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At least 19 recordsLinked to original sources

PubMed1985

Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein.

The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate. The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG). The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy. Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. Lysine-25 was identified by peptide mapping as being the major protected residue. Modification of this residue does abolish DNA-binding activity. Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA. Peptide mapping established that, in this case, lysine-17 was the major protected residue. The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA.

Amino Acids↗

PubMed2005

Structural basis of the temperature transition of Pf1 bacteriophage.

The filamentous bacteriophage Pf1 undergoes a reversible temperature-dependent transition that is also influenced by salt concentrations. This structural responsiveness may be a manifestation of the important biological property of flexibility, which is necessary for long, thin filamentous assemblies as a protection against shear forces. To investigate structural changes in the major coat protein, one- and two-dimensional solid-state NMR spectra of concentrated solutions of Pf1 bacteriophage were acquired, and the structure of the coat protein determined at 0 degrees C was compared with the structure previously determined at 30 degrees C. Despite dramatic differences in the NMR spectra, the overall change in the coat protein structure is small. Changes in the orientation of the C-terminal helical segment and the conformation of the first five residues at the N-terminus are apparent. These results are consistent with prior studies by X-ray fiber diffraction and other biophysical methods.

Amino Acid Sequence↗

PubMed1993

Identification of a compact DNA-binding domain in the gene 5 protein of Pf1 bacteriophage.

The structure of the gene 5 protein of filamentous bacteriophage Pf1 and its interaction with viral DNA have been investigated by a series of limited proteolysis experiments. The ability of purified proteolytic fragments of the Pf1 gene 5 protein to bind oligonucleotides and polynucleotides was monitored by gel retardation and fluorescence. The results show the presence of a compact DNA-binding "core" domain consisting of residues 1-112 of the protein, which is protected from proteolysis in the nucleoprotein complex. Digestion of the free gene 5 protein with subtilisin produces a smaller fragment (residues 7-102) which can no longer bind DNA. Although the N-terminal "core" domain shows full DNA binding activity by fluorescence, the gel retardation experiments suggest reduced kinetic stability of this domain in complexes with oligonucleotides, resulting from the removal of residues 113-144 from the C-terminus of the protein. The sequence of the C-terminal 32 amino acid residues is unusual, with a high proportion of alanine, glutamine, and proline residues which may be related to the role of this sequence in stabilizing the complex.

Amino Acid Sequence↗

PubMed1991

Membrane-mediated assembly of filamentous bacteriophage Pf1 coat protein.

Filamentous bacteriophage Pf1 assembles by a membrane-mediated process during which the viral DNA is secreted through the membrane while being encapsulated by the major coat protein. Neutron diffraction studies showed that in the virus most of the coat protein consists of two alpha-helical segments arranged end-to-end with an intervening mobile surface loop. Nuclear magnetic resonance studies of the coat protein in the membrane-bound form have shown that the secondary structure is essentially identical to that in the intact virus. A comparison indicates that during membrane-mediated viral assembly, while the secondary structure of the coat protein is largely conserved, its tertiary structure changes substantially.

Bacteriophages↗

PubMed1987

Cloning and expression of the filamentous bacteriophage Pf1 major coat protein gene in Escherichia coli. Membrane protein processing and virus assembly.

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the replicative form (RF) DNA of the class II filamentous bacteriophage Pf1, which infects Pseudomonas aeruginosa. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter. In transformed Escherichia coli JM101 cells, in the presence of the inducer isopropyl-beta-D-thiogalactoside, the bacteriophage Pf1 gene was strongly expressed. The bacteriophage Pf1 coat protein displays the same pattern of negatively charged N-terminal region, hydrophobic middle region and positively charged C-terminal region as that of its counterpart in the class I bacteriophage fd, which infects E. coli, but otherwise the two proteins have no sequence homology. However, the Pf1 procoat protein was found to undergo processing and insertion into the E. coli cell inner membrane, like its fd counterpart, demonstrating that this part of the assembly process is the same for these different bacteriophages. The complete transcriptional unit, incorporating the tac promoter and rrnB transcription terminators flanking the Pf1 coat protein gene, was excised from the expression plasmid and cloned into the intergenic space of bacteriophage R252, an fd bacteriophage that carries an amber mutation in its own major coat protein gene. The Pf1 coat protein gene was again well expressed in infected E. coli cells but the chimeric bacteriophage had growth properties identical to those of the parent bacteriophage R252 on suppressor and non-suppressor strains of E. coli. The class I bacteriophage Pf1 coat protein evidently cannot be recognized by the class I bacteriophage assembly complex at or in the E. coli cell inner membrane, either at the point of initiation of assembly or during the elongation process.

Amino Acid Sequence↗

PubMed2000

The molecular structure and structural transition of the alpha-helical capsid in filamentous bacteriophage Pf1.

The major coat protein in the capsid of Pf1 filamentous bacteriophage (Inovirus) forms a helical assembly of about 7000 identical protein subunits, each of which contains 46 amino-acid residues and can be closely approximated by a single gently curved alpha-helix. Since the viral DNA occupies the core of the tubular capsid and appears to make no significant specific interactions with the capsid proteins, the capsid is a simple model system for the study of the static and dynamic properties of alpha-helix assembly. The capsid undergoes a reversible temperature-induced structural transition at about 283 K between two slightly different helix forms. The two forms can coexist without an intermediate state, consistent with a first-order structural phase transition. The molecular model of the higher temperature form was refined using improved X-ray fibre diffraction data and new refinement and validation methods. The refinement indicates that the two forms are related by a change in the orientation of the capsid subunits within the virion, without a significant change in local conformation of the subunits. On the higher temperature diffraction pattern there is a region of observed intensity that is not consistent with a simple helix of identical subunits; it is proposed that the structure involves groups of three subunits which each have a slightly different orientation within the group. The grouping of subunits suggests that a change in subunit libration frequency could be the basis of the Pf1 structural transition; calculations from the model are used to explore this idea.

Amino Acid Sequence↗

PubMed1991

DNA sequence of the filamentous bacteriophage Pf1.

The genome of the class II filamentous bacteriophage Pf1 has been sequenced by a combination of the chain termination and chemical degradation methods. It consists of 7349 nucleotides in a closed, circular loop of single-stranded DNA. The size and position of its open reading frames (ORFs) in general resemble those of other filamentous bacteriophage genomes. The size and position of the spaces between the ORFs have not been conserved, however, and six short reading frames (2 of which overlap) occupy a region corresponding to that filled by genes 2 and 10 in the Ff genome. Most of the ORFs are preceded by sequences resembling ribosome binding sites from the phage's host. Pseudomonas aeruginosa, that appear to differ somewhat from their counterparts in Escherichia coli. A search for sequences related to known pseudomonad promoters suggests that the promoters in this bacteriophage may well be ntr-dependent, with the two strongest preceding the gene for the major coat protein (gene 8) and another ORF (430). Gene 8 is followed by a sequence with the properties of a rho-independent terminator of transcription, like that at the same position in the genome of Ff. The Pf1 genome contains no collection of potential stem-and-loop structures corresponding to those that initiate replication of Ff DNA and assembly of the Ff virion, although isolated structures of this kind are present. The available evidence suggests that at least 13 of the 14 major ORFs are expressed. Overall, the organization of the Pf1 genome differs from that of the other class II filamentous phage whose genome has been sequenced, Pf3, as much as it does from that of the class I phages Ff and IKe.

Amino Acid Sequence↗

PubMed2001

Characterization of molecular alignment in aqueous suspensions of Pf1 bacteriophage.

The phase diagram of Pf1 solutions has been studied indirectly by observation of 2H quadrupole splittings of the solvent signal and measurement of dipolar couplings in solute macromolecules. At low volume fractions of Pf1 and at high ionic strength, alignment of both the phage and the solute depends strongly on the strength of the magnetic field. Both the theoretical and experimentally determined phase diagram of Pf1 show that at low concentrations and high ionic strengths the solution becomes isotropic. However, just below the nematic phase boundary the behavior of the system is paranematic, with cooperative alignment which depends on the strength of the applied magnetic field. Above 16 mg/ml Pf1 is fully nematic up to 600 mM NaCl. Alignment of proteins with a significant electric dipole moment, which tends to be strong in Pf1, can be reduced by either high ionic strength or low phage concentration. Because ionic strength modulates both the orientation and magnitude of the alignment tensor in Pf1 medium, measurement at two ionic strengths can yield linearly independent alignment tensors.

Antigens, Bacterial↗

PubMed1999

Conformational studies of the C-terminal domain of bacteriophage Pf1 gene 5 protein.

The gene 5 protein (g5p) of the bacteriophage Pf1 is a 144 residue single-stranded (ss) DNA binding protein involved in replication and packaging of the viral DNA. Compared to the gene 5 proteins of other filamentous bacteriophages, such as fd, the Pf1 g5p has an additional C-terminal sequence ( approximately 40 residues) with an unusual amino acid composition, being particularly rich in proline, glutamine and alanine. This C-terminal sequence is susceptible to limited proteolysis, in contrast to the globular N-terminal domain of the protein. The C-terminal sequence has been shown to play a role in the stabilisation of the protein-ssDNA complex. In the present study, the DNA sequence corresponding to the 38 amino acid residue C-terminal peptide has been cloned and expressed. A variety of biophysical techniques suggest that this peptide has a largely irregular conformation in solution, in contrast to the N-terminal globular domain that is principally beta-sheet. However, circular dichroism (CD) spectroscopy indicates that the peptide can be induced to form a structure that resembles a left-handed polyproline-like (P(II)) helix, suggesting that the C-terminal tail of the protein may adopt a more structured conformation in the appropriate physiological environment.

Amino Acid Sequence↗

PubMed1986

The ionic properties of the filamentous bacteriophages Pf1 and fd.

The surface charge of the filamentous bacteriophages Pf1 and fd was determined by polyelectrolyte titration covering a range from pH 3 to 9. The isoelectric point was measured at pH 4.0 for Pf1 and at pH 4.2 for fd. Close agreement of experimental and calculated data was found, confirming the hypothesis that charged residues in the DNA are neutralized by oppositely charged residues of the coat proteins. Almost perfect consistence is reached when structural details of the viruses are included.

Coliphages↗

PubMed2004

Line shape analyses for water 17O NMR quintet observed in a bacteriophage Pf1 solution at different temperatures.

Partially resolved 17O NMR quintet was observed in a filamentous bacteriophage Pf1 solution at 70 degrees C with a quadrupole splitting approximately 100 Hz. As the temperature decreased, the resolution was reduced but the line shapes were still indicative of residual quadrupole splitting. Line shape analyses were performed using the quadrupolar relaxation theory for spin 5/2. The contribution to the residual quadrupole splitting from the electric field gradients stemming from the phage filaments, which were oriented in the magnet, was taken into account. As a result, the observed 17O spectra at different temperatures were simulated and the hydration number of the phage DNA was determined.

Bacteriophage Pf1↗

PubMed2006

Nuclear spin relaxation of sodium cations in bacteriophage Pf1 solutions.

The nuclear magnetic resonance (NMR) spectra for the I=3/2 23Na cation dissolved into filamentous bacteriophage Pf1 solutions display line splittings and relaxation times consistent with an interaction between the 23Na nuclear quadrupole moment and the electric field gradient produced by the negatively charged Pf1 particles. The 23Na NMR line splittings and relaxation rates corresponding to magnetization recovery and single, double, and triple quantum coherence decays are measured in Pf1 solutions and compared to theoretical values. The deviation of the observed dc spectral density J0 from the equal first harmonic J(omega0) and second harmonic J(2omega0) values as J(omega0)=J(2omega0) not equal to J0 in these solutions suggests that ion migration in the electric field gradient of the Pf1 particles produces an anisotropic relaxation mechanism. Correlation functions and thus spectral densities for this process are calculated from solutions to the Fokker-Planck equation for radial motion in an electric potential and used to estimate measured relaxation rates. Appropriate electric potentials are generated from the solutions to the Poisson-Boltzmann equation for a charged Pf1 particle in aqueous phase, functions that lead to theoretical estimates of NMR line splittings consistent with experimental observations.

Bacteriophage Pf1↗

PubMed1987

Dynamic light-scattering study on changes in flexibility of filamentous bacteriophage Pf1 with temperature.

The temperature dependence of the flexibility of bacteriophage Pf1 was investigated by dynamic light scattering, and the following results were obtained: The gamma/K2 values measured at 1 degree-25 degrees C and at various K values were T/eta-scaled to 20 degrees C, where gamma is the first cumulant of the field correlation function of scattered light, K is the length of the scattering vector, T is the absolute temperature, and eta is the solvent viscosity at T. And it was found that the scaled gamma/K2 values at low K values were independent of temperature, whereas those at high K values increased sigmoidally and reversibly against temperature. This suggests that the virion is more flexible at temperatures above the transition temperature Tt. This characteristic temperature Tt depended on the pH of the suspension: Tt = 11 degrees C at pH 6.9 and Tt = 8 degrees C at pH 8.2.

Bacteriophages↗

PubMed1991

NMR studies of the structure and dynamics of membrane-bound bacteriophage Pf1 coat protein.

Filamentous bacteriophage coat protein undergoes a remarkable structural transition during the viral assembly process as it is transferred from the membrane environment of the cell, where it spans the phospholipid bilayer, to the newly extruded virus particles. Nuclear magnetic resonance (NMR) studies show the membrane-bound form of the 46-residue Pf1 coat protein to be surprisingly complex with five distinct regions. The secondary structure consists of a long hydrophobic helix (residues 19 to 42) that spans the bilayer and a short amphipathic helix (residues 6 to 13) parallel to the plane of the bilayer. The NH2-terminus (residues 1 to 5), the COOH-terminus (residues 43 to 46), and residues 14 to 18 connecting the two helices are mobile. By comparing the structure and dynamics of the membrane-bound coat protein with that of the viral form as determined by NMR and neutron diffraction, essential features of assembly process can be identified.

Capsid↗

PubMed1988

Conformation of the coat protein of filamentous bacteriophage Pf1 determined by neutron diffraction from magnetically oriented gels of specifically deuterated virions.

The structure of filamentous bacteriophage Pf1 has been studied using neutron diffraction from magnetically oriented gels of native and valine-deuterated phage. Neutron diffraction intensities were measured to approximately 8 A resolution along the equator and first six layer-lines, and differences due to the deuterated valine residues were apparent. Analysis of equatorial data indicate that one valine residue is located at a radius of about 13 A, three are in the hydrophobic center of the protein coat at an average of about 22 A radius, and one is near the outer surface of the virion at about 28 A radius. Analysis of the three-dimensional data was initiated using the rod model for the alpha-helices of the coat protein derived from earlier X-ray diffraction studies. This model was refined against the neutron diffraction intensities from native phage to obtain a phase set that was used to calculate a difference map between the valine-deuterated and native phage. The difference map exhibits peaks that correspond to the positions of the five valine residues in the coat protein. From the amino acid sequence and the alpha-helical conformation of the coat protein, the five valine residues can be unambiguously assigned to the difference peaks. This assignment indicates that the two alpha-helices of the coat protein are parallel to one another, connected by a short stretch of non-helical peptide. The valine positions also indicate that the helical surface lattice of the phage particle is right-handed.

Amino Acid Sequence↗

PubMed1983

Pf1 bacteriophage replication--assembly complex. X-ray fibre diffraction of the high humidity form.

The helical intracellular nucleoprotein complex of Pf1 bacteriophage has been studied by X-ray fibre diffraction in various hydration states. The helix pitch changes from 44 A in dry fibres to 55 A in wet fibres, whereas the unit rise between subunits in the helix apparently does not change with humidity. This result indicates that the nucleoprotein assembly twists more readily than it stretches. This is consistent with its biological role of tightening the viral DNA into a more compact form for packaging in the virion.

Coliphages↗

PubMed2004

Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy.

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.

Bacteriophage Pf1↗

PubMed1991

Neutron diffraction studies of the structure of filamentous bacteriophage Pf1. Demonstration that the coat protein consists of a pair of alpha-helices with an intervening, non-helical surface loop.

The structure of filamentous bacteriophage Pf1 has been studied using neutron diffraction from magnetically oriented gels of native and specifically deuterated phage. These methods have been used to determine the positions of the two methionine, two tyrosine and six isoleucine residues of the coat protein. Combined with the positions of the five valine residues previously determined, they represent one third (15 of 46) of the residues of the coat protein. These 15 amino acid residue positions have been used as the basis for constructing a model for the protein consisting of two alpha-helices with an intervening surface loop. The first helix extends from near the amino terminus to Ile12. The second helix extends from Lys20 to at least Met42, and may contain a bend between Ile32 and Val35. The two helices are tilted by about 15 degrees relative to one another, and are positioned in such a way that they appear to be bound end-to-end by main-chain hydrogen bonds. The intervening, non-helical loop, made up of Thr13 to Met19, connects the two helices without disrupting the pattern of main-chain hydrogen bonding, but does not result in a bend in the otherwise continuous helical structure. This model is used to predict the approximate positions of all amino acid residues in the Pf1 protein coat, providing a basis for further understanding of a number of viral properties including the symmetry transitions, the non-isomorphism of heavy-atom derivatives, and the protein-protein and protein-DNA interactions in the virion.

Amino Acid Sequence↗