The complex protein capsids of many viruses exhibit dramatic reorganizations at critical stages in their life-cycle. Here, time-resolved solution X-ray scattering was used to study a dynamic, ...large-scale conformational maturation of the 420 subunit, 13
MDa, icosahedrally symmetric HK97 bacteriophage capsid. Isoscattering points in the time-resolved scattering patterns and singular value decomposition revealed that the expansion occurs as a cooperative, two-state reaction. The analysis demonstrates that the population shift from Prohead-II to Expansion Intermediate I, EI-I (60
Å larger than Prohead-II) occurs in minutes, but does not reveal the time required for individual transitions that occur stochastically. Any intermediate forms that may be traversed during this conversion are unstable and do not constitute an appreciable population of the ensemble of particles. In an energetic landscape view, particles must undergo an energy barrier-crossing event in order to successfully convert from Prohead-II to EI-I. This implies that the particles “hop” over the energy barrier stochastically as they individually attain an expansion-active state. Interestingly, systematic deviations from single-exponential kinetics were observed for the population shift. This may indicate that in undergoing the irreversible conversion from Prohead-II to EI-I, particles are subject to a complex energy landscape that links the initial and final particle forms.
Radical structural changes drive the maturation of the capsid of HK97, a λ-like, dsDNA bacteriophage of
Escherichia coli. These include expansion from ∼560 to ∼660 Å 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. Expansion causes residues on three different subunits to move up to 35 Å to form 420 active sites that each catalyze the formation of a lysine-asparagine crosslink between adjacent subunits, making crosslink formation an indirect reporter of structural change. Intermediate crosslinking patterns support a previously proposed model of expansion, while hydrophobic properties aid in distinguishing discrete intermediates. A structure derived from cryo-EM images reveals the free intermediate conformation of penton arms, supporting our model for coordinated movement of hexons and pentons on the capsid lattice.
Neutral oxygen in the saturnian system shows variability, and the total number of oxygen atoms peaks at 4 x 10³⁴. Saturn's aurora brightens in response to solar-wind forcing, and the auroral spectrum ...resembles Jupiter's. Phoebe's surface shows variable water-ice content, and the data indicate it originated in the outer solar system. Saturn's rings also show variable water abundance, with the purest ice in the outermost A ring. This radial variation is consistent with initially pure water ice bombarded by meteors, but smaller radial structures may indicate collisional transport and recent renewal events in the past 10⁷ to 10⁸ years.
Bacteriophage capsids are a striking example of a robust yet dynamic genome delivery vehicle. Like most phages, HK97 undergoes a conformational maturation that converts a metastable Prohead into the ...mature Head state. In the case of HK97, maturation involves a significant expansion of the capsid and concomitant cross-linking of capsid subunits. The final state, termed Head-II, is a 600
Å diameter icosahedral structure with catenated subunit rings. Cryo-EM, small angle X-ray scattering (SAXS), and biochemical assays were used previously to characterize the initial (Prohead-II) and final states (Head-II) as well as four maturation intermediates. Here we extend the characterization of the acid-induced expansion of HK97
in vitro by monitoring changes in intrinsic fluorescence, circular dichroism (CD), and SAXS. We find that the greatest changes in all observables occur at an early stage of maturation. Upon acidification, fluorescence emissions from HK97 exhibit a blueshift and decrease in intensity. These spectral changes reveal two kinetic phases of the expansion reaction. The early phase exhibits sensitivity to pH, increasing in rate nearly 200-fold when acidification pH is lowered from 4.5 to 3.9. The second, slower phase reported by fluorescence is relatively insensitive to pH. Time-resolved SAXS experiments report an increase in overall particle dimension that parallels the fluorescence changes for the early phase. Native agarose gel assays corroborated this finding. By contrast, probes of CD at far-UV indicate that secondary structural changes precede the early expansion phase reported by SAXS and fluorescence. Based on the crystallographic structure of Head-II and the pseudo-atomic model of Prohead-II, we interpret these changes as reflecting the conversion of subunit N-terminal arms (N-arm) from unstructured polypeptide to the mixture of β-strand and β-turn observed in the Head-II crystal structure. Refolding of the N-arm may thus represent the conformational trigger that initiates the irreversible expansion of the phage capsid.
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 order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.
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, which illuminate the assembly of Prohead I. The mutant capsid protein is capable of going through all of the biochemically and morphologically defined steps of capsid maturation, and when it is expressed by itself from a plasmid it assembles efficiently into a Prohead I that is morphologically indistinguishable from the wild-type Prohead I, with a full complement of both hexamers and pentamers. Unlike the wild-type Prohead I, when the mutant structure is dissociated into capsomers
in vitro, only hexamers are found. When such preparations are put under assembly conditions, these mutant hexamers assemble into “Whiffleballs”, particles that are identical with Prohead I except that they are missing the 12 pentamers. These Whiffleballs can even be converted to Prohead I by specifically binding wild-type pentamers. We argue that the ability of the mutant hexamers to assemble in the absence of pentamers implies that they retain a memory of their earlier assembled state, most likely as a conformational difference relative to assembly-naive hexamers. The data therefore favor a model in which Prohead I assembly is regulated by conformational switching of the hexamer.
N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the ...prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. Here, we describe the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.
BackgroundAcetabular osteolysis is a major problem affecting long-term survival of total hip prostheses. Since lytic lesions may be asymptomatic until extensive bone loss has occurred, early ...detection of lytic lesions is important. The purposes of this study were to determine the efficacy and potential role of high-resolution helical (or spiral) computed tomography with metal-artifact minimization in the early detection of osteolysis of the pelvis and to use the method to determine if there was a relationship between the extent of osteolysis and the amount of polyethylene wear.MethodsForty patients (fifty hips) who had undergone primary cementless total hip arthroplasty between 1988 and 1994 were evaluated as part of an ongoing prospective study. These patients had a history of high-level activity that was believed to place them at increased risk for accelerated polyethylene wear. The most recent follow-up radiographs were compared with the three-month postoperative radiographs. Helical computed tomography scans with metal-artifact minimization were made, and evidence of osteolytic lesions on these scans was compared with that on the radiographs. Two-dimensional wear analysis was performed with use of digitized radiographs, and the results were compared with loss of bone volume as calculated from the computed tomography scans.ResultsAcetabular lysis was identified on the radiographs of sixteen hips and on the computed tomography scans of twenty-six hips. Radiographs underestimated the extent of the lysis in thirteen of the sixteen hips. There was no correlation (r = 0.036) between linear wear and the measured volume of bone loss, with the numbers available. On the basis of the amount of lysis seen on the computed tomography scans, one patient underwent a revision procedure.ConclusionsHelical computed tomography with metal-artifact minimization is more sensitive for identifying and quantifying osteolysis after total hip arthroplasty than is plain radiography. Since computed tomography scans show both the extent and the location of lytic lesions, they are useful to guide treatment decisions as well as to assist in planning for surgical intervention, when needed, in patients with suspected osteolysis.
Mycobacteriophage D29 is a lytic phage that infects both fast and slow-growing mycobacterial species. The complete genome sequence of D29 reveals that it is a close relative of the temperate ...mycobacteriophage L5, whose sequence has been described previously. The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens. Comparison of the two genomes shows that they are punctuated by a large number of insertions, deletions, and substitutions of genes, consistent with the genetic mosaicism of lambdoid phages.
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