Head Of T4 Research Articles

Gated Ethidium- and Bleomycin-Loading in Phage T4 That Is Subsequently Purified Leak-Free

Chemotherapy-inhibiting tumor cell evolution to drug-resistance is potentially suppressed by using a drug delivery vehicle (DDV) that has gating. Gating would be used to increase tumor-selectivity of delivery of DDV packaged drug. Tumor-selectivity increase would make possible increase in tumor-delivered drug dose, which would suppress opportunities to evolve drug resistance. Currently used DDVs do not have gating but gating is a natural feature of some bacteriophages (phages). Phage T4, which has recently been found highly persistent in murine blood, is a potential gated DDV. Thus, here, we proceed towards a T4-DDV by developing (1) improved procedure for generating high concentrations and amounts of phage T4, (2) elevated temperature-driven gate-opening and ethidium- and bleomycin-loading, and (3) purification of loaded T4 by rate zonal centrifugation. We test for loading by native agarose gel electrophoresis (AGE) with fluorescence detection. We observe loading in both phage T4 and T4 (tail-free) heads. The loaded particles have an openable, closed gate. Stored, mature T4 phages and phage heads do not release ethidium during at least a month at 4 °C and 6 days at 37 and 42 °C. Tumor-specific T4 phage delivery is projected via both the EPR effect and high T4 persistence.

Biphasic Packing of DNA and Internal Proteins in Bacteriophage T4 Heads Revealed by Bubblegram Imaging.

The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins (“ejection proteins”) have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions; it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100–110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.

EPIDEMIOLOGY STUDY OF VIRAL NERVOUS NECROSIS (VNN) IN MALAYSIAN GROUPERS: SEQUENCE ANALYSIS STRATEGIES AND QUASISPECIES DETERMINANT’S

EPIDEMIOLOGY STUDY OF VIRAL NERVOUS NECROSIS (VNN) IN MALAYSIAN GROUPERS: SEQUENCE ANALYSIS STRATEGIES AND QUASISPECIES DETERMINANT’S

Functional and Structural Characterization of PaeM, a Colicin M-like Bacteriocin Produced by Pseudomonas aeruginosa

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg(2+) ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

Molecular Mimicry Between Spermatozoa and Bacteria

Sperm immobilization factor isolated from Staphylococcus aureus immobilizes human spermatozoa as well as motile bacteria, showing that sperm immobilization factor receptor might be shared by bacteria and human spermatozoa. Thus, we sought to identify a common sperm immobilization factor binding receptor on spermatozoa and bacteria. Sperm immobilization factor was isolated from S. aureus. Sperm immobilization factor binding receptors were isolated from spermatozoa and bacteria. Antisperm antibodies in humans cross-reacted with bacteria antibodies. Thus, molecular similarities between determinants of spermatozoa and pathogenic microorganisms can be inferred. Sperm immobilization factor isolated from S. aureus immobilized spermatozoa (150 μg/ml) and motile bacteria, including Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis (500 μg/ml). This showed the presence of a common sperm immobilization factor binding conformation on spermatozoa and bacteria. Furthermore, receptors binding sperm immobilization factor were extracted and purified from spermatozoa, E. coli, P. aeruginosa and P. mirabilis. Molecular mimicry between spermatozoa and bacteria was confirmed by observing the blockage of spermatozoa immobilization by sperm immobilization factor in the presence of receptors isolated from spermatozoa, E. coli, P. aeruginosa or P. mirabilis. Also, a higher concentration of sperm immobilization factor (200 μg/ml) caused sperm death. Blocking the death of spermatozoa induced by sperm immobilization factor in the presence of these receptors provided further evidence for a common receptor. Results provide evidence for molecular similarity between bacteria and spermatozoa.

Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

Investigation of bacteriophage T4 by atomic force microscopy.

Bacteriophage T4 was visualized using atomic force microscopy (AFM). The images were consistent with, and complementary to electron microscopy images. Head heights of dried particles containing DNA were about 75 nm in length and 60 nm in width, or about 100 nm and 85 nm respectively when scanned in fluid. The diameter of hydrated tail assemblies was 28 nm and their lengths about 130 nm. Seven to eight pronounced, right-handed helical turns with a pitch of 15 nm were evident on the tail assemblies. At the distal end of the tail was a knob shaped mass, presumably the baseplate. The opposite end, where the tail assembly joins the head, was tapered and connected to the portal complex, which was also visible. Phage that had ejected their DNA revealed the internal injection tube of the tail assembly. Heads disrupted by osmotic shock yielded boluses of closely packed DNA that unraveled slowly to expose threads composed of multiple twisted strands of nucleic acid. Assembly errors resulted in the appearance of several percent of the phage exhibiting two rather than one tail assemblies that were consistently oriented at about 72° to one another. No pattern of capsomeres was visible on native T4 heads. A mutant that is negative for the surface proteins hoc and soc, however, clearly revealed the icosahedral arrangement of ring shaped capsomeres on the surface. The hexameric rings have an outside diameter of about 14 nm, a pronounced central depression, and a center-to-center distance of 15 nm. Phage collapsed on cell surfaces appeared to be dissolving, possibly into the cell membrane.

Engineered T4 Viral Nanoparticles for Cellular Imaging and Flow Cytometry

Viruses are of particular interest as scaffolds for biotechnology applications given their wide range of shapes and sizes and the possibility to modify them with a variety of functional moieties to produce useful virus-based nanoparticles (VNPs). In order to develop functional VNPs for cell imaging and flow cytometry applications, we used the head of the T4 bacteriophage as a scaffold for bioconjugation of fluorescent dyes. Bacteriophage T4 is a double-stranded DNA virus with an elongated icosahedron head and a contractile tail. The head is ∼100 nm in length and ∼90 nm in width. The large surface area of the T4 head is an important advantage for the development of functional materials since it can accommodate significantly larger numbers of functional groups, such as fluorescent dyes, in comparison with other VNPs. In this study, Cy3 and Alexa Fluor 546 were chemically incorporated into tail-less T4 heads (T4 nanoparticles) for the first time, and the fluorescent properties of the dye-conjugated nanoparticles were characterized. The T4 nanoparticles were labeled with up to 19 000 dyes, and in particular, the use of Cy3 led to fluorescent enhancements of up to 90% compared to free Cy3. We also demonstrate that the dye-conjugated T4 nanoparticles are structurally stable and that they can be used as molecular probes for cell imaging and flow cytometry applications.

The measurement of lysosomal phospholipase A2 activity in plasma

A deficiency of lysosomal phospholipase A2 (LPLA2) causes macrophage-associated phospholipidosis, suggesting that the enzyme is important in the lipid catabolism. Because LPLA2 is secreted by macrophages, extracellular LPLA2 activity may potentially reflect a change in macrophage activation. In this report, the detection of LPLA2 activity in plasma was established by the measurement of the transacylase activity of LPLA2 under acidic conditions. No transacylase activity of LPLA2 was detected in normal human plasma when the plasma was incubated with liposomes consisting of 1,2-dioleoylphosphatidylcholine/sulfatide/N-acetylsphingosine (NAS) at pH 4.5. However, the transacylase activity in the plasma was detected when liposomes consisting of 1,2-dioleoylphosphatidylglycerol/NAS were used as a substrate. To establish the specificity of the assay, ceramide transacylase activity was detected in the plasma of wild-type mice. By contrast, the plasma obtained from LPLA2-knockout mice had no measurable transacylase activity under the same conditions. The enzymatic activity of recombinant LPLA2 was inhibited by treatment with methylarachidonylfluorophosphonate. The inhibitor also suppressed the transacylase activity observed in both normal human and wild-type mouse plasma, establishing that the transacylase activity observed in plasma is due to LPLA2. Plasma LPLA2 activity may be a useful bioassay marker for the identification of LPLA2-related disorders.

Abnormal degradation of erythrocyte membrane proteins in Down's syndrome

HereditasVolume 106, Issue 1 p. 151-153 Open Access Abnormal degradation of erythrocyte membrane proteins in Down's syndrome M. GACZYNSKA, M. GACZYNSKA Chair of Biophysics, University of Lódź, 90–237 Lódź Department of BiochemistrySearch for more papers by this authorJ. ROSIN, J. ROSIN Institute of Occupational Medici, Department of Physiology, Institute of Fundamental SciencesSearch for more papers by this authorG. BARTOSZ, G. BARTOSZ Chair of Biophysics, University of Lódź, 90–237 Lódź Department of BiochemistrySearch for more papers by this authorJ. KeDZIORA, Corresponding Author J. KeDZIORA WAM and 4th Clinical Hospital*Department of Physiology Institute of Fundamental Sciences “WAM” Medical Academy Plac 9 Maja No. 1 90–647 Lódź. PolandSearch for more papers by this authorJ. JESKE, J. JESKE Medical Academy of LódźSearch for more papers by this author M. GACZYNSKA, M. GACZYNSKA Chair of Biophysics, University of Lódź, 90–237 Lódź Department of BiochemistrySearch for more papers by this authorJ. ROSIN, J. ROSIN Institute of Occupational Medici, Department of Physiology, Institute of Fundamental SciencesSearch for more papers by this authorG. BARTOSZ, G. BARTOSZ Chair of Biophysics, University of Lódź, 90–237 Lódź Department of BiochemistrySearch for more papers by this authorJ. KeDZIORA, Corresponding Author J. KeDZIORA WAM and 4th Clinical Hospital*Department of Physiology Institute of Fundamental Sciences “WAM” Medical Academy Plac 9 Maja No. 1 90–647 Lódź. PolandSearch for more papers by this authorJ. JESKE, J. JESKE Medical Academy of LódźSearch for more papers by this author First published: March 1987 https://doi.org/10.1111/j.1601-5223.1987.tb00248.xAboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Literature cited Asano, K., Osawa, Y., Yanagisawa, N., Takahashi, Y. and Oshima, M. 1985. Erythrocyte membrane abnormalities in patients with amyotrophic chorea with acanthocythosis. Part 2. Abnormal degradation of membrane proteins. J. Neurol. Sci. 68: 161– 173. Bartosz, G. and Kȩdziora, J. 1982. Aging of the erythrocyte. XX. Decreased red cell life span in Down's syndrome. Cell Biol. Inl. Rep. 6: 1119– 1122. Bartosz, G., Soszynski, M. and Kȩdziora J. 1982. Aging of the erythrocyte. VI. Accelerated red cell membranes aging in Down's syndrome Cell Biol. Int. Rep. 6: 73– 77. Gaczyńska, M., Bartosz, G., Rosin, J. and Soszyński, M. 1985. Proteolytic self-digestion of bovine erythrocyte membranes. Int. J. Biochem. 17: 1237– 1245. Kay, M.M.B., Goodman, S., Sörensen, K., Whitfield, C., Wong, P., Zaki, L. and Rudloff, V. 1983. Senescent cell antigen is immunologically related to band 3. Proc. Natl. Acad. Sci. U.S.A 80: 1631– 1635. Kȩdziora, J., Hly7nacute;czak, A. J., Jeske. J. and Kański, M. 1972. Physicochemical properties and metabolism of erythro-cytes in children with Down's syndrome. Pol. Endocrinol. 23: 63– 70. Kȩdziora, J., Bartosz, G., Koter, M. and Leyko, W. 1982a. Erythrocyte membrane acetylcholinesterase in Down's syndrome. IRCS Med. Sci. 10: 785. Kȩdziora, J., Koter, M. and Bartosz, G. 1982b. Influence of acrocentric chromosome G-21 on the structural changes of erythrocyte membranes. Stud. Biophys. 90: 119– 120. Koter, M., Kȩdziora, J., Bartosz, G. and Leyko, W. 1982. Down's syndrome: permeability of the erythrocyte membrane for spin-labeled non-electrolytes Experientia 38: 1447. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680– 685. Melloni. E., Salamino, F., Sparatore, B., Michetti, M. and Pontremoli, S., 1982. Cooperation between soluble and membrane bound proteinases in the degradation of β-hemoglobin chains in intact human erythrocytes. Archs. Biochem. Biophys. 216: 495– 502. Volume106, Issue1March 1987Pages 151-153 ReferencesRelatedInformation

The neck of bacteriophage T4 is a ring-like structure formed by a hetero-oligomer of gp13 and gp14

After packaging of DNA into the head of bacteriophage T4 is completed, a neck is formed at the portal vertex of the head to be ready for the tail attachment. The main components of the neck are gp13 and gp14 (gp: gene product), which consist of 309 and 256 amino acid residues, respectively. In order to elucidate the structure and subunit arrangement in the neck, overexpression systems of gene 13 and gene 14 were constructed and purified to homogeneity. Far-UV circular dichroism (CD) spectra of gp13 and gp14 indicated that gp13 is rich in α-helices whereas gp14 is rich in β-sheets. Sedimentation velocity analysis of gp13 and gp14 revealed that both proteins are present as monomers in solution. The frictional ratios ( f/ f 0) of the two proteins indicated that gp14 has a more elongated shape than gp13. Although isolated gp13 and gp14 do not interact with each other when mixed under physiological conditions, they form a hetero-oligomer complex with the stoichiometry of 10:5 after treatment with ammonium sulfate. Electron microscopy of this complex has shown that it forms a ring-like structure of 15 nm in diameter.

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

Characterization of Gel-separated Glycoproteins Using Two-step Proteolytic Digestion Combined with Sequential Microcolumns and Mass Spectrometry

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.

Molecular architecture of the prolate head of bacteriophage T4

The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached. The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-A resolution by using cryo-electron microscopy. The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers T(end) = 13 laevo for the icosahedral caps and T(mid) = 20 for the midsection. Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein (hoc) and small outer capsid protein (soc), are clearly evident in the reconstruction. The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97. The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc(-) and hoc(-)soc(-) T4 structures.

Green fluorescent protein as a probe of rotational mobility within bacteriophage T4

Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads and proheads as a probe of the internal environment. Targeting was accomplished with internal protein III (IPIII) fusion proteins or capsid targeting sequence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII. Recombinant phage T4[CTS▾IPIII▾GFP], T4[CTS▾IPIII TGFP], and T4[CTS▾GFP] packaged GFP fusion proteins and processed them at cleavage sites designated ▾. Steady-state and time-resolved fluorescence measurements suggest that packaged GFP is concentrated to a high density, that fusion protein IPIII TGFP occurs in a tightly clustered arrangement, and that the internal milieu of the phage head reduces rotational mobility of GFP. Phage, but not proheads, packaged with fusion protein IPIII TGFP gave an unexpectedly lower anisotropy than phage and proheads packaged with GFP, which suggests IPIII TGFP is bound to DNA in a manner that causes close associations between GFP molecules resulting in homotransfer between fluorophores within packaged phage. Targeting of reporter proteins into active virions is a promising approach for determining the structure of the condensed DNA, and properties of encapsidated viral enzymes.

Concentration evaluation of chromatin in unstained resin-embedded sections by means of low-dose ratio-contrast imaging in STEM

Quantitative STEM with the imaging mode of ratio-contrast was investigated in order to evaluate the local concentration of DNA in situ for different kinds of DNA plasms in terms of intracellular packing densities (p.d.). The ability of ratio imaging to suppress thickness variations provided the basis to use unstained sections from cryofixed and freeze-substituted material. The DNA p.d. within the nucleoid of E. coli was determined to be about 100 mg ml -1. Quantitative data concerning the p.d. of DNA in condensed eukaryotic chromatin assuming equal amounts of DNA and protein were evaluated for the first time: approximately 400 mg ml -1 chromatin which corresponds to 200 mg ml -1 DNA. The p.d. of DNA in chromosomes from the dinoflagellate Amphidinium carterae, a eukaryote devoid of histones and with only small relative amounts of histone-like protein, was also found to be of the order of 200 mg ml -1. The highest p.d. of DNA was measured for the head of the bacteriophage T4 with more than 800 mg ml -1, in fair agreement with previous calculations. The results provide further support for a condensation mode of low protein chromatins that involves a liquid-crystalline organization of the DNA filaments.

A proposed structure of bacteriophage T4 gene product 22—A major prohead scaffolding core protein

Gene 22 of bacteriophage T4 encodes a major prohead scaffolding core protein of 269 amino acid residues. From its nucleotide sequence the gene product (gp) 22 has a predicted M r of 29.9 and a p I of 4.3. The protein is rich in charged residues (glutamic acid and lysine) and contains low amounts of proline and glycine and no cysteine residues. We suggest that gp22 undergoes limited proteolytic processing which eliminates the short C-terminal piece from the molecule during the early steps of prohead assembly. Most amino acid residues of the gp22 polypeptide chain (80%) have an α-helical conformation and form seven peculiar α-helices. A model suggesting the spatial organization of gp22 is presented. Three long α-helices numbered 1 (1A and 1B), 3, and 5 (5A and 5B) are packed in an antiparallel fashion along the major axis of the road-shaped molecule. Two rather short α-helices (2 and 4) are located at the distal and proximal ends of the protein molecule, respectively. Helix number 2, which is a proteolytic fragment of gp22 found in mature T4 heads, is packed with helices 1A and 3, similar to a novel element of supersecondary structure, the αα-corner. Helix number 4 probably interacts with the gp20 connector of the prohead. The implications of the structure of the gp22 molecule for the assembly of the prohead core are discussed.

Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured, Deep-Etched Replicas

The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.

Head structure of bacteriophages T2 and T4

The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods. The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis. Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*.