Identification of the major proteins of the virions of bacteriophage ZF40 Pectobacterium carotovorum.

The vast variety of bacteriophages and the uniqueness of their individual representatives dictate to perform the detailed study of the actual phage-cell interactions, the virion morphogenesis and morphopoiesis in particular. An analysis of the complete genome sequence of the temperate phage ZF40 Pectobacterium carotovorum has shown that it is a representative of a unique group of phages of the Myoviridae family [Comeau A. M, Tremblay D., Moineau S., Rattei T., Kushkina A. I, Tovkach F I., H.M. Krisch, H.W. Ackermann Phage Morphology Recapitulates Phylogeny: The Comparative Genomics of a New Group of Myoviruses // PLoS ONE.--July 2012. - 7. - N 7. - e40102]. Characteristic features of these viruses are a small length of the tail compared with the diameter of the capsid and a complicated pattern of the tail sheath, leading to its criss-cross striation. In the presented article the major proteins were identified by means of the SDS-PAGE method: the head proteins (mp2: 33.9 kDa), the sheath (mp1: 39.2 kDa) and the tail tube ones (mp3: 19.9 kDa). It was proved that the mp2 molecular weight is the same with the gp46, the putative major capsid protein derived from the results of the genome sequencing. Therefore, it is still not determined whether the gp46 (mp2) of the virulent mutant 421 of the phage ZF40 is exposed to post-translational modification in the course of the phage particle maturation during its development in the cells of the strain M2-4/50RI P. carotovorum. To study the morphogenetic development pathways it was proposed to use the phage variants that form an excess of individual components of the virion: capsids, procapsids and separate tails propagated on different hosts.

[The beta-helical domain of bacteriophage T4 controls the folding of the fragment of long tail fibers in a chimeric protein].

The key stage of the infection of the Escherichia coli cell with bacteriophage T4, the binding to the surface of the host cell, is determined by the specificity of the long tail fiber proteins of the phage, in particular, gp37. The assembly and oligomerization of this protein under natural conditions requires the participation of at least two additional protein factors, gp57A and gp38, which strongly hinders the production of the recombinant form of gp37. To overcome this problem, a modern protein engineering strategy was used, which involves the construction of a chimeric protein containing a carrier protein that drives the correct folding of the target protein. For this purpose, the trimeric beta-helical domain of another protein of phage T4, gp5, was used. It was shown that this domain, represented as a rigid trimeric polypeptide prism, has properties favorable for use as a protein carrier. A fragment of protein gp37 containing five pentapeptides repeats, Gly-X-His-X-His, which determine the binding to the receptors on the bacterial cell surface, was fused in a continuous reading frame to the C-terminus of the domain of gp5. The resulting chimeric protein forms a trimer that has the native conformation of gp37 and exhibits biological activity.

The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein.

The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.

Two-chaperone assisted soluble expression and purification of the bacteriophage T4 long tail fibre protein gp37.

Bacteriophage T4 recognises its host cells through its long tail fibre protein gene product (gp) 37. Gp37 is a protein containing 1026 amino acids per monomer, forming a fibrous parallel homotrimer at the distal end of the long tail fibres. The other distal half-fibre protein, gp36, is much smaller, forming a trimer of 221 amino acids per monomer. Functional and structural studies of gp37 have been hampered by the inability to produce suitable amounts of it. We produced soluble gp37 by co-expression with two bacteriophage T4-encoded chaperones in a two-vector system; co-expression with each chaperone separately did not lead to good amounts of correctly folded, trimeric protein. An expression vector for the bacteriophage T4 fibrous protein chaperone gp57 was co-transformed into bacteria with a compatible bi-cistronic expression vector containing bacteriophage T4 genes 37 and 38. A six-histidine tag is encoded amino-terminal to the gp37 gene. Recombinant trimeric gp37, containing the histidine tag and residues 12-1026 of gp37, was purified from lysed bacteria by subsequent nickel-affinity, size exclusion and strong anion exchange column chromatography. Yields of approximately 4 mg of purified protein per litre of bacterial culture were achieved. Electron microscopy confirmed the protein to form fibres around 63 nm long, presumably gp36 makes up the remaining 11 nm in the intact distal half-fibre. Purified, correctly folded, gp37 will be useful for receptor-binding studies, high-resolution structural studies and for specific binding and detection of bacteria.

The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria.

The contractile tail of bacteriophage T4 is a molecular machine that facilitates very high viral infection efficiency. Its major component is a tail sheath, which contracts during infection to less than half of its initial length. The sheath consists of 138 copies of the tail sheath protein, gene product (gp) 18, which surrounds the central non-contractile tail tube. The contraction of the sheath drives the tail tube through the outer membrane, creating a channel for the viral genome delivery. A crystal structure of about three quarters of gp18 has been determined and was fitted into cryo-electron microscopy reconstructions of the tail sheath before and after contraction. It was shown that during contraction, gp18 subunits slide over each other with no apparent change in their structure.

Foldon-guided self-assembly of ultra-stable protein fibers.

A common objective in protein engineering is the enhancement of the thermodynamic properties of recombinant proteins for possible applications in nanobiotechnology. The performance of proteins can be improved by the rational design of chimeras that contain structural elements with the desired properties, thus resulting in a more effective exploitation of protein folds designed by nature. In this paper, we report the design and characterization of an ultra-stable self-refolding protein fiber, which rapidly reassembles in solution after denaturation induced by harsh chemical treatment or high temperature. This engineered protein fiber was constructed on the molecular framework of bacteriophage P22 tail needle gp26, by fusing its helical core to the foldon domain of phage T4 fibritin. Using protein engineering, we rationally permuted the foldon upstream and downstream from the gp26 helical core and characterized gp26-foldon chimeras by biophysical analysis. Our data demonstrate that one specific protein chimera containing the foldon immediately downstream from the gp26 helical core, gp26(1-140)-F, displays the highest thermodynamic and structural stability and refolds spontaneously in solution following denaturation. The gp26-foldon chimeric fiber remains stable in 6.0 M guanidine hydrochloride, or at 80 degrees C, rapidly refolds after denaturation, and has both N and C termini accessible for chemical/biological modification, thereby representing an ideal platform for the design of self-assembling nanoblocks.

An intersubunit active site between supercoiled parallel beta helices in the trimeric tailspike endorhamnosidase of Shigella flexneri Phage Sf6.

Sf6 belongs to the Podoviridae family of temperate bacteriophages that infect gram-negative bacteria by insertion of their double-stranded DNA. They attach to their hosts specifically via their tailspike proteins. The 1.25 A crystal structure of Shigella phage Sf6 tailspike protein (Sf6 TSP) reveals a conserved architecture with a central, right-handed beta helix. In the trimer of Sf6 TSP, the parallel beta helices form a left-handed, coiled-beta coil with a pitch of 340 A. The C-terminal domain consists of a beta sandwich reminiscent of viral capsid proteins. Further crystallographic and biochemical analyses show a Shigella cell wall O-antigen fragment to bind to an endorhamnosidase active site located between two beta-helix subunits each anchoring one catalytic carboxylate. The functionally and structurally related bacteriophage, P22 TSP, lacks sequence identity with Sf6 TSP and has its active sites on single subunits. Sf6 TSP may serve as an example for the evolution of different host specificities on a similar general architecture.

Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil.

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.

Maximizing recovery of native protein from aggregates by optimizing pressure treatment.

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 +/- 0.9 to 47.4 +/- 19.6 microg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.

Structural organisation of the head-to-tail interface of a bacterial virus.

In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-naïve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.

Pulse-chase analysis of the in vivo assembly of the bacteriophage T4 tail.

The in vivo assembly pathway of the complex tail of bacteriophage T4 virus was determined using pulse-chase analysis as a non-invasive alternative to the in vitro experiments previously used to map assembly. Bacteriophage T4 mutants defective in head assembly were used to infect cultures of Escherichia coli in order to study tail assembly in isolation. Beginning with the onset of late protein synthesis, the cultures were labeled continuously with [(3)H]leucine to normalize against subsequent sample losses. After completed tails had begun to accumulate at a constant rate, the cultures were pulsed with [(35)S]methionine, and then chased. Completed tails were purified at one minute intervals for the next 30 minutes and their proteins separated electrophoretically and counted by liquid scintillation. Total (35)S incorporation into each protein rose and then leveled off as the chase of unlabeled methionine flushed the label through the pools of soluble proteins and assembly intermediates and into completed tails. The inflection point in the sigmoidal (35)S-incorporation curve of each protein marks the maximal uptake of (35)S within that pool just before the effect of the chase becomes apparent and the curve begins to level off. The length of the delay in the apparent chase time reflects the position of that protein in the pathway. The closer the assembly point to the end of the pathway, the sooner the chase appears, revealing the relative order of assembly. As predicted, tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest inflection, while those earlier in the pathway take longer to chase. Of the 17 tail proteins analyzed, 14 are in agreement with the established in vitro pathway. The other three, gp15, gp10 and gp53, have helped us to develop a model that offers a plausible explanation for their altered chase times.

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.

Properties of recombinant bacteriophage T4 tail sheath protein and its deletion fragments.

A vector for expression of recombinant bacteriophage T4 tail sheath protein (gp18) under control of phage T7 promoter in Escherichia coli cells has been constructed. The entire length recombinant gp18 (659 amino acids) polymerizes in vivo into extended polysheaths. To study gp18 folding mechanisms, six vectors for expression of deletion mutants have been constructed. Three proteins--1N, 2N, and 3N--contain, respectively, 268, 316, and 372 amino acids of the gp18 N-tail region. The other three fragments--1C, 2C, and 3C--contain, respectively, 455, 356, and 288 amino acids of the gp18 C-tail. The fragments 1N, 2N, 1C, 2C, and 3C form insoluble aggregates during expression. However, fragment 3N accumulates in soluble form in the cellular cytoplasm and does not form polymeric structures; this has allowed an effective purification method to be developed for it. The interaction of monoclonal antibodies against recombinant gp18 with protein fragments and with phage sheath before and after contraction has been studied. The fragment 3N seems to be a stable domain of native phage sheath gp18.

Stoichiometry and domainal organization of the long tail-fiber of bacteriophage T4: a hinged viral adhesin.

The long-tail fibers (LTFs) form part of bacteriophage T4's apparatus for host cell recognition and infection, being responsible for its initial attachment to susceptible bacteria. The LTF has two parts, each approximately 70 to 75 nm long; gp34 (140 kDa) forms the proximal half-fiber, while the distal half-fiber is composed of gp37 (109 kDa), gp36(23 kDa) and gp35 (30 kDa). LTFs have long been thought to be dimers of gp34, gp37 and gp36, with one copy of gp35. We have used mass mapping by scanning transmission electron microscopy (STEM), quantitative SDS-PAGE, and computational sequence analysis to study the structures of purified LTFs and half-fibers of both kinds. These data establish that the LTF is, in fact, trimeric, with a stoichiometry of gp34: gp37: gp36: gp35 = 3:3:3:1. Averaged images of stained and unstained molecules resolve the LTF into a linear stack of 17 domains. At the proximal end is a globular domain of approximately 145 kDa that becomes incorporated into the baseplate. It is followed by a rod-like shaft (33 x 4 mm; 151 kDa) which correlates with a cluster of seven quasi repeats, each 34 to 39 residues long. The proximal half-fiber terminates in three globular domains. The distal half-fiber consists of ten globular domains of variable size and spacing, preceding a needle-like end domain (15 x 2.5 nm; 31 kDa). The LTF is rigid apart from hinges between the two most proximal domains, and between the proximal and distal half-fibers. The latter hinge occurs at a site of local non-equivalence (the "kneecap") at which density, correlated with the presence of gp35, bulges asymmetrically out on one side. Several observations indicate that gp34 participates in the sharing of conserved structural modules among coliphage tail-fiber genes to which gp37 was previously noted to subscribe. Two adjacent globular domains in the proximal half-fiber match a pair of domains in the distal half-fiber, and the rod domain in the proximal half-fiber resembles a similar domain in the T4 short tail-fiber (gp12). Finally, possible structures are considered; combining our data with earlier observations, the most likely conformation for most of the LTF is a three-stranded beta-helix.

Structural and physicochemical analysis of the contractile MM phage tail and comparison with the bacteriophage T4 tail.

The three-dimensional (3-D) structure of the bacteriophage MM extended tail has been determined from electron micrographs of negatively stained specimens and compared with 3-D models of coprocessed extended bacteriophage T4 tails. Accordingly, the phage MM extended tail exhibits an axial repeat of 3.8 nm and can be indexed according to the integer helical selection rule l = -3n + 7m (n = 6n') compared to 4.1 nm and l = -2n + 7m (n = 6n') for the T4 phage tail. Compared to the T4 tail sheath, which reveals a stacked-disk-like appearance, the MM tail exhibits a more open structure, yielding an arrow-head-like appearance. Although the phage MM extended tail sheath is more stable than the T4 tail sheath under low-ionic-strength conditions, various chemical treatments of the MM tail sheath revealed responses, notably disassembly and contraction, similar to those previously described for the T4 tail sheath. Extended tails and their structural components contained in phage lysates or prepared by chemical degradation were compared in the EM, and the mass-per-length values of extended tails and tail tubes were determined by quantitative scanning transmission electron microscopy and compared to the corresponding values computed from the respective 3-D mass density maps. Accordingly, masses of 111 and 135 kDa/nm were obtained for the MM and T4 phage tail sheaths, respectively, with the corresponding tail tubes calculated at 19.3 and 25.5 kDa/nm, respectively. Although negative staining and freeze drying/metal shadowing of the two tails revealed different extended tail sheath structures, freeze-dried/metal-shadowed specimens of their contracted tails revealed very similar 6-fold symmetric axial repeats, with the subunits arranged on a pseudo-12-fold symmetric surface lattice following the integer helical selection rule l = n + 11m. In both cases tail contraction started at the baseplate and propagated headward as a wave forming a contraction gradient with a sharp boundary.