral assembly may have potential as a therapeutic strategy.     

Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket engineering

D -Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of  D -amino acids, making it one of the most promising routes for synthesizing optically pure  L -amino acids, including  L -phosphinothricin ( L -PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate  D -PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward  D -PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M_3rd-SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M_3rd-SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M_3rd-SHVG reshapes the tunnel-pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M_3rd-SHVG as biocatalyst, 500 mM D, L -PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.     

Determination of the critical concentration required for desmin assembly.

The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx. 80 amino acid residues. These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits. A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product. A method has been devised for purifying the three types of independently folded domain from crude extracts of E. coli, based on their pH-(and heat-)stabilities. The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity. This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution.