Inquiry learning. Teaching scientific inquiry.
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Adolescent↗
Biomedical subjects
Roger W Hendrix
Publications and source records attributed to Roger W Hendrix.
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-domain (11kDa) and gp5* (31 kDa): gp5* forms the contiguous shell from which clusters of Delta-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 degrees C with an enthalpy change of 14 kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 degrees C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11A resolution, visible changes are confined to the gp5 hexamers. Their Delta-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5* hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Delta-domains restrain their gp5* rings from switching to a lower free energy, EI-I-like, state; above 53 degrees, this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation.
Bacteriophages↗
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 energy. The procapsid represents a kinetically accessible local minimum of free energy. In maturation, it progresses to lower minima in a cascade punctuated by irreversible processes ("locks"), i.e. proteolysis and crosslinking, that lower kinetic barriers and prevent regression. We infer that Delta-domains not only guide assembly but also restrain the procapsid from premature expansion; their removal by proteolysis is conducive to initiating expansion and to its proceeding to completion. We also analyzed the mutant E219K, whose capsomers reassemble in vitro into procapsids with vacant vertices called "whiffleballs". E219K assemblies all have markedly reduced stability compared to wild-type gp5 (DeltaT(p) approximately -7 degrees C to -10 degrees C; where T(p) is the denaturation temperature). As the mutated residue is buried in the core of gp5, we attribute the observed reduction in stability to steric and electrostatic perturbations of the packing of side-chains in the subunit interior. To explain the whiffleball phenotype, we suggest that these effects propagate to the capsomer periphery in such a way as to differentially affect the stability or solubility of dissociated pentamers, leaving only hexamers to reassemble.
Bacteriophages↗
Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform.
Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.
DNA, Viral↗
Recoding in bacteriophages and bacterial IS elements.
Dynamic shifts between open reading frames and the redefinition of codon meaning at specific sites, programmed by signals in mRNA, permits versatility of gene expression. Such alterations are characteristic of organisms in all domains of life and serve a variety of functional purposes. In this article, we concentrate on programmed ribosomal frameshifting, stop codon read-through and transcriptional slippage in the decoding of phage genes and bacterial mobile elements. Together with their eukaryotic counterparts, the genes encoding these elements are the richest known source of nonstandard decoding. Recent analyses revealed several novel sequences encoding programmed alterations in gene decoding and provide a glimpse of the emerging picture.
Bacteriophages↗
Shared architecture of bacteriophage SPO1 and herpesvirus capsids.
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Bacillus Phages↗
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 transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 A translations and 6 degrees rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.
Bacteriophage lambda↗
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 requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.
Capsid↗
Cooperative reorganization of a 420 subunit virus capsid.
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 A 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.
Capsid Proteins↗
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, 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.
Bacteriophages↗
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 crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.
Bacteriophages↗
Bacteriophage HK97: assembly of the capsid and evolutionary connections.
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Bacteriophages↗
The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy.
The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.
Base Sequence↗
Conserved translational frameshift in dsDNA bacteriophage tail assembly genes.
A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.
Amino Acid Sequence↗
Evidence that a local refolding event triggers maturation of HK97 bacteriophage capsid.
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 angstroms 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 beta-strand and beta-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.
Bacteriophages↗
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. Expansion causes residues on three different subunits to move up to 35 A 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.
Asparagine↗
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 signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.
Asparagine↗
Hot new virus, deep connections.
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Evolution, Molecular↗