In vivo suppression of phytochrome aggregation by the GroE chaperonins in Escherichia coli

When expressed in Escherichia coli, a truncated form of phytochrome (oat PHYA AP3 residues 464-1129) self associates to form a series of products ranging in size from monomers to aggregates of greater than 20 subunits. When these same phytochrome sequences are coexpressed with the chaperonins GroEL and GroES, the truncated phytochrome migrates as a native-like dimer in size exclusion chromatography and no higher-order aggregates were detected. GroEL and GroES inhibition of phytochrome aggregation in E. coli presumably occurs via the suppression of folding pathways leading to incorrectly folded phytochrome.     

Identification of the lipoprotein initiating domain of apolipoprotein B

We have explored the minimum sequence requirement for the initiation of apolipoprotein B (apoB)-mediated triglyceride-rich lipoprotein assembly. A series of apoB COOH-terminal truncation mutants, spanning a range from apoB34 (amino acid residues 1-1544 of apoB100) to apoB19 (residues 1-862) were transfected into COS cells with and without coexpression of the microsomal triglyceride transfer protein (MTP). ApoB34, -25, -23, -21, -20.5, and -20.1 underwent efficient conversion to buoyant lipoproteins when coexpressed with MTP. ApoB19.5 (amino acids 1-884) also directed MTP-dependent particle assembly, although at reduced efficiency. When apoB19.5 was truncated by another 22 amino acids to form apoB19, MTP-dependent lipoprotein assembly was abolished. Analysis of the lipid stoichiometry of secreted lipoproteins revealed that all apoB truncation mutants formed spherical particles containing a hydrophobic core. Even highly truncated assembly-competent forms of apoB, such as apoB19.5 and 20.1, formed lipoproteins with surface:core lipid ratios of <1. We conclude that the translation of the first approximately 884 amino acids of apoB completes a domain capable of initiating nascent lipoprotein assembly. The composition of lipids recruited into lipoproteins by this initiating domain is consistent with formation of small emulsion particles, perhaps by simultaneous desorption of both polar and neutral lipids from a saturated bilayer.     

Effect of nucleocapsid on multimerization of <Emphasis Type="Italic">gypsy</Emphasis> structural protein GAG

The structural protein (Gag) of Drosophila retrovirus gypsy contains capsid and nucleocapsid domains. Gag forms virus-like particles in a bacterial cell; furthermore, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid vary in size and are less organized than particles formed by a full-length Gag. The nucleocapsid determines the organization and structure of the particles, which is ensured by the amino acid residues at its N-terminal (a nucleocapsid proximal part). The assembly of the particle occurs in the presence of any RNAs or single-stranded DNA oligonucleotides.     

Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis

The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated. Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified. The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco). Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity. In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function. All mutants were also severely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains.To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli strain CG712 resulting in an inability to assemble -phage was exploited in a complementation assay. Transformation with plasmids directing the expression of mutant chloroplast cpn10 proteins in two cases restored -phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein. In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn 10 showed significantly reduced complementation efficiencies.     

Afp14 is involved in regulating the length of Anti‐feeding prophage (Afp)

The anti‐feeding prophage (Afp), a phage‐tail‐like particle that causes cessation of feeding in the New Zealand grass grub, Costelytra zealandica, is encoded by 18 open reading frames (afp1–18). C‐terminal truncations of afp14 resulted in shortened Afp particles, suggesting that Afp14 is involved in Afp length determination. We constructed an Afp assembly system (afp1–18), wherein Afp14 was truncated after the N‐terminal 88 residues. This construct, when expressed in trans in Escherichia coli expressing a N‐terminal 98‐amino acid Afp14 construct, yielded fully assembled Afp but no assembled Afp was detected in the case of a N‐terminal 96‐amino acid Afp14 construct. These results suggested that the 98 N‐terminal, amino acid residues of Afp14 is crucial for the initiation of Afp assembly via baseplate formation. Trans‐based expression of wild‐type afp14 resulted in Afp particles of varying lengths, all of which were shorter than the wild‐type Afp particle. On the other hand, similar expression of Afp14 harboring a C‐terminal extension (KLLEH₆) resulted in elongated Afp particles. This information, combined with bioinformatics data, allowed us to propose a model delineating the mechanism and role of Afp14 in the maturation of the Afp particle.     

Expression of a Highly Toxic Protein, Bax, in Escherichia coli by Attachment of a Leader Peptide Derived from the GroES Cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384–11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication

We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.     

Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates

Chaperonin 10 protein from Rattus norvegicus (Rat cpn10) has been reported to bind chaperonin 60 from Escherichia coli (GroEL) in an ATP-dependent manner. Chemically synthesized Rat cpn10 was immobilized in a defined orientation to agarose-bound monomeric avidin using a reversible biotinylated affinity label ( 1 ), attached to the N^α-terminal residue. The resulting affinity chromatographic matrix was then used to isolate binding proteins from a crude cell lysate. Following affinity separation the bound ligand and ligate was released by treatment with organic base. Rat cpn10 was prepared using a highly effective synthetic protocol involving HBTU/HOBt activation and capping with N-(2-chlorobenzyloxycarbonyloxy) succinimide to terminate unreacted amino groups. The biotinylated Fmoc-based molecule ( 1 ) was introduced specifically onto the N^α-terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of side-chain protecting groups using a low-TFMSA/high-HF procedure. Crude biotinylated Rat cpn10 (Rat cpn10+ 1 ) was immobilized on monomeric avidin with a binding efficiency of approximately 75% and unlabelled truncated/capped impurities eluted off the column with buffer. The biotinylated Rat cpn10–avidin affinity matrix was then used to isolate GroEL from a crude cell lysate. The identity of the purified protein was confirmed by SDS–PAGE and binding to a specific anti-GroEL monoclonal antibody (MoAb). These results extend the applicability of the biotinylated label ( 1 ), providing a reversible non-covalent anchor for immobilization of peptide and protein ligands, thus simplifying isolation of ligates and enabling recovery of synthetic material under mild conditions. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Non‐housekeeping,non‐essential GroEL (chaperonin) has acquired novel structure and function beneficial under stress in cyanobacteria

GroELs which are prokaryotic members of the chaperonin (Cpn)/Hsp60 family are molecular chaperones of which Escherichia coli GroEL is a model for subsequent research. The majority of bacterial species including E. coli and Bacillus subtilis have only one essential groEL gene that forms an operon with the co‐chaperone groES gene. In contrast to these model bacteria, two or three groEL genes exist in cyanobacterial genomes. One of them, groEL2, does not form an operon with the groES gene, whereas the other(s) does. In the case of cyanobacteria containing two GroEL homologs, one of the GroELs, GroEL1, substitutes for the native GroEL in an E. coli cell, but GroEL2 does not. Unlike the E. coli GroEL, GroEL2 is not essential, but it plays an important role which is not substitutable by GroEL1 under stress. Regulation of expression and biochemical properties of GroEL2 are different/diversified from GroEL1 and E. coli GroEL in many aspects. We postulate that the groEL2 gene has acquired a novel, beneficial function especially under stresses and become preserved by natural selection, with the groEL1 gene retaining the original, house‐keeping function. In this review, we will focus on difference between the two GroELs in cyanobacteria, and divergence of GroEL2 from the E. coli GroEL. We will also compare cyanobacterial GroELs with the chloroplast Cpns (60α and 60β) which are thought to be evolved from the cyanobacterial GroEL1. Chloroplast Cpns appear to follow the different path from cyanobacterial GroELs in the evolution after gene duplication of the corresponding ancestral groEL gene.     

Expression and Crystallization of an N-Terminally Activated Form of the Bacillus thuringiensis Cry1Ca Toxin

When the active form of the Bacillus thuringiensis delta-endotoxin Cry1Ca was expressed in E. coli severe growth retardation was observed. The absence of a short peptide from the N-terminus of the protoxin was responsible for this effect. The introduction of a mutation at an amino acid previously reported as being involved in the initial stages of pore formation within the natural insect target partially abolished the growth retardation effect. We suggest that removal of the N-terminal peptide is a necessary step in toxin activation, the presence of this peptide preventing proper interaction of the toxin with the target membrane. Expression of the truncated toxin in Bacillus thuringiensis also prevented the formation of Cry1Ca crystals. Received: 7 March 2001 / Accepted: 12 April 2001     

Transmission Electron Microscopy of GroEL, GroES, and the Symmetrical GroEL/ES Complex

Two new 2-D crystal forms of the Escherichia coli chaperone GroEL (cpn60) 2 × 7-mer have been produced using the negative staining-carbon film (NS-CF) technique. These 2-D crystals, which contain the cylindrical GroEL in side-on and end-on orientations, both possess p21 symmetry, with two molecules in the respective unit cells. The crystallographically averaged images correlate well with those obtained by other authors from single particle analysis of GroEL and our own previous crystallographic analysis. 2-D crystallization of the smaller chaperone GroES (cpn10) 7-mer has also been achieved using the NS-CF technique. Crystallographically averaged images of GroES single particle images indicate considerable variation in molecular shape, which is most likely due to varying molecular orientation on the carbon support film. The quaternary structure of GroES does, nevertheless, approximate to a ring-like shape. The complex formed by GroEL and GroES in the presence of ATP at room temperature has been shown to possess a symmetrical hollow ellipsoidal conformation. This symmetrical complex forms in the presence of a 2:1 or greater molar ratio of GroES:GroEL. At lower molar ratios linear chains of GroEL form, apparently linked by GroES in a 1:1 manner, which provide supportive evidence for the ability of both ends of the GroEL cylinder to interact with GroES. The apparent discrepancy between our data and that of other groups who have described an asymmetrical "bullet-shaped" (holo-chaperone) GroEL/ES complex is discussed in detail.     

A carboxy-terminal deletion impairs the assembly of GroEL and confers a pleiotropic phenotype in Escherichia coli K-12.

A series of COOH-terminal deletions of the chaperonin GroEL have been examined for effects in vivo at haploid copy number on the essential requirement of GroEL for cell growth. Strains with a deletion of up to 27 COOH-terminal amino acids were viable, but not viable strain could be isolated with a deletion of 28 or more codons. When substitutions were placed in the COOH-terminal amino acid Val-521 of the 27-amino-acid-deleted (delta 27) mutant, we found variable effect--Trp and Glu led to inviability, whereas Arg and Gly were viable but slow growing. The effects of the Arg substitution plus deletion (V521R delta) were examined in more detail. Whereas the delta 27 mutant with the wild-type residue Val-521 grew as well as a strain with wild-type GroEL, the V521R delta mutant strain (groEL202) exhibited a broad range of phenotypic defects. These include slow growth; filamentous morphology; a defect in plating lambda; absence of activity of expressed human ornithine transcarbamylase, as seen in other GroEL mutants; and several newly observed defects, such as absence of motility, sensitivity to UV light and mitomycin, a defect in one mode of specialized transduction, and inability to grow on rhamnose. Sucrose gradient analysis of extracts from the V521R delta cells showed a substantially reduced level of GroEL sedimenting at the normal 20S position of the assembled tetradecamer and a relatively large amount of more lightly sedimenting subunits. This indicates that the substitution-deletion mutation interferes with oligomeric assembly of GroEL into its functional form. This is discussed in light of the recently determined crystal structure of GroEL.     

Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid

The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.     

Modulation of interleukin-8 activity by gingipains from Porphyromonas gingivalis: implications for pathogenicity of periodontal disease

Gingipains are the major cysteine proteinases synthesized by Porphyromonas gingivalis which, in soluble form, are able to initially convert IL-8 (77 amino acid residues) to a more potent species truncated at the amino terminus, followed by slow degradation and destruction of chemokine biological activity. In contrast, the same enzymes when associated with bacterial outer-membrane blebs (vesicles), instantly degrade this chemokine. This division of enhancing and inactivating activity between soluble and membrane-bound gingipains can cause the compartmentalization of pro- and anti-inflammatory reactions to distal and proximal positions from bacterial plaque, respectively, which may explain why, despite the massive neutrophil accumulation at periodontitis sites, there is no elimination of infection.     

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.     

Expression of truncated phosphoproteins of Nipah virus and Hendra virus in <Emphasis Type="Italic">Escherichia coli</Emphasis> for the differentiation of henipavirus infections

The genus Henipavirus in the family Paramyxoviridae compromises two newly identified dangerous pathogens, Nipah virus and Hendra virus. Phosphoprotein of the two viruses is one of the major immunodominant antigens and the most divergent protein in the viral genomes. We have now expressed two pairs of truncated phosphoproteins of the two viruses in Escherichia coli in a soluble form using a vector tailored from pET32a. The truncated recombinant phosphoproteins were purified with His-Tag affinity chromatography and their antigenicity was determined by western blotting and ELISA. The longer pair of truncated recombinant phosphoproteins, covering amino acid residues 4–550, was more antigenic than the shorter one and of potential utility in the serological differentiation of henipavirus infections.     

Molecular characterization of GroES and GroEL homologues from Clostridium botulinum

We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947). The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies. Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue. The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced. The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins. Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage.     

Structure and Expression of a GroE-homologous Operon of a Macronucleus-Specific Symbiont Holospora obtusa of the Ciliate Paramecium caudatum

ABSTRACT The reproductive form of a macronucleus-specific symbiont Holospora obtuse , when harbored by the macronucleus of the ciliate Paramecium caudatum , selectively synthesized a 63-kDa protein which is immunologically related to GroEL, or HSP60, of Escherichia coli. Heat shock treatment of isolated cells of the reproductive and infectious form of the bacterium also induced the synthesis of the GroEL homolog. Immunoblotting showed that the amount of this protein per cell, whether the reproductive or infectious form, is roughly constant. Cloning and sequencing of a gene coding for the GroEL homolog suggested that the protein is 55.2% identical to GroEL of E. coli at the amino acid sequence level, and that the gene is preceded by an open reading frame which encodes a protein 39.6% identical to GroES of E. coli. Northern blot hybridization showed that the GroEL homologous gene is highly expressed in the reproductive form, but only in a trace amount in the intermediate and infectious form. Immunoelectron microscopy revealed that the GroEL homolog is localized in the cytoplasm of the reproductive and infectious form.     

27 amino acid residues can be deleted from the N-terminus of human lymphotoxin without impairment of its cytotoxic activity

In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LTδ37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LTδ28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LTδ27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.     

Dimeric rous sarcoma virus capsid protein structure relevant to immature Gag assembly

The structure of the N-terminal domain (NTD) of Rous sarcoma virus (RSV) capsid protein (CA), with an upstream 25 amino acid residue extension corresponding to the C-terminal portion of the Gag p10 protein, has been determined by X-ray crystallography. Purified Gag proteins of retroviruses can assemble in vitro into virus-like particles closely resembling in vivo-assembled immature virus particles, but without a membrane. When the 25 amino acid residues upstream of CA are deleted, Gag assembles into tubular particles. The same phenotype is observed in vivo. Thus, these residues act as a “shape determinant” promoting spherical assembly, when they are present, or tubular assembly, when they are absent. We show that, unlike the NTD on its own, the extended NTD protein has no β-hairpin loop at the N terminus of CA and that the molecule forms a dimer in which the amino-terminal extension forms the interface between monomers. Since dimerization of Gag has been inferred to be a critical step in assembly of spherical, immature Gag particles, the dimer interface may represent a structural feature that is essential in retrovirus assembly.