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J F Conway

Publications and source records attributed to J F Conway.

At least 37 records · Page 2Linked to original sources
PubMed1997

Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA.

The capsid protein of hepatitis B virus, consisting of an "assembly" domain (residues 1-149) and an RNA-binding "protamine" domain (residues 150-183), assembles from dimers into icosahedral capsids of two different sizes. The C terminus of the assembly domain (residues 140-149) functions as a morphogenetic switch, longer C termini favoring a higher proportion of the larger capsids, it also connects the protamine domain to the capsid shell. We now have defined the location of this peptide in capsids assembled in vitro by engineering a mutant assembly domain with a single cysteine at its C terminus (residue 150), labeling it with a gold cluster and visualizing the cluster by cryo-electron microscopy. The labeled protein is unimpaired in its ability to form capsids. Our density map reveals a single undecagold cluster under each fivefold and quasi-sixfold vertex, connected to sites at either end of the undersides of the dimers. Considering the geometry of the vertices, the C termini must be more crowded at the fivefolds. Thus, a bulky C terminus would be expected to favor formation of the larger (T = 4) capsids, which have a greater proportion of quasi-sixfolds. Capsids assembled by expressing the full-length protein in Escherichia coli package bacterial RNAs in amounts equivalent to the viral pregenome. Our density map of these capsids reveals a distinct inner shell of density-the RNA. The RNA is connected to the protein shell via the C-terminal linkers and also makes contact around the dimer axes.

Capsid↗
PubMed1997

Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy.

Despite the development of vaccines, the hepatitis B virus remains a major cause of human liver disease. The virion consists of a lipoprotein envelope surrounding an icosahedral capsid composed of dimers of a 183-residue protein, 'core antigen' (HBcAg). Knowledge of its structure is important for the design of antiviral drugs, but it has yet to be determined. Residues 150-183 are known to form a protamine-like domain required for packaging RNA, and residues 1-149 form the 'assembly domain' that polymerizes into capsids and, unusually for a capsid protein, is highly alpha-helical. Density maps calculated from cryo-electron micrographs show that the assembly domain dimer is T-shaped: its stem constitutes the dimer interface and the tips of its arms make the polymerization contacts. By refining the procedures used to calculate the map, we have extended the resolution to 9 A, revealing major elements of secondary structure. In particular, the stem, which protrudes as a spike on the capsid's outer surface, is a 4-helix bundle, formed by the pairing of alpha-helical hairpins from both subunits.

Capsid↗
PubMed1997

The making and breaking of symmetry in virus capsid assembly: glimpses of capsid biology from cryoelectron microscopy.

Virus capsids constitute a diverse and versatile family of protein-bound containers and compartments ranging in diameter from approximately 200 A (mass approximately 1 MDa) to >1500 A (mass>250 MDa). Cryoelectron microscopy of capsids, now attaining resolutions down to 10 A, is disclosing novel structural motifs, assembly mechanisms, and the precise locations of major epitopes. Capsids are essentially symmetric structures, and icosahedral surface lattices have proved to be widespread. However, many capsid proteins exhibit a remarkable propensity for symmetry breaking, whereby chemically identical subunits in distinct lattice sites have markedly different structures and packing relationships. Temporal differences in the conformation of a given subunit are also manifested in the large-scale conformational changes that accompany capsid maturation. Larger and more complex capsids, such as DNA bacteriophages and herpes simplex virus, are formed not by simple self-assembly, but under the control of tightly regulated programs that may include the involvement of viral scaffolding proteins and cellular chaperonins, maturational proteolysis, and conformational changes on an epic scale. In addition to its significance for virology, capsid-related research has implications for biology in general, relating to the still largely obscure assembly processes of macromolecular complexes that perform many important cellular functions.

Antigens, Viral↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Dimorphism of hepatitis B virus capsids is strongly influenced by the C-terminus of the capsid protein.

Hepatitis B virus (HBV) is an enveloped virus with an icosahedral capsid. Its homodimeric capsid protein ("core antigen") assembles into particles of two sizes, one with T = 3 icosahedral symmetry (90 dimers) and the other with T = 4 symmetry (120 dimers). We have investigated this assembly process in vitro, using a variety of purified, bacterially expressed, capsid proteins. All of our constructs lacked the predominantly basic C-terminal 34 amino acids of the full-length capsid protein (183 amino acids) and were further truncated to terminate at specific points between residues 138 and 149. While the smallest construct (138 residues) did not assemble into capsids, those terminating at residue 140, and beyond, assembled into mixtures of T = 3 and T = 4 particles. The two kinds of capsids could be separated on sucrose gradients and did not interconvert upon protracted storage. The proportion of T = 3 capsids, assayed by sucrose gradient fractionation, analytical ultracentrifugation, and cryoelectron microscopy, was found to increase systematically with larger deletions from the C-terminus. The variant terminating at residue 149 formed approximately 5% of T = 3 capsids, while the 140-residue protein produced approximately 85% of this isomorph. For the 147-residue capsid protein, the structures of both capsids were determined to 17 A resolution by three-dimensional reconstruction of cryoelectron micrographs. In these density maps, the boundaries of the constituent dimers can be clearly seen and the quaternary structures of the two capsids compared. The arrangement of dimers around their icosahedral five-fold axes is almost identical, whereas the quasi-six-fold arrangements of dimers are distinctly different.

Amino Acid Sequence↗
PubMed1995

Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system.

Bacteriophage capsid assembly pathways provide excellent model systems to study large-scale conformational changes and other mechanisms that regulate the formation of macromolecular complexes. These capsids are formed from proheads: relatively fragile precursor particles which mature by undergoing extensive remodeling. Phage HK97 employs novel features in its strategy for building capsids, including assembly without a scaffolding protein, and the formation of a network of covalent cross-links between neighboring subunits in the mature virion. In addition, proteolytic cleavage of the capsid protein from 42 kDa to 31 kDa is essential for maturation. To investigate the structural bases for proteolysis and cross-linking, we have used cryo-electron micrographs to reconstruct the three-dimensional structures of purified particles from four discrete stages in the assembly pathway: Prohead I, Prohead II, Head I and Head II. Prohead I has icosahedral T = 7 packing of blister-shaped pentamers and hexamers. The pentamers are 5-fold symmetric, but the hexamers exhibit an unusual departure from 6-fold symmetry, as if two trimers had undergone a shear dislocation of about 25 A. Proteolytic conversion to Prohead II leaves the outer surface largely unchanged, but a major loss of density from the inner surface is observed, which we infer to represent the excision of the amino-terminal domains of the capsid protein. Upon expansion to the Head I state, the capsid becomes markedly larger, thinner walled, and more polyhedral: moreover, the capsomer shapes change radically; especially notable is the disappearance of the large hexon dislocation. No differences between Head I and the covalently cross-linked Head II could be observed at the current resolution of about 25 A, from which we infer that it is the conformational rearrangements effected by expansion that create the micro-environments needed for the autocatalytic formation of the isodipeptide bonds found in the mature virions ("pseudo-active sites").

Amino Acid Sequence↗
PubMed1994

Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns.

The filamentous hemagglutinin (FHA) of Bordetella pertussis is an adhesin that binds the bacteria to cells of the respiratory epithelium in whooping-cough infections. Mature FHA is a 220 kDa secretory protein that is highly immunogenic and has been included in acellular vaccines. We have investigated its structure by combining electron microscopy and circular dichroism spectroscopy (CD) with computational analysis of its amino acid sequence. The FHA molecule is 50 nm in length and has the shape of a horseshoe nail: it has a globular head that appears to consist of two domains; a 35 nm-long shaft that averages 4 nm in width, but tapers slightly from the head end; and a small, flexible, tail. Mass measurements by scanning transmission electron microscopy establish that FHA is a monomer. Its sequence contains two regions of tandem 19-residue pseudo-repeats: the first, of 38 cycles, starts at residue 344; the second, of 13 cycles, starts at residue 1440. The repeat motifs are predicted to consist of short beta-strands separated by beta-turns, and secondary structure measurements by CD support this prediction. We propose a hairpin model for FHA in which the head is composed of the terminal domains; the shaft consists mainly of the repeat regions conformed as amphipathic, hyper-elongated beta-sheets, with their hydrophobic faces apposed; and the tail is composed of the intervening sequence. Further support for the model was obtained by immuno-labeling electron microscopy. The 19-residue repeats of FHA have features in common with the leucine-rich repeats (LRRs) that are present in many eukaryotic proteins, including some adhesion factors. The model is also compared with the two other classes of filamentous proteins that are rich in beta-structure, i.e. viral adhesins and two beta-helical secretory proteins. Our proposed structure implies how the functionally important adhesion sites and epitopes of FHA are distributed: its tripeptide (RGD) integrin-binding site is assigned to the tail; the putative hemagglutination site forms part of the head; and two classes of immunodominant epitopes are assigned to opposite ends of the molecule. Possible mechanisms are discussed for two modes of FHA-mediated adhesion.

Adhesins, Bacterial↗
PubMed1994

Finding a needle in a haystack: detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus).

Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly.

Capsid↗
PubMed1993

The short tail-fiber of bacteriophage T4: molecular structure and a mechanism for its conformational transition.

Electron microscopy, image processing and computational sequence analysis were used to investigate the structure of the short tail-fiber of bacteriophage T4. This molecule, an oligomer of gp12, is an adhesin that binds the virion irreversibly to the bacterial surface. Short tail-fibers were isolated from mutant-infected cells in which gp12 is synthesized and assembled correctly, but not incorporated into virions. Visualized in negative stain, these filamentous molecules are approximately 38 nm in total length, with an arrowhead-shaped head (approximately 10 nm long by 6 nm wide), a 24-nm shaft of uniform width (approximately 3.8 nm), and a small, seemingly flexible, tail. The primary sequence contains a domain consisting of tandem quasi-repeats, each about 40 residues long, extending from approximately residue 50 to residue 320. Molecular mass analyses by scanning transmission electron microscopy confirm that the molecule is a trimer. The masses of the head, shaft, and tail domains are consistent with (trimers of) the carboxy-terminus, the repeat region, and the amino-terminus, respectively. When short tail-fibers are visualized extending from baseplates, their heads are distal, i.e., detached, implying that it is the tail that remains in contact with the baseplate. Analysis of the molecules' curvature properties detects three hinge-sites: these suggest how the short tail-fiber may be initially accommodated in a compact conformation in the "hexagon" state of the baseplate, from which it converts to the extended conformation when the baseplate switches into its "star" state.

Amino Acid Sequence↗
PubMed1991

Three-stranded alpha-fibrous proteins: the heptad repeat and its implications for structure.

Amino acid sequence data have been collected for the coiled-coil rod domains of three-stranded alpha-fibrous proteins--fibrinogen, laminin, tenascin, macrophage scavenger receptor protein and the leg fibre protein from bacteriophage. Such domains are characterized by a heptad substructure in which apolar residues occur alternately three and four residues apart. The distribution of residues in each position of the heptad has been analysed, and the results compared with those obtained for the two-stranded alpha-fibrous proteins, which include the intermediate filament and myosin families. Distinctions can be drawn between the sequences in two- and three-stranded coiled-coil structures and these provide criteria that will prove useful in predicting secondary and tertiary structure purely from sequence data.

Amino Acid Sequence↗
PubMed1990

Structural features in the heptad substructure and longer range repeats of two-stranded alpha-fibrous proteins.

Considerable sequence data have been collected from the intermediate filament proteins and other alpha-fibrous proteins including myosin, tropomyosin, paramyosin, desmoplakin and M-protein. The data show that there is a clear preference for some amino acids to occur in specific positions within the heptad substructure that characterizes the sequences which form the coiled-coil rod domain in this class of proteins. The results also indicate that although there are major similarities between the various proteins there are also key differences. In all cases, however, significant regularities in the linear disposition of the acidic and the basic residues in the coiled-coil segments can be related to modes of chain and molecular aggregation. In particular a clear trend has been observed which relates the mode of molecular aggregation to the number of interchain ionic interactions per heptad pair.

Amino Acids↗
PubMed1989

An estimate of the mean length of collagen fibrils in rat tail-tendon as a function of age.

A theoretical expression has been derived for the mean collagen fibril length in tendon based on the assumption that collagen fibrils originate in cell surface invaginations and terminate either at some remote cell surface or another collagen fibril bundle. The expression thus determined requires knowledge of the effective lengths of the fibrocytes (or fibrocyte assemblies) and the cellular content of the tendon. Both of these parameters have been measured experimentally as a function of age for rat-tail tendon using a combined light microscope and electron microscope approach. The results obtained for immature tendon suggest that the mean collagen fibril length is at least equal to the critical length required to maintain the appropriate tensile properties. In the most mature tissue studied, however, the mean-collagen fibril length is in excess of 100 times the critical length.

Aging↗
PubMed1988

Safety and efficacy of electrohydraulic lithotripsy by ureteroscopy.

Electrohydraulic lithotripsy, when performed blindly for ureteral calculi in the past, resulted in frequent ureteral perforation and, therefore, it was considered to be unsafe. A total of 33 patients from 5 institutions underwent electrohydraulic lithotripsy for ureteral calculi under direct vision. There were only 3 minor ureteral perforations treated by ureteral catheter drainage. All patients were free of stones after treatment and there were no long-term complications. Electrohydraulic lithotripsy by ureteroscopy is considered to be a safe and effective method for fragmentation and removal of large ureteral calculi.

Adult↗