Determination of the Near-Atomic Structure of Non-Purified Oligomeric E. coli Proteins.

The roughly purified extract of E. coli proteins has been studied by cryoelectron microscopy, the class-sums containing 2D projections of two proteins (ß-galactosidase and 2-oxoglutarate dehydrogenase complex catalytic domain (ODC-CD)), identified in an extract by tandem mass spectrometry, have been distinguished. The structures of these proteins have been solved at near-atomic resolution. De novo simulation of the ODC-CD structure yielded an atomic model that revealed differences in the positions of some amino acid residues of the active center, in comparison with the known crystal structures.

The chaperonin TRiC is blocked by native and glycated prion protein.

Eukaryotic double-ring chaperonin TRiC is an ATP-dependent protein-folding machine. Most of its substrates are known to form large ordered structures from multiple polypeptide chains. Since these structures are similar to fibrillar and oligomeric forms of amyloidogenic proteins, we hypothesized that TRiC may play a role in the development of neurodegenerative diseases of amyloid nature including prion diseases. Enzyme-linked immunosorbent assay showed that monomeric, oligomeric and fibrillar forms of prion protein (PrP) bind strongly to chaperonin TRiC, whereas glycation reduces the prion protein affinity for chaperonin. Nevertheless, dynamic light scattering, electron microscopy and thioflavin T fluorescence confirmed that all studied forms of PrP undergo an amyloid transformation after interaction with chaperonin, but different forms of prion protein are capable of having different effects on the functional state of TRiC. For example, prion protein monomers completely block its ability to reactivate the chaperonin's natural substrate - sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS). At the same time, PrP oligomers and fibrils only partially prevent the reactivation of GAPDS upon the action of TRiC. The monomeric forms of prion protein glycated by methylglyoxal do not inhibit, but only slow down the chaperone-dependent reactivation of GAPDS. Thus, the interaction of amyloidogenic proteins with chaperonins could cause cell malfunction.

Effect of chaperonin encoded by gene 146 on thermal aggregation of lytic proteins of bacteriophage EL Pseudomonas aeruginosa.

Investigation of the chaperonin encoded by gene 146 of bacteriophage EL Pseudomonas aeruginosa that we characterized earlier has been continued. To reveal the mechanism of its functioning, new recombinant substrate proteins, fragments of gene product (gp) 183 containing the lysozyme domain were prepared. Their interaction with gp146 was studied. The influence of the phage chaperonin on the thermal aggregation of one of these gp183 fragments and endolysin (gp188) was investigated in both the presence and the absence of ATP by dynamic light scattering. In the absence of ATP, the phage chaperonin forms stable complexes with substrate proteins, thereby protecting them against thermal aggregation. Experimental data obtained for different substrate proteins are analyzed.

Influence of distamycin, chromomycin, and UV-irradiation on extraction of histone H1 from rat liver nuclei by polyglutamic acid.

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A(3) (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this difference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and ~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV-stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.

[Pseudomonas aeruginosa bacteriophage SN: 3D-reconstruction of the capsid and identification of surface proteins by electron microscopy].

The virulent P. aeruginosa bacteriophage SN belongs to the PB1-like species of the Myoviridae family. The comparatively small (66391 bp) DNA genome of this phage encodes 89 predicted open reading frames and the proteome involves more than 20 structural proteins. A 3D model of the phage capsid to approximately 18 A resolution reveals certain peculiarities of capsomer structure typical of only this bacteriophage species. In the present work recombinant structural proteins SN gp22 and gp29 were expressed and purified; and specific polyclonal antibodies were obtained. Immune-electron microscopy of purified phage SN using secondary gold-conjugated antibodies has revealed that gp29 forms a phage sheath, and gp22 decorates the capsid. Precise identification of multicopy major capsid proteins is essential for subsequent construction of gene-engineered phages bearing non-native peptides on their surfaces (phage display).

Role of the C-terminus in folding and oligomerization of bacteriophage T4 gene product 9.

Bacteriophage T4 gene product 9 (gp9) is a structural protein of baseplate that plays a key role at the beginning of the infection process. Biologically active gp9 is a trimer that consists of three domains. It is a convenient model to study folding and oligomerization mechanisms of complex multidomain proteins. The influence of deletions and mutations of several amino acid residues in the C-terminal part of molecule on protein folding, oligomerization, and functional activity has been studied. It was determined that gp9 trimerization occurs post-translationally. It was shown that Gln282 and Ile284 are essential for gp9 trimer stabilization. The disruption of hydrogen bonds formed by Gln282 with Leu203 and Thr205 of neighboring chain has effect not only on interaction between monomers within trimer but also on folding of the polypeptide chain. Tsf (temperature sensitive for folding) and su (suppressor) mutations in the C-terminal region of the polypeptide chain affecting protein folding have been found.

Functional role of the N-terminal domain of bacteriophage T4 gene product 11.

Bacteriophage T4 late gene product 11 (gp11), the three-dimensional structure of which has been solved by us to 2.0 A resolution, is a part of the virus' baseplate. The gp11 polypeptide chain consists of 219 amino acid residues and the functionally active protein is a three-domain homotrimer. In this work, we have studied the role of gp11 N-terminal domain in the formation of a functionally active trimer. Deletion variants of gp11 and monoclonal antibodies recognizing the native conformation of gp11 trimer have been selected. Long deletions up to a complete removal of the N-terminal domain, containing 64 residues, do not affect the gp11 trimerization, but considerably change the protein structure and lead to the loss of its ability to incorporate into the baseplate. However, the deletion of the first 17 N-terminal residues results in functionally active protein that can complete the 11(-)-defective phage particles in in vitro complementation assay. This region of the polypeptide chain is probably essential for gp11-gp10 stable complex formation at the early stages of phage baseplate assembly in vivo. A study of the gp10 deletion variants suggests that the central domain of gp10 trimer is responsible for the interaction with gp11.

[Comparison of the genome for phylogenetically related bacteriophages phiKZ and EL of Pseudomonas aeruginosa: evolutionary aspects and minimal genome size].

Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phiKZ and EL, which belong to a group of (phiKZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phiKZ and EL genomes.

Molecular architecture of bacteriophage T4.

In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device--one of the most complex multiprotein components--has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.

[Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa]. / Fenogeneticheskaia kharakteristika gruppy gigantskikh Phi KZ-podobnykh bakteriofagov Pseudomonas aeruginosa.

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.

Chaperonin-mediated folding of bacteriophage T4 major capsid protein. II. Production of gene product 23 deletion mutants.

Folding of bacteriophage T4 major capsid protein, gene product 23 (534 a.a.), is aided by two proteins: E. coli GroEL chaperonin and viral gp31 co-chaperonin. In the present work a set of mutants with extensive deletions inside gene 23 using controlled digestion with Bal31 nuclease has been constructed. Proteins with deletions were co-expressed from plasmid vectors with phage gp31 co-chaperonin. Deletions from 8 to 33 a.a. in the N-terminal region of the gp23 molecule covering the protein proteolytic cleavage site during capsid maturation have no influence on the mutants' ability to produce in E. coli cells proteins which form regular structures--polyheads. Deletions in other regions of the polypeptide chain (187-203 and 367-476 a.a.) disturb the correct folding and subsequent assembly of gp23 into polyheads.

Engineering of bacteriophage T4 tail sheath protein.

Gene product 18 (gp18, 659 amino acids) forms bacteriophage T4 contractile tail sheath. Recombinant protein assembles into different length polysheaths during expression in the cell, which complicates the preparation of protein crystals for its spatial structure determination. To design soluble monomeric gp18 mutants unable to form polysheaths and useful for crystallization, we have used Bal31 nuclease for generation deletions inside gene 18 encoding the Ile507-Gly530 region. Small deletions in the region of Ile507-Ile522 do not affect the protein assembly into polysheaths. Protein synthesis termination occurs because of reading frame failure in the location of deletions. Some fragments of gp18 containing short pseudo-accidental sequence in the C-terminal, while being soluble, have lost the ability for polysheath assembly. For the first time we succeeded in obtaining crystals of a soluble gp18 fragment containing 510 amino acids which, according to trypsin resistance, is similar to native protein monomer.

[Autoplaque formation in a Pseudomonas fluorescens strain: phage-like particles and transactivation of the defective phage]. / Avtoplakirovanie u shtammma Pseudomonas fluorescens: fagopodobnye chastitsy i transaktivatsiia defektnogo profaga.

Natural bacteriophages of Pseudomonas fluorescens are rare and its temperate phages have not been described so far. In search for these phages, we have found that one of the P. fluorescens strains forms numerous small transparent autoplaques of different size and shape, which contained material reproducible on the same strains. When centrifuged in a cesium chloride gradient, this material yielded a band in the density zone of about 1.3 g/cm3, where protein components or bacteriophages with a relatively low content of nucleic acid are usually located. In the band material, electron microscopy revealed phagelike particles with empty and mostly undamaged heads and tails carrying in their distal region a formation resembling contracted sheath. DNA isolated from the preparation consisted of two components: a distinct 54-kb fragment, and a diffuse fragment ranging in size from 20 to 9.5 kb. Treatment of the large DNA fragment with various endonucleases yielded 42.2- and 29.5-kb fragments (on average for different endonucleases); whereas the same treatment of the diffuse fragment yielded two- to three distinct fragments with the overall molecular sizes of 8.9 and 6.2 kb (for different nucleases). We have suggested that cells harbor two different genetic elements whose interaction results in the autoplaque appearance and in the formation of negative colonies after infection with the autoplaque material. One of the two elements displays properties of a defective prophage with disturbed DNA synthesis and assembly, whereas the other exhibits the properties of a transposable phage. After complementation or some other interaction between these elements (transactivation, prophage induction caused by repressor inactivation), a bulk of defective phage particles devoid of DNA and a few DNA-containing particles were produced. It remains unclear whether both DNA types are contained in the same or different particles. The phage (or a system of elements) referred to as PT3 is noninducible. The phage mutants forming larger negative colonies (NCs) were also revealed. Some of bacterial mutants resistant to PT3 infection produce the mutant phage with small and turbid NCs. PT3 produces no NCs on the lawns of other strains of the same or other pseudomonade species. This is the first case of describing a natural temperate bacteriophage in P. fluorescens. The two different elements of this phage may represent the same genome of the defective prophage divided into two portions within a bacterial chromosome, each of which is capable of packaging into the phage head.

Expression and properties of bacteriophage T4 gene product 11.

A plasmid vector for expression of bacteriophage T4 gene product 11 (gp11) in E. coli cells has been constructed. Gp11 is a baseplate protein that connects short tail fibers providing irreversible adsorption of the virus on a cell. A method based on chromatography on hydroxyapatite has been developed for purification of recombinant gp11. The protein is active in an in vitro complementation assay and transforms defective phage particles lacking gp11 into infective ones. Gel filtration data suggest that the biologically active protein is a trimer. According to CD spectroscopy and sequence analysis data, the polypeptide chain of gp11 contains not less than 20% alpha-helical segments, about 30% beta-structure, and belongs to the class of alpha/beta structural proteins.

Structure and folding of bacteriophage T4 gene product 9 triggering infection. II. Study Of conformational changes of gene product 9 mutants using monoclonal antibodies.

Gene product 9 (gp9) of bacteriophage T4, whose spatial structure we have recently solved to 2.3 A resolution, is a convenient model for studying the folding and oligomerization mechanisms of complex proteins. The gp9 polypeptide chain consists of 288 amino acids forming three domains. Three monomers, packed in parallel, assemble to a functionally active protein. The main aim of this work was to study conformational changes and trimerization of gp9 deletion mutants using monoclonal antibodies (mAbs). We selected a set of mAbs interacting with the amino, middle, and carboxyl regions of the protein, respectively. Eighteen mAbs bind to native as well as to denatured protein, and two mAbs bind to denatured protein only. Using mAbs, we found that deletions of the gp9 N-terminal region result in conformational changes in the middle and C-terminal domains. The study of mAb binding to the CDelta. truncated mutant by competitive ELISA and immunoblotting shows that the C-terminus of the gp9 sequence is essential for protein trimerization and stability. A single point substitution of the Gln282 residue causes formation of a labile trimer that has significant conformational changes in the protein domains. The results of our study show that folding and trimerization of gp9 is a cooperative process that involves all domains of the protein.

Structure of bacteriophage T4 gene product 11, the interface between the baseplate and short tail fibers.

Bacteriophage T4, like all other viruses, is required to be stable while being transmitted from host to host, but also is poised to eject efficiently and rapidly its double-stranded DNA genome to initiate infection. The latter is coordinated by the recognition of receptors on Escherichia coli cells by the long tail fibers and subsequent irreversible attachment by the short tail fibers. These fibers are attached to the baseplate, a multi-subunit assembly at the distal end of the tail. Recognition and attachment induce a conformational transition of the baseplate from a hexagonal to a star-shaped structure. The crystal structure of gene product 11 (gp11), a protein that connects the short tail fibers to the baseplate, has been determined to 2.0 A resolution using multiple wavelength anomalous dispersion with Se. This structure is compared to the trimeric structure of gp9, which connects the baseplate with the long tail fibers. The structure of gp11 is a trimer with each monomer consisting of 218 residues folded into three domains. The N-terminal domains form a central, trimeric, parallel coiled coil surrounded by the middle "finger" domains. The fingers emanate from the carboxy-terminal beta-annulus domain, which, by comparison with the T4 whisker "fibritin" protein, is probably responsible for trimerization. The events leading from recognition of the host to the ejection of viral DNA must be communicated along the assembled trimeric (gp9)(3) attached to the long tail fibers via the trimeric baseplate protein (gp10)(3) to the trimeric (gp11)(3) and the trimeric short tail fibers.

Structure and folding of bacteriophage T4 gene product 9 triggering infection. I. Production and properties of recombinant protein.

Gene product 9 (gp9, 288 amino acid residues per monomer, molecular weight 30.7 kD) of bacteriophage T4 triggers the baseplate reorganization and the sheath contraction after interaction of the long tail fibers with the receptors of the bacterial cell. In this work we have produced the recombinant protein and determined that gp9 is a stable homotrimer and active in in vitro complementation assay completing the defective phage particles which lack gp9. According to CD-spectroscopy data, the gp9 polypeptide chain contains 65-73% beta-structure and 11-16% alpha-helical segments, this being in good agreement with secondary structure prediction results. Additionally, we have constructed a set of plasmid vectors for expression of gp9 deletion mutants. The fragments with consecutive truncations of the N-terminus of the molecule, as well as the full-length protein, are trimers resistant to SDS treatment and decrease infective phage particle formation in in vitro complementation assay with native gp9. The deletion of the molecule C-terminal region results in failure of trimerization and decreases the stability of the protein.

The structure of bacteriophage T4 gene product 9: the trigger for tail contraction.

BACKGROUND: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the Escherichia coli host. The tail has a baseplate to whïch are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates DNA ejection. The baseplate is a multisubunit complex of proteins encoded by 15 genes. Gene product 9 (gp9) is the protein that connects the long tail fibers to the baseplate and triggers the tail contraction after virus attachment to a host cell. RESULTS: The crystal structure of recombinant gp9, determined to 2.3 A resolution, shows that the protein of 288 amino acid residues assembles as a homotrimer. The monomer consists of three domains: the N-terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded beta sandwich with a topology not previously observed; and the C-terminal domain is an eight-stranded, antiparallel beta sandwich having some resemblance to 'jelly-roll' viral capsid protein structures. CONCLUSIONS: The biologically active form of gp9 is a trimer. The protein contains flexible interdomain hinges, which are presumably required to facilitate signal transmission between the long tail fibers and the baseplate. Structural and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil domain is associated with the long tail fibers.

Polymerization of bacteriophage T4 tail sheath protein mutants truncated at the C-termini.

Gene 18 of bacteriophage T4 encodes the contractile protein of the tail sheath. Previous work has shown that the full-length recombinant gene product (gp) 18 of 658 amino acid residues assembles in Escherichia coli cells into a long polysheath structure. However, the gp18 mutants truncated at the N-termini form insoluble aggregates similar to inclusion bodies. In this study, six plasmid vectors expressing the recombinant gp18 proteins truncated at the C-termini have been constructed. The CDelta58, CDelta129, CDelta152, C[g1]72, CDelta248, and CDelta287 proteins contain 600, 529, 506, 486, 410, and 371 residues of the full-length gp18 molecule, respectively. All the recombinant proteins were soluble and, except for the CDelta287 mutant, were assembled into polysheath-related structures. Electron microscopy of negatively stained purified proteins was performed and the resulting images were analyzed by computing their Fourier transforms. The CDelta58 and CDelta129 mutants, in addition to forming common contracted-type polysheath structures, assembled into thinner filaments that we called "noncontracted polysheaths" (NCP). The CDelta152, CDelta172, and CDelta248 proteins assembled into the NCP type only. Image processing showed that the NCP filaments significantly differ from both extended sheaths of T4 particle and polysheaths. The structure of the NCP filaments might correspond to the transitional helices postulated by Moody (J. Mol. Biol., 1973, 80, 613-636) that appeared during the process of tail contraction. Our results suggest that a short region at the C-terminus of the CDelta129 protein determines the contractile properties of the gp18 molecule. The shortest, the CDelta287 protein, does not assemble into regular structures, thus indicating that a sequence's stretch at the C-end of the CDelta248 mutant might be responsible for polymerization of gp18.

Co-expression of gene 31 and 23 products of bacteriophage T4.

Folding of the major capsid protein of bacteriophage T4 encoded by gene 23 is aided by Escherichia coli GroEL chaperonin and phage co-chaperonin gp31. In the absence of gene product (gp) 31, aggregates of recombinant gp23 accumulate in the cell similar to inclusion bodies. These aggregates can be solubilized with 6 M urea. However, the protein cannot form regular structures in solution. A system of co-expression of gp31 and gp23 under the control of phage T7 promoter in E. coli cells has been constructed. Folding of entire-length gp23 (534 amino acid residues) in this system results in the correctly folded recombinant gp23, which forms long regular structures (polyheads) in the cell.