Co-expression of gene 31 and 23 products of bacteriophage T4

Folding of the major capsid protein of bacteriophage T4 encoded by gene 23 is aided by Escherichia coli GroEL chaperonin and phage co-chaperonin gp31. In the absence of gene product (gp) 31, aggregates of recombinant gp23 accumulate in the cell similar to inclusion bodies. These aggregates can be solubilized with 6 M urea. However, the protein cannot form regular structures in solution. A system of co-expression of gp31 and gp23 under the control of phage T7 promoter in E. coli cells has been constructed. Folding of entire-length gp23 (534 amino acid residues) in this system results in the correctly folded recombinant gp23, which forms long regular structures (polyheads) in the cell.     

Proteolysis of the major capsid protein T4 bacteriophage polyheads limited by quaternary structure.

Bacteriophage T4 carrying an amber mutation in gene 22 plus an amber mutation in gene 21 form aberrant, tubular structures termed rough polyheads, instead of complete phage when they infect Escherichia coli B. These rough polyheads consist almost entirely of the major capsid protein in its uncleaved form (gp23). When rough polyheads are treated under mild conditions with any of the five proteases, trypsin, chymotrypsin, thermolysin, pronase, or the protease from Staphylococcus aureus V8, the gp23 is rapidly hydrolyzed at a limited number of peptide bonds. In contrast, cleaved capsid protein (gp23) in mature phage capsids is completely resistant to proteolysis under the same conditions. A major project in this laboratory requires determining the primary structure of gp23, a large protein (Mr = 58,000) quite rich in those amino acids at which cleavages are achieved by conventional means. Recovery of peptides from the complex mixtures resulting from such cleavages proved to be extremely difficult. The limited proteolysis of gp23 in rough polyheads had yielded a set of large, easily purified fragments which are greatly simplifying the task of determining the primary structure of this protein.     

The maturation-dependent conformational change of the major capsid protein of bacteriophage T4 involves a substantial change in secondary structure

We have investigated the conformational basis of the expansion transformation that occurs upon maturation of the bacteriophage T4 prohead, by using laser Raman spectroscopy to determine the secondary structure of the major capsid protein in both the precursor and the mature states of the surface lattice. This transformation involves major changes in the physical, chemical, and immunological properties of the capsid and is preceded in vivo by processing of its major protein, gp23 (56 kDa), to gp23* (49 kDa), by proteolysis of its N-terminal gp23-delta domain. The respective secondary structures of gp23 in the unexpanded state, and of gp23* in the expanded state, were determined from the laser Raman spectra of polyheads, tubular polymorphic variants of the capsid. Similar measurements were also made on uncleaved polyheads that had been expanded in vitro and, for reference, on thermally denatured polyheads. We find that, with or without cleavage of gp23, expansion is accompanied by substantial changes in secondary structure, involving a major reduction in alpha-helix content and an increase in beta-sheet. The beta-sheet contents of gp23* or gp23 in the expanded state of the surface lattice, and even of gp23 in the unexpanded state, are sufficient for a domain with the "jellyroll" fold of antiparallel beta-sheets, previously detected in the capsid proteins of other icosahedral viruses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembly-dependent conformational changes in a viral capsid protein. Calorimetric comparison of successive conformational states of the gp23 surface lattice of bacteriophage T4

Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (t*m) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23* prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta t*m = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals' and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23* in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.     

Structure of T4 polyheads: II. A pathway of polyhead transformations as a model for T4 capsid maturation

We have studied the aberrant tubular polyheads of bacteriophages T4D and T2L as a model system for capsid maturation. Six different types of polyhead surface lattice morphology, and the corresponding protein compositions are reported and discussed. Using in vitro systems to induce transformations between particular polyhead types, we have deduced that the structural classes represent successive points in a transitional pathway. In the first step, coarse polyheads (analogous to the prohead τ-particle) are proteolytically cleaved by a phagecoded protease, a fragment of the gene 21 product. This cleavage of P23 to P23¹ induces a co-operative lattice transformation in the protein of the surface shell, to a conformation equivalent to that of T2L giant phage capsids. These polyheads (derived either from T4 or T2L lysates) can accept further T4-coded proteins. In doing so, they pass through intermediate structural states, eventually reaching an end point whose unit cell morphology is indistinguishable from that of the giant T4 capsids. At least one protein (called soc (Ishii & Yanagida, 1975)) is bound stoichiometrically to P23¹ in the end-state conformation. The simulation of several aspects of capsid maturation (cleavage of P23 to P23¹, stabilization, and lattice expansion) in the polyhead pathway suggest that it parallels the major events of phage T-even capsid maturation, decoupled from any involvement of DNA packaging.     

High-level expression of a polypeptides encoded by a large segment of the envelope gene of HIV-1 in E. coli by directed evolution

A random mutagenesis library of gp120-801 (a large segment of the envelope protein gene of HIV-1) was constructed using error-prone PCR and DNA shuffling methods, and one mutant of gp120-801 was selected from this library using a green fluorescent protein (GFP) fusion vector. After being cloned into pEX31b and expressed in E. coli, the expressed fusion protein reached to 15% of total bacterial proteins for the mutant but was just 1–2% for the wild type.     

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.     

Specific labeling of protein domains with antibody fragments

Monovalent antibody Fab fragments, prepared from antisera raised against two different types of crystalline arrays made of either intact, or a proteolytic fragment of bacteriophage T4 major capsid protein, gp23*, were employed to stoichiometrically label different gp23* protein domains on the outer surface of a tubular variant (i.e., "polyheads") of bacteriophage T4 capsids. Computer filtrations of both negatively stained and freeze-dried/metal-shadowed specimens permitted approximate mapping of the Fab binding sites within the capsomere of the polyheads.     

Bacteriophage T4 bypass31 mutations that make gene 31 nonessential for bacteriophage T4 replication: mapping bypass31 mutations by UV rescue experiments.

The product of gene 31 is normally required for assembly of the T4 capsid. Two mutations that each bypass that requirement are shown to be located at separate sites in gene 23, which encodes the major structural protein of the capsid. A second phenotypic effect that characterizes both bypass31 mutant strains is the ability to multiply in host-defective strains, such as hdB3-1 and groEL mutants, on which wild-type T4 is unable to assemble capsids. The genetic data indicate that both phenotypic effects are due to the bypass31 mutation. Elimination of the requirement for both the phage protein, gp31, and the host protein, GroEL, by either of two single mutations in gene 23 indicates that GroEL and gp31 are normally needed to interact with gp23 in capsid assembly of wild-type T4.     

Letter: Capsid structure of bacteriophage lambda

The arrangement of capsomers in the capsid of phage λ has been investigated by electron micrography of negatively stained fragments of empty capsids, polyheads, and intact virions. The proposed structure is a composite T = 7 levo lattice, with hexamer and pentamer clustering of the D protein and trimer clustering of the E protein. Such a lattice requires that the λ capsid contain 420 copies of the D and E proteins, a number compatible with recent chemical determinations.     

Conformational changes of a viral capsid protein. Thermodynamic rationale for proteolytic regulation of bacteriophage T4 capsid expansion, co-operativity, and super-stabilization by soc binding.

We have used differential scanning calorimetry in conjunction with cryo-electron microscopy to investigate the conformational transitions undergone by the maturing capsid of phage T4. Its precursor shell is composed primarily of gp23 (521 residues): cleavage of gp23 to gp23* (residues 66 to 521) facilitates a concerted conformational change in which the particle expands substantially, and is greatly stabilized. We have now characterized the intermediate states of capsid maturation; namely, the cleaved/unexpanded, state, which denatures at tm = 60 degrees C, and the uncleaved/expanded state, for which tm = 70 degrees C. When compared with the precursor uncleaved/unexpanded state (tm = 65 degrees C), and the mature cleaved/expanded state (tm = 83 degrees C, if complete cleavage precedes expansion), it follows that expansion of the cleaved precursor (delta tm approximately +23 degrees C) is the major stabilizing event in capsid maturation. These observations also suggest an advantage conferred by capsid protein cleavage (some other phage capsids expand without cleavage): if the gp23-delta domains (residues 1 to 65) are not removed by proteolysis, they impede formation of the stablest possible bonding arrangement when expansion occurs, most likely by becoming trapped at the interface between neighboring subunits or capsomers. Icosahedral capsids denature at essentially the same temperatures as tubular polymorphic variants (polyheads) for the same state of the surface lattice. However, the thermal transitions of capsids are considerably sharper, i.e. more co-operative, than those of polyheads, which we attribute to capsids being closed, not open-ended. In both cases, binding of the accessory protein soc around the threefold sites on the outer surface of the expanded surface lattice results in a substantial further stabilization (delta tm = +5 degrees C). The interfaces between capsomers appear to be relatively weak points that are reinforced by clamp-like binding of soc. These results imply that the "triplex" proteins of other viruses (their structural counterparts of soc) are likely also to be involved in capsid stabilization. Cryo-electron microscopy was used to make conclusive interpretations of endotherms in terms of denaturation events. These data also revealed that the cleaved/unexpanded capsid has an angular polyhedral morphology and has a pronounced relief on its outer surface. Moreover, it is 14% smaller in linear dimensions than the cleaved/expanded capsid, and its shell is commensurately thicker.     

Repetitive versus monomeric antigen presentation: direct visualization of antibody affinity and specificity

The concept of presenting antigens in a repetitive array to obtain high titers of specific antibodies is increasingly applied by using surface-engineered viruses or bacterial envelopes as novel vaccines. A case for this concept was made 25 years ago, when producing high-titer antisera against ordered arrays of gp23, the major capsid protein of bacteriophage T4 (Aebi et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5514-5518). In view of the current interest in this concept we thought it useful to employ this system to directly visualize the dependence of antibody affinity and specificity on antigen presentation. We compared antibodies raised against T4 polyheads, a tubular variant of the bacteriophage T4 capsid, which have gp23 hexamers arranged in a crystalline lattice (gp23(repetitive)), with those raised against the hexameric gp23 subunits (gp23(monomeric)). The labeling patterns of Fab-fragments prepared from these antibodies when bound to polyheads were determined by electron microscopy and image enhancement. Anti-gp23(repetitive) bound in a monospecific, stoichiometric fashion to the gp23 units constituting the polyhead surface. In contrast, anti-gp23(monomeric) decorated the polyhead surface randomly and with a 40-fold lower occupancy. These results concur with the difference in titers established by ELISA for the antisera against the repetitively displayed form of antigen (anti-gp23(repetitive)) and the randomly presented antigen (gp23(monomeric)), and they constitute a compelling visual documentation of the concept of repetitive antigen presentation to elicite a serotype-like immune response.     

Effect of N-terminal deletions on the activity of pokeweed antiviral protein expressed in E. coli

Pokeweed antiviral protein (PAP) from Phytolacca americana is a highly specific N-glycosidase removing adenine residues (A⁴³²⁴ in 28S rRNA and A²⁶⁶⁰ in 23S rRNA) from intact ribosomes of both eukaryotes and prokaryotes. Due to the ribosome impairing activity the gene coding for mature PAP has not been expressed so far in bacteria whereas the full-length gene (coding for the mature 262 amino acids plus two signal peptides of 22 and 29 amino acids at both N- and C-termini, respectively) has been expressed in Escherichia coli. In order to determine: 1) the size of the N-terminal region of PAP which is required for toxicity to E. coli; and 2) the location of the putative enzymatic active site of PAP, 5′-terminal progressive deletion of the PAP full-length gene was carried out and the truncated forms of the gene were cloned in a vector containing a strong constitutive promoter and a consensus Shine-Dalgarno ribosome binding site. The ribosome inactivation or toxicity of the PAP is used as a phenotype characterized by the absence of E. coli colonies, while the mutation of PAP open reading frames in the small number of survived clones is used as an indicator of the toxicity to E. coli cells. Results showed that the native full-length PAP gene was highly expressed and was not toxic to E. coli cells although in vitro ribosome inactivating activity assay indicated it was active. However, all of the N-terminal truncated forms (removal of seven to 107 codons) of the PAP gene were toxic to E. coli cells and were mutated into either out of frame, early termination codon or inactive form of PAP (i.e., clone PAPΔ107). Deletion of more than 123 codons restored the correct gene sequence but resulted in the loss of the antiviral and ribosome inactivating activities and by the formation of a large number of clones. These results suggest that full-length PAP (with N- and C-terminal extensions) might be an inactive form of the enzyme in vivo presumably by inclusion body formation or other unknown mechanisms and is not toxic to E. coli cells. However, it is activated by at least seven codon deletions at the N-terminus. Deletions from seven through to 107 amino acids were lethal to the cells and only mutated forms (inactive) of the gene were obtained. But deletion of more than 123 amino acids resulted in the loss of enzymatic activity and made it possible to express the correct PAP gene in E. coli. Because deletion of Tyr94 and Va195, which are involved in the binding of the target adenine base, did not abolish the activity of PAP, it is concluded that the location previously proposed for PAP enzymatic active site should be reassessed.     

The maturation-dependent conformational change of phage T4 capsid involves the translocation of specific epitopes between the inner and the outer capsid surfaces

After polymerization of the phage T4 prohead is complete, its capsid expands by approximately 16%, is greatly stabilized, and acquires the capacity to bind accessory proteins. These effects are manifestations of a large-scale, irreversible, conformational change undergone by the major capsid protein, gp23 (521 residues) which is cleaved to gp23* (residues 66-521) during this maturation process. In order to explore its structural basis, we have performed immunoelectron microscopy with antibodies raised against synthetic peptides that correspond to precisely defined segments of the amino acid sequence of gp23. These antibodies were used to label purified polyheads (tubular polymorphic variants of the normal icosahedral capsid), in experiments designed to impose constraints on the possible foldings of the gp23/gp23* polypeptide chains in their successive conformational states. Peptide 1 (residues 48-57), part of the gp23-delta domain that is excised when gp23 is converted to gp23*, resides on the inner surface of the precursor surface lattice, but--if not proteolyzed--is found on the outer surface of the mature surface lattice. Peptide 2 (residues 65-73), immediately distal to the cleavage site, is located on the inside of the precursor surface lattice, and remains there subsequent to expansion. Peptide 3 (residues 139-146) is translocated in the opposite direction from peptide 1, i.e., from the outer to the inner surface upon expansion; moreover, expansion greatly increases the polyheads' affinity for these antibodies. Peptide 5 (residues 301-308) is located on the inside in both the precursor and the mature states. Taking into account data from other sources, these observations imply that the conformational change that underlies capsid expansion involves a radical reorganization of the proteins' structure, in which at least three distinct epitopes, situated in widely differing parts of the polypeptide chain, are translocated from one side to the other. Moreover, the amino-terminal portion of gp23/gp23*, around the cleavage site, is particularly affected.     

Improved soluble expression of the gene encoding amylolytic enzyme Amo45 by fusion with the mobile-loop-region of co-chaperonin GroES in Escherichia coli

Abstract

The gene encoding the amylolytic enzyme Amo45, originating from a metagenomic project, was retrieved by a consensus primer-based approach for glycoside hydrolase (GH) family 57 enzymes. Family 57 contains mainly uncharacterized proteins similar to archaeal thermoactive amylopullulanases. For characterization of these family members soluble, active enzymes have to be produced in sufficient amounts. Heterologous expression of amo45 in E.coli resulted in low yields of protein, most of which was found in inclusion bodies. To improve protein production and to increase the amount of soluble protein, two different modifications of the gene were applied. The first was fusion to an N-terminal His-tag sequence which increased the yield of protein, but still resulted in high amounts of inclusion bodies. Co-expression with chaperones enhanced the amount of soluble protein 4-fold. An alternative modification was the attachment of a peptide consisting of the amino acid sequence of the mobile-loop of the co-chaperonin GroES of E.coli. This sequence improved the soluble protein production 5-fold compared to His₆-Amo45 and additional expression of chaperones was unnecessary.     

Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: the evolution of pentameric vertex proteins in icosahedral viruses

Many large viral capsids require special pentameric proteins at their fivefold vertices. Nevertheless, deletion of the special vertex protein gene product 24 (gp24) in bacteriophage T4 can be compensated by mutations in the homologous major capsid protein gp23. The structure of such a mutant virus, determined by cryo-electron microscopy to 26 angstroms, shows that the gp24 pentamers are replaced by mutant major capsid protein (gp23) pentamers at the vertices, thus re-creating a viral capsid prior to the evolution of specialized major capsid proteins and vertex proteins. The mutant gp23* pentamer is structurally similar to the wild-type gp24* pentamer but the insertion domain is slightly more distant from the gp23* pentamer center. There are additional SOC molecules around the gp23* pentamers in the mutant virus that were not present around the gp24* pentamers in the wild-type virus.     

The control region of the F sex factor DNA transfer cistrons: Physical mapping by deletion analysis

Summary A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules. The method involves transformation of E. coli cells with linear plasmid DNAs generated by restriction enzyme cleavage. We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA. Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro. Deletion analysis has also allowed the identification of the traK gene product.     

Studies on the morphopoiesis of the head of phage T-even

Summary The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T₄ amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.     

Genetic studies on capsid-length determination in bacteriophage T4. II. Genetic evidence that specific protein-protein interactions are involved.

A bacteriophage T4 mutation (ptg19-80c) located in gene 23, which encodes the major structural protein of the T4 capsid, results in the production of capsids of abnormal length. Mutations outside gene 23 which partially suppress ptg19-80c have been described in the accompanying paper (D. H. Doherty, J. Virol. 43:641-654, 1982). Characterization of these suppressors was extended. A complementation test suggested that the suppressors were in genes 22 and 24. These genes coded for the major component of the morphogenetic core of the capsid precursor and the vertex protein of the capsid, respectively. The suppressor mutations were found to have no obvious phenotype in the absence of ptg19-80c. Suppression was shown to be allele specific: other ptg mutations at different sites in gene 23 were not suppressed by the suppressors of ptg19-80c. These results indicated that specific interactions among the three proteins gp22, gp23, and gp24 may play a role in the regulation of T4 capsid-length determination. Current models for capsid-length determination are considered in the light of these results.     

Genetic studies of the ribosomal proteins in Escherichia coli. 7. Mapping of several ribosomal protein components by transduction experiments between different strains of Escherichia coli

Summary Two 50s (50-10 and 50-12) and two 30s (30-4 and 30-7) ribosomal proteins could be distinguished between Shigella dysenteriae Sh/s and Escherichia coli K-12 JC411 with CMC column chromatography. On the other hand, E. coli K-12 AT2472 was shown to have a 30s ribosomal protein, 30-6(AT), which is specific to this strain and distinguishable from 30-6 of other E. coli K-12 strains. Transduction experiments by phage Plkc between Sh. dysenteriae Sh/s and E. coli ATSPCO1, a spectinomycin resistant mutant derived from AT2472 in which the 30-4 protein is altered, indicated that the genes specifying the above five ribosomal protein components are located in the streptomycin region on the E. coli chromosome.The gene order for three 50s (50-8, 50-10 and 50-12) and three 30s [str (30-?), 30-4 and 30-6] ribosomal proteins on the chromosome was determined by transduction technique between Sh. dysenteriae Sh/s and E. coli ATSPC01, between E. coli ATSPC01 and E. coli ER05 (an erythromycin resistant strain in which the 50-8 protein is altered), and between Sh. dysenteriae Sh/s and E. coli ERSPC14 (str ^s spc ^r ery ^r), respectively. It was found that these protein genes are arranged on the chromosome in the order of str (30-?)-30-4-30-6-50-8-50-10-50-12.