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bacteriophage hk97 structure: wholesale covalent cross-linking between the major head shell subunits.we describe initial genetic and structural characterizations of hk97, a temperate bacteriophage of escherichia coli. we isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic dna. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hk97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too l ...19911709700
assembly in vitro of bacteriophage hk97 proheads.bacteriophage hk97 is a lambdoid phage with a head assembled from 415 copies of a 42 kda subunit arranged in an icosahedrally symmetrical lattice with a triangulation number of 7. prohead i, the first shell structure in the assembly pathway, is composed of 42 kda coat protein subunits that have not yet undergone the proteolytic cleavage, conformational changes, and covalent cross-linking steps that occur later in the assembly of mature heads. prohead i can be efficiently dissociated into capsome ...19957473718
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 be ...19957473720
complexes between chaperonin groel and the capsid protein of bacteriophage hk97.the 42 kda capsid protein of bacteriophage hk97 requires the groel and groes chaperonin proteins of its escherichia coli host to facilitate correct folding, both in vivo and in vitro. in the absence of groes and atp, denatured gp5 forms a stable complex with the 14 subunit groel molecule. we characterized the electrophoretic and biochemical properties of this complex. in electrophoresis on a native (nondenaturing) gel, the band of the gp5-groel complex shifts to a slower migrating position relat ...19957578104
bacteriophage hk97 head assembly.the head assembly pathway of bacteriophage hk97 shares many features with head assembly pathways determined for other dsdna phages, and it also provides examples of novel variations on the basic theme. we describe aspects of two specific steps in the assembly pathway, the covalent cross-linking among the assembled head protein subunits and the cleavage of those subunits that takes place earlier in the pathway. comparisons of head assembly pathways among different phages, as well as comparisons o ...19957669350
structural transitions during bacteriophage hk97 head assembly.bacteriophage hk97 builds its head shell from a 42 kda major head protein, but neither this 42 kda protein nor its processed, 31 kda form is found in the mature head. instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed prohead i and prohead ii. prohead i is asse ...19957723019
genetic basis of bacteriophage hk97 prohead assembly.we report studies to determine which bacteriophage genes are required for assembly of phage hk97 proheads and what roles they play. we identify the gene encoding the major capsid protein of phage hk97 and report its dna sequence, together with the dna sequences of the two genes immediately upstream from it. when the capsid protein is expressed from a plasmid in the absence of other phage-encoded proteins, it assembles, with good efficiency and accuracy into prohead-like structures composed of th ...19957723020
bacteriophage hk97 head assembly: a protein ballet. 19989521001
crystallization and preliminary x-ray analysis of the dsdna bacteriophage hk97 mature empty capsid.hk97 is a temperate dsdna bacteriophage of escherichia coli that is structurally similar to phage lambda, with an icosahedral head of triangulation (7) number 7. although the capsids of several large dsdna phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available. we have grown crystals of mature but empty bacteriophage hk97 capsids that diffract to at least 3.5 a using synchrotron radiation. the hk97 head ii crystals are the first ...19989527920
protein chainmail: catenated protein in viral capsids.the capsid shells of bacteriophage hk97 and several other phages contain polypeptides that are covalently linked into complexes so large that they do not enter polyacrylamide gels after denaturation. the enormous apparent size of these protein complexes in hk97 derives from a novel protein topology. hk97 subunits cross-link via isopeptide bonds into oligomers that are closed rings of five or six members. however, polypeptides from neighboring pentamer and hexamer rings intertwine before the cova ...19989674427
arrangement and functional identification of genes in the regulatory region of lambdoid phage h-19b, a carrier of a shiga-like toxin.h-19b is a lambdoid phage that carries the genes (stx-i) encoding the two toxin subunits of a shiga-like toxin; escherichia coli lysogens of h-19b are converted to toxin producers. based on the determination of a 17-kb region of the h-19b genome and functional studies, we have identified the early regulatory region and associated genes of h-19b, as well as the location of the late regulatory region and the toxin and lysis genes. a comparative analysis of the sequence of the h-19b genome reveals ...19989858702
imaging rna and dynamic protein segments with low-resolution virus crystallography: experimental design, data processing and implications of electron density maps.single crystal diffraction data were collected from virus crystals in the resolution range of 270 to 14 a using a synchrotron x-ray source and a small-angle scattering instrument adapted for single crystal measurements. reflections were measured from single crystals of the capsid of the double-stranded dna bacteriophage hk97 and synthetic flock house virus-like particles (sfhv). the quality of the low-resolution measurements was confirmed by excellent scaling statistics for both data sets. the s ...19989878362
crystallographic analysis of the dsdna bacteriophage hk97 mature empty capsid.hk97 is a member of the siphovirus family of dsdna bacteriophages. it is similar in architecture to bacteriophage lambda, the type member of this family, with an icosahedral capsid of triangulation number t = 7. no high-resolution structural information is available for the dsdna phages, and hk97 is the only dsdna bacteriophage capsid to produce crystals which diffract x-rays. at 650 a in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. t ...199910089306
topologically linked protein rings in the bacteriophage hk97 capsid.the crystal structure of the double-stranded dna bacteriophage hk97 mature empty capsid was determined at 3.6 angstrom resolution. the 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold. the final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins. each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356. this generates 12 pentameric and 60 hexameric rings of covalen ...200011000116
virus maturation involving large subunit rotations and local refolding.large-scale conformational changes transform viral precursors into infectious virions. the structure of bacteriophage hk97 capsid, head-ii, was recently solved by crystallography, revealing a catenated cross-linked topology. we have visualized its precursor, prohead-ii, by cryoelectron microscopy and modeled the conformational change by appropriately adapting head-ii. rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two m ...200111326105
an elastic network model of hk97 capsid maturation.the structure of the capsid of bacteriophage hk97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. typically the capsid assembles through polymerization and maturation processes. maturation is composed of proteolytic cleavages to the precursor capsid (called prohead ii), expansion triggered by dna packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links ...200312972347
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 o ...200415071181
domain structures and roles in bacteriophage hk97 capsid assembly and maturation.head assembly in the double-stranded dna coliphage hk97 involves initially the formation of the precursor shell prohead i from approximately 420 copies of a 384-residue subunit. this is followed by proteolytic removal of residues 2-103 to create prohead ii, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make head ii. here, we report and structurally interpret solution raman spectra of prohead i, prohead ii, and head ii particles. the raman signa ...200415122908
control of crosslinking by quaternary structure changes during bacteriophage hk97 maturation.radical structural changes drive the maturation of the capsid of hk97, a lambda-like, dsdna bacteriophage of escherichia coli. these include expansion from approximately 560 to approximately 660 a in diameter, metamorphosis from a round to an angular shape, and formation of covalent crosslinks between adjacent capsomers. analogous transformations also occur in unrelated viruses and protein complexes. we find that expansion and crosslinking happen concurrently during maturation at low ph. expansi ...200415175152
molecular dynamics of protein complexes from four-dimensional cryo-electron microscopy.cryo-electron microscopy of single particles offers a unique opportunity to detect and quantify conformational variation of protein complexes. different conformers may, in principle, be distinguished by classification of individual projections in which image differences arising from viewing geometry are disentangled from variability in the underlying structures by "multiple particle analysis"--mpa. if the various conformers represent dynamically related states of the same complex, mpa has the po ...200415450298
maturation dynamics of bacteriophage hk97 capsid.maturation of the bacteriophage hk97 capsid requires a large conformational change of the virus capsid. experimental studies have identified several intermediates along this maturation pathway. to gain insights into the molecular mechanisms of capsid maturation, we examined the fluctuation dynamics of the procapsid and mature capsid using a residue-level computational approach. the most cooperative motions of the procapsid are found to be consistent with the observed change in configuration that ...200515766543
crosslinking renders bacteriophage hk97 capsid maturation irreversible and effects an essential stabilization.in hk97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue k169 in the 'e-loop' of each subunit with n356 on another subunit. we show by complementation experiments with the k169y mutant, which cannot crosslink, that crosslinking is an essential function. the precursor prohead-ii passes through three expansion intermediate (ei) states en route to the end state, head-ii. we investigated the effects of expansion and crosslinkin ...200515775971
control of virus assembly: hk97 "whiffleball" mutant capsids without pentons.the capsid of escherichia coli bacteriophage hk97 assembles as a 420 subunit icosahedral shell called prohead i which undergoes a series of maturation steps, including proteolytic cleavage, conformational rearrangements, and covalent cross-linking among all the subunits to yield the highly stable mature head ii shell. prohead i have been shown to assemble from pre-formed hexamers and pentamers of the capsid protein subunit. we report here the properties of a mutant of the capsid protein, e219k, ...200515808861
conservation of the capsid structure in tailed dsdna bacteriophages: the pseudoatomic structure of phi29.bacteriophage phi29 is one of the smallest and simplest known dsdna phages, making it amenable to structural investigations. the three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 a resolution by cryo-electron microscopy (cryo-em), allowing the identification of alpha helices and beta sheets. their arrangement indicates that the folds of the phi29 and bacteriophage hk97 capsid proteins are similar except for an additional immunoglobulin-like domain of the ph ...200515837419
bacteriophage hk97: assembly of the capsid and evolutionary connections. 200516139590
crosslinking in viral capsids via tiling theory.a vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. it has been shown in twarock [2004. a tiling approach to vius capsids assembly explaining a structural puzzle in virology. j. theor. biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. this theory is extended here to multi-level tilings in ord ...200616337970
time-resolved molecular dynamics of bacteriophage hk97 capsid maturation interpreted by electron cryo-microscopy and x-ray crystallography.the bacteriophage hk97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. immature empty capsids, termed prohead ii, assemble in vivo in an escherichia coli expression system. maturation of these particles may be induced in vitro, converting them into head ii capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. one method of in vitro maturation require ...200616427314
a free energy cascade with locks drives assembly and maturation of bacteriophage hk97 capsid.we investigated the thermodynamic basis of hk97 assembly by scanning calorimetry and cryo-electron microscopy. this pathway involves self-assembly of hexamers and pentamers of the precursor capsid protein gp5 into procapsids; proteolysis of their n-terminal delta-domains; expansion, a major conformational change; and covalent crosslinking. the thermal denaturation parameters convey the changes in stability at successive steps in assembly, and afford estimates of the corresponding changes in free ...200617007875
capsid conformational sampling in hk97 maturation visualized by x-ray crystallography and cryo-em.maturation of the bacteriophage hk97 capsid from a precursor (prohead ii) to the mature state (head ii) involves a 60 a radial expansion. the mature particle is formed by 420 copies of the major capsid protein organized on a t = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. well-characterized ph 4 expansion intermediates make hk97 valuable for investigating quaternary structural dynamics. here, we use x-ray crystallography and cryo-em to demonstrate that in the final ...200617098191
a thermally induced phase transition in a viral capsid transforms the hexamers, leaving the pentamers unchanged.scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-em can reveal the accompanying structural changes. the procapsid of bacteriophage hk97 (prohead i) is a 450a-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. gp5 consists of the n-terminal delta-d ...200717188892
solution nmr studies of the maturation intermediates of a 13 mda viral capsid.dynamic regions of proteins often play essential roles in function, assembly, or maturation of macromolecular complexes. when x-ray crystallography and cryo-electron microscopy are used to investigate macromolecular structures, information about these dynamic regions is lost because of conformational disorder or flexibility. structural studies on the precursor capsid conformations of the lambdoid bacteriophage hk97, a model system for macromolecular maturation, reveal that essential regions tend ...200717536799
structure of the hypothetical protein pf0899 from pyrococcus furiosus at 1.85 a resolution.the hypothetical protein pf0899 is a 95-residue peptide from the hyperthermophilic archaeon pyrococcus furiosus that represents a gene family with six members. p. furiosus orf pf0899 has been cloned, expressed and crystallized and its structure has been determined by the southeast collaboratory for structural genomics (http://www.secsg.org). the structure was solved using the sca2structure pipeline from multiple data sets and has been refined to 1.85 a against the highest resolution data set col ...200717620707
unfolding thermodynamics of the delta-domain in the prohead i subunit of phage hk97: determination by factor analysis of raman spectra.an early step in the morphogenesis of the double-stranded dna (dsdna) bacteriophage hk97 is the assembly of a precursor shell (prohead i) from 420 copies of a 384-residue subunit (gp5). although formation of prohead i requires direct participation of gp5 residues 2-103 (delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and dna packaging. the prohead i delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-helic ...200918983851
mutational analysis of a conserved glutamic acid required for self-catalyzed cross-linking of bacteriophage hk97 capsids.the capsid of bacteriophage hk97 is stabilized by approximately 400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. isopeptide cross-links form between asparagine and lysine side chains on two subunits. the carboxylate of glutamic acid 363 (e363) from a third subunit is found ap ...200919091865
an unexpected twist in viral capsid maturation.lambda-like double-stranded (ds) dna bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. the extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from whic ...200919204733
structure and energetics of encapsidated dna in bacteriophage hk97 studied by scanning calorimetry and cryo-electron microscopy.encapsidation of duplex dna by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of dna. the hk97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. to characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. we found that, as in other phages, hk97 dna is organized in coaxially wound nested shells. when dna-filled cap ...200919540242
the crystal structure of bacteriophage hk97 gp6: defining a large family of head-tail connector proteins.the final step in the morphogenesis of long-tailed double-stranded dna bacteriophages is the joining of the dna-filled head to the tail. the connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for dna from the head during infection. here, we report the determination of a 2.1 a crystal structure of gp6 of bacteriophage hk97. through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined ...201019895817
assembly of bacteriophage p2 capsids from capsid protein fused to internal scaffolding protein.most tailed bacteriophages with double-stranded dna genomes code for a scaffolding protein, which is required for capsid assembly, but is removed during capsid maturation and dna packaging. the gpo scaffolding protein of bacteriophage p2 also doubles as a maturation protease, while the scaffolding activity is confined to a 90 residue c-terminal "scaffolding" domain. bacteriophage hk97 lacks a separate scaffolding protein; instead, an n-terminal "delta" domain in the capsid protein appears to ser ...201020063181
virus particle maturation: insights into elegantly programmed nanomachines.similar modes of virus maturation have been observed in dsdna bacteriophages and the structurally related herpes viruses and some type of maturation occur in most animal viruses. recently a variety of biophysical studies of maturation intermediates of bacteriophages p22, lambda, and hk97 have suggested an energy landscape that drives the transitions and structure-based mechanisms for its formation. near-atomic resolution models of subunit tertiary structures in an early intermediate of bacteriop ...201020149636
critical salt bridges guide capsid assembly, stability, and maturation behavior in bacteriophage hk97.hk97 is a double-stranded dna bacteriophage that undergoes dramatic conformational changes during viral capsid maturation and for which x-ray structures, at near atomic resolution, of multiple intermediate and mature capsid states are available. both amide h/(2)h exchange and crystallographic comparisons between the pre-expanded prohead ii particles and the expanded head ii of bacteriophage hk97 revealed quaternary interactions that remain fixed throughout maturation and appear to maintain inter ...201020332083
structure and assembly of bacteriophage t4 head.the bacteriophage t4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which dna enters during packaging and exits during infection. the past twenty years of research has greatly elevated the understanding of phage t4 head assembly and dna packaging. the atomic s ...201021129201
a conformational switch involved in maturation of staphylococcus aureus bacteriophage 80α capsids.bacteriophages are involved in many aspects of the spread and establishment of virulence factors in staphylococcus aureus, including the mobilization of genetic elements known as s. aureus pathogenicity islands (sapis), which carry genes for superantigen toxins and other virulence factors. sapis are packaged into phage-like transducing particles using proteins supplied by the helper phage. we have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the pro ...201021129380
generation of random numbers on graphics processors: forced indentation in silico of the bacteriophage hk97.the use of graphics processing units (gpus) in simulation applications offers a significant speed gain as compared to computations on central processing units (cpus). many simulation methods require a large number of independent random variables generated at each step. we present two approaches for implementation of random number generators (rngs) on a gpu. in the one-rng-per-thread approach, one rng produces a stream of random numbers in each thread of execution, whereas the one-rng-for-all-thr ...201021194190
the prohead-i structure of bacteriophage hk97: implications for scaffold-mediated control of particle assembly and maturation.virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. many viruses encode scaffolding proteins to shift the equilibrium toward particle formation by promoting intersubunit interactions and stabilizing assembly intermediates. bacteriophage hk97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like n-terminal segment termed the delta domain. when gp5 ...201121276801
assembly mechanism is the key determinant of the dosage sensitivity of a phage structural protein.altering the expression level of proteins that are subunits of complexes has been proposed to be particularly detrimental because the resulting stoichiometric imbalance among components would lead to misassembly of the complex. here we show that assembly of the phage hk97 connector complex is severely inhibited by the overexpression of one of its component proteins, gp6. however, this effect is a result of the unusual mechanism by which the oligomerization and assembly of gp6 are controlled. alt ...201121646545
survival of the fittest: a role for phage-encoded eukaryotic-like kinases.phages are often thought of as mortal enemies of bacteria. this dynamic relationship has led to the evolution of a number of processes in bacteria designed to defeat these attacks. examples of these include blocking phage attachment, crispr, and restriction modification systems. temperate phages provide another source of protection by excluding infection of heterologous phage, thwarting phage production and further infection. this strategy protects the rest of the bacterial population from attac ...201121992081
activation of a prophage-encoded tyrosine kinase by a heterologous infecting phage results in a self-inflicted abortive infection.bacteria in their struggle for survival have evolved or acquired defences against attacking phage. however, phage often contribute to this defence through mechanisms in which a prophage protects the bacterial population from attack by another, often unrelated, phage. the 933w prophage, which carries shiga toxin genes that enhance pathogenicity of enterohaemorrhagic escherichia coli strain o157:h7, also carries the stk gene encoding a eukaryotic-like tyrosine kinase that excludes (aborts) infecti ...201121985444
transferrin-mediated targeting of bacteriophage hk97 nanoparticles into tumor cells.next-generation targeted nanodevices are currently under development for imaging and therapeutic applications. we engineered hk97 viral nanoparticles (vnps) for tumor cell-specific targeting.201121182418
the moron comes of age.prophage-encoded genes can provide a variety of benefits for their bacterial hosts. these beneficial genes are often contained within "moron" elements. morons, thus termed as the insertion of the dna encoding them adds "more on" the genome in which they are found, are independent transcriptional units disseminated among phage genomes through horizontal gene transfer. morons have been identified in the majority of phage genomes and they have been found to play diverse roles in bacterial physiolog ...201223739268
bacteriophage hk97 capsid assembly and maturation.the escherichia coli phage hk97 has provided a productive experimental system for investigating how virus capsids are assembled from their protein components and how the assembled capsids mature to their final form. aspects of the process for which the hk97 system has been particularly informative include assembly of the icosahedral capsid shell from the component proteins, structure of the capsid subunits and of the entire capsid as it progresses through its maturation, and the mechanism of the ...201222297521
on the catalytic mechanism of bacteriophage hk97 capsid crosslinking.during maturation of the phage hk97 capsid, each of the 415 capsid subunits forms covalent bonds to neighboring subunits, stabilizing the capsid. crosslinking is catalyzed not by a separate enzyme but by subunits of the assembled capsid in response to conformational rearrangements during maturation. this report investigates the catalytic mechanism. earlier work established that the crosslinks are isopeptide (amide) bonds between side chains of a lysine on one subunit and an asparagine on another ...201728359902
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