Different arrangements of protein subunits and single-stranded circular DNA in the filamentous bacterial viruses fd and Pf1

A single-stranded circular DNA molecule of 6690 ± 450 nucleotides accounts for 5.5 ± 0.3% of the mass of Pf1 virus. The remaining mass is contributed almost entirely by subunits of the major coat protein. A non-integral nucleotide to subunit ratio of 0.87 ± 0.05 is calculated from the DNA content, the average nucleotide mass (309), and the known mass of one protein subunit (4609). There are therefore 7690 ± 680 major coat protein subunits in the virus. The virus length determined by electron microscopy is 1960 ± 70 nm. The data give an average axial distance of 2.55 ± 0.24 Å between protein subunits in dry virus. Since there is an up strand and a down strand of the circular DNA within the virus filament, an axial distance between bases in a given strand of 5.9 ± 0.5 Å is calculated. Available X-ray data show that an axial repeat of 72 Å, or slightly less, would be expected for dry Pf1 virus (0% relative humidity). A structural model in which 27 protein subunits and 24 nucleotides are contained in this repeat would be consistent with our data. The DNA conformation and the subunit packing in Pf1 differ considerably from those in fd, even though both are filamentous viruses containing single-stranded circular DNA. The uncertainties cited are 95% confidence limits.     

Different packaging of DNA in the filamentous viruses Pf1 and Xf

Xf Virus DNA, like Pf1 DNA, is a single-stranded circular molecule and contains, within experimental error, the same number of nucleotides, 7400. This was unexpected since Pf1 virus is 2 μm long while Xf virus is only 1 μm long. The ratio of nucleotides to major coat protein subunits has been found to be nearly unity in Pf1 and nearly two in Xf, but it is not certain that the ratios have exactly integer values. Calculations give the average axial internucleotide separation in Pf1virus as 5.3 Å whereas in Xf virus, the calculated separation is only 2.6 Å. The protein subunits in both Pf1 and Xf have calculated axial separations close to 2.6 Å. The results provide a solution to a problem encountered in the interpretation of X-ray diffraction patterns of these viruses concerning the number of protein subunits per helical turn.     

Hydrodynamic properties and structure of fd virus

A length of 8950 ± 200 Å and a diameter of 90 ± 10 Å have been obtained for fd virus from a simultaneous solution of the Broersma equations relating the length and diameter of a rod-like particle to its rotational, D_R, and translational, D_T, diffusion coefficients. Measurements of D_R were by transient electric birefringence, and of D_T by low-angle intensity fluctuation spectroscopy. A mass of (16.4 ± 0.6) × 10⁶ daltons was calculated from the Svedberg equation using our measured values of D_T, the sedimentation coefficient and the density increment. These results, together with the molecular weight of fd DNA, give a total number of major coat protein subunits of 2710 ± 110 and a ratio of nucleotides to protein subunits which is definitely non-integral, 2.30 ± 0.11. These measurements help delineate significant structural differences between fd and other filamentous viruses. Also included in this paper is an Appendix (by L. A. Day & S. A. Berkowitz) concerning the number of nucleotides, 6370 ± 140, and the density and refractive index increments of fd DNA.     

Pf1 bacteriophage replication-assembly complex: X-ray fibre diffraction and scanning transmission electron microscopy

X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and M_r = 29.6 × 10⁶. It consists of 1770 protein subunits, each of 15,400 M_r, and one viral DNA molecule of 2.3 × 10⁶M_r: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion.     

Assembly of the head of bacteriophage P22: X-ray diffraction from heads,proheads and related structures

We have used electron microscopy and small-angle X-ray diffraction to study the three principal structures found in the head assembly pathway of Salmonella phage P22. These structures are, in order of their appearance in the pathway: proheads, unstable filled heads (which lose their DNA and become empty heads upon isolation), and phage. In addition, we can convert proheads to an empty head-like form (the empty prohead) in vitro by treating them with 0.8% sodium dodecyl sulfate at room temperature.We have shown that proheads are composed of a shell of coat protein with a radius of 256 Å, containing within it a thick shell or a solid ball (outer radius 215 Å) of a second protein, the scaffolding protein, which does not appear in phage. The three other structures studied are all about 10% larger than proheads, having outer shells with radii of about 285 Å. Empty heads and empty proheads appear identical by small-angle X-ray diffraction to a resolution of 25 Å, both being shells about 40 Å thick. Phage appear to be made up of a protein shell identical to that in empty heads and empty proheads, within which is packed the DNA.Some details of the DNA packing are also revealed by the diffraction pattern of phage. The inter-helix distance is about 28 Å, and the hydration is about 1.5 g of water per g of DNA. Certain aspects of the pattern suggest that the DNA interacts in a specific mariner with the coat protein subunits on the inside edge of the protein shell.Thus, the prohead-to-head transformation involves, in addition to the loss of an internal scaffold and its replacement by DNA, a structural transition in the outer shell. Diffraction from features of the surface organization in these structures indicates that the clustering of the coat protein does not change radically during the expansion. The fact that the expansion occurs in vitro during the formation of empty proheads shows that it is due to the bonding properties of the coat protein alone, although it could be triggered in vivo by DNA -protein interactions. The significance of the structural transition is discussed in terms of its possible role in the control of head assembly and DNA packaging.     

Filamentous Bacterial viruses

The filamentous bacterial virus is a simple and well-characterized model system for studying how genetic information is transformed into molecular machines. The viral DNA is a single-stranded circle coding for about 10 proteins. The major viral coat protein is largely α-helical, with about 46 amino acid residues. Several thousand identical copies of this protein in a helical array form a hollow cylindrical tube 1–2μ long, of outer diameter 60 Å and inner diameter 20 Å, with the twisted circular DNA extending down the core of the tube. Before assembly, the viral coat protein spans the cell membrane, and assembly involves extrusion of the coat from the membrane. X-ray fibre diffraction patterns of the Pf 1 species of virus at 4°C, oriented in a strong magnetic field, give three-dimensional data to 4 Å resolution. An electron density map calculated from native virus and a single iodine derivative, using the maximum entropy technique, shows a helix pitch of 5.9 Å. This may indicate a stretched A-helix, or it may indicate a partially 3₁₀ helix conformation, resulting from the fact that the coat protein is an integral membrane protein before assembly, and is still in the hydrophobic environment of other coat proteins after assembly.     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Pf1 filamentous bacterial virus: X-ray fibre diffraction analysis of two heavy-atom derivatives

Specific chemical reactions have been used to prepare and characterize two different heavy-atom derivatives of Pfl filamentous bacterial virus. Two atoms of iodine were bound to Tyr25 of the coat protein using immobilized lactoperoxidase. One atom of mercury was introduced by first attaching a thiol group to the amino terminus of the protein. High quality X-ray fibre diffraction patterns of the virus were obtained using a strong magnetic field to orient the virions during preparation of fibres. Bessel functions were resolved by preparing native fibres at 4 °C, which induces layer-line “splitting” and thereby gives three-dimensional data to 4 Å resolution. Analysis of the intensity changes caused by the heavy atoms on the diffraction patterns at 10 Å resolution showed that the virus has 5.4 protein subunits per 15 Å pitch. The iodine atoms were found at a mean radius of 26 to 28 Å and the mercury at a radius of 31 to 33 Å.     

Assembly in vitro of bacteriophage P22 procapsids from purified coat and scaffolding subunits

The coat and scaffolding proteins of bacteriophage P22 procapsids have been purified in soluble form. By incubating both purified proteins with a mutant-infected cell extract lacking procapsids, but competent for DNA packaging in vitro (Poteete et al., 1979), we were able to obtain assembly of biologically active procapsids in vitro. The active species for complementation in vitro in both protein preparations copurified with the soluble subunits, indicating that these subunits represent precursors in procapsid polymerization.When the purified coat and scaffolding subunits were mixed directly, they polymerized into double-shelled procapsid-like structures during dialysis from 1.5 m-guanidine hydrochloride to buffer. When dialyzed separately under the same conditions, the scaffolding subunits did not polymerize but remained as soluble subunits, as did most of the coat subunits. No evidence was found for self-assembly of the scaffolding protein in the absence of the coat protein.The unassembled coat subunits sedimented at 3.9 S and the unassembled scaffolding subunits sedimented at 2.4 S in sucrose gradients. The Stokes' radius, determined by gel filtration, was 25 Å for the coat subunits and 34 Å for the scaffolding subunits. These results indicate that the scaffolding subunits are relatively slender elongated molecules, whereas the coat subunits are more globular.The experiments suggest that the procapsid is built by copolymerization of the two protein species. Their interaction on the growing surface of the shell structure, and not in solution, appears to regulate successive binding interactions.     

Filamentous bacterial viruses. XI. Molecular architecture of the class II (Pf1, Xf) virion

The diffraction patterns of the Pf 1 and Xf strains of filamentous bacterial viruses (class II) can be interpreted in terms of a simple helix of protein subunits with 15Åpitch, having 22 units in five turns. The protein subunits are each elongated in an axial direction, and also slope radially, so as to overlap each other, giving an arrangement of subunits reminiscent of scales on a fish. The protein helix forms a tube with inner diameter about 20Åand outer diameter about 60Å. The single-stranded circular DNA is contained within this tube, with two DNA strands running the length of the tube.The diffraction patterns of fd, If 1 and IKe (class I) can be interpreted in terms of a perturbed version of the class II simple helix.     

Subunit size and paracrystal structure of avian liver acetyl-CoA carboxylase.

The subunit molecular weight of chicken liver acetyl-CoA carboxylase has been redetermined by immunoprecipitation and sodium dodecyl sulfate gel electrophoresis. In the presence of parotid trypsin inhibitor, the immunoprecipitate gave a single band corresponding to a molecular weight of 230,000, which was also found to contain bound biotin. From the biotin content of the protomer (1.0 prosthetic group per 480,000 daltons) it appears that it consists of two non identical subunits, both with molecular weights of approximately 230,000.Electron microscopy has been carried out on the active filamentous form of the enzyme and on paracrystals formed under high-salt conditions. These indicate that the filaments are readily distortable helical ribbons, with an approximate axial repeat of 1100 Å, containing eight protomers. The paracrystals are made up of a staggered lateral packing of filaments.     

Filamentous bacterial viruses. XIL. Molecular architecture of the class I (fd, If1, IKe) virion

The X-ray diffraction patterns of the fd, If1 and IKe strains of filamentous bacterial viruses (class I) indicate that the arrangement of capsid proteins in the virion approximates a left-handed helix of 15 Å pitch with 4.5 units per turn. The protein molecules are each elongated in an axial direction, and also slope radially, so as to overlap each other and give an arrangement of molecules reminiscent of scales on a fish. This helix of capsid proteins is related to the class II helix by a small twist about the helix axis. The protein molecules are also perturbed (by a few Ångström units) away from the positions that they would occupy in a simple 4.5 units per turn helix. The perturbation repeats about every five protein molecules, and is mainly axial. This arrangement of proteins forms a tube with inner diameter about 20 Å and outer diameter about 60 Å, encapsulating the DNA.     

Structural and enzymatic studies of the T4 DNA replication system. I. Physical characterization of the polymerase accessory protein complex

In this study, we have investigated the structural and physical properties of the bacteriophage T4 DNA polymerase accessory proteins. We find that T4 gene 44 and 62 proteins associate to form a tight, highly homogeneous complex, containing four gene 44 protein subunits and one gene 62 protein subunit. The molecular mass of the complex is 163,700 daltons. Sedimentation results suggest that the complex is quite asymmetric, with a prolate ellipsoid axial ratio of about 5:1. This protein complex is known to carry a DNA-dependent ATPase activity; we show by photoaffinity labeling that the ATP-binding sites reside in the gene 44 protein subunits of the complex. Equilibrium sedimentation and chemical cross-linking studies indicate that the T4 gene 45 protein self-associates to form a trimer in solution. This trimer species also appears to be quite asymmetric, showing an axial ratio for a prolate ellipsoid of about 6:1, assuming normal hydration.     

X-ray scattering of the non-isometric Bacillus subtilis phage phi29.

The non-isometric virus φ29 and its empty capsid have enough elements of symmetry so that their size and approximate shape can be determined by low-angle X-ray scattering. The scattering curve of the virus can be simulated by a cylinder of 390 Å diameter and 460 Å height. The protein coat has a thickness of about 30 Å. The DNA is packed tightly and regularly in the phage head.     

Filamentous bacteriophage Pf1 structure determined at 7 Å resolution by refinement of models for the α-helical subunit

The molecular structure of filamentous bacteriophage Pf1 has been determined to 7 Å resolution by analysis of X-ray diffraction data from partially oriented fibers of virus particles. The continuous intensity distribution along layer-lines was measured by numerically separating contributions from overlapping layer-lines. The data were phased by an iterative refinement technique that used the known spatial extent and high α-helical content of the virus particle to refine a structural model. This refinement converges to a unique structural solution that is consistent with the X-ray data and with information derived from physical and chemical studies. The coat protein consists of two α-helical segments: one, almost parallel to the particle axis, is centered at a radius of about 15 Å; the other, at about 25 Å radius, is tilted by about 25 ° to the particle axis. This structure is consistent with every generalization about α-helical packing. The inner and outer segments interlock along most of their length with a crossing angle of 20.5 °. The inner α-helical segments also interact with symmetry-related copies of themselves, as do the outer segments. The double layer of tightly packed, intricately interlocked α-helices forms a stable, 20 Å thick protein coat around the viral DNA.     

Structure and assembly of filamentous bacterial viruses.

Filamentous bacterial viruses are flexible nucleoprotein rods, about 6 nm in diameter by 1000-2000 nm in length (depending on the virus strain). A protein shell encloses a central core of single-stranded circular DNA. The coat protein subunits forming the shell are largely alpha-helix, elongated in an axial direction, and also sloping radially, so as to overlap each other and give an arrangement of subunits reminiscent of scales on a fish. This arrangement of alpha-helices is rather like some models of myosin filaments. An early step in assembly of the virion is the formation of a complex between the viral DNA and an intracellular packaging protein that is not found in completed virions. Newly synthesized coat protein becomes associated with the plasma membrane of the cell. During the final steps of assembly, the packaging protein is displaced from the DNA and replaced by coat protein as the virion passes out through the plasma membrane of the host cell.     

Gene V protein of fd bacteriophage. Dimer formation and the role of tyrosyl groups in DNA binding.

Gene V protein exists principally as a dimer in neutral buffers which are 0.15 M in NaCl, NaF, or NaClO4. Higher concentrations of NaClO4 or NaCl disrupt the dimers, but higher concentrations of NaF do not. There is a single sulfhydryl group per gene V protein monomer in native monomers, dimers, and DNA-protein complexes. Disulfide formation leads to loss of protein solubility and DNA binding capacity. The fluorescence of tyrosyl groups is the same for monomers and dimers in NaCl and NaClO4 solutions, but it is extensively quenched on binding to poly(dT) and fd-DNA. Complexes of gene V protein and fd-DNA isolated from lysates of infected cells were found to contain 4.70- +/- 0.13 nucleotides per monomer of gene V protein whereas complexes formed in vitro contain 4.05 +/- 0.17 nucleotides/monomer. It is postulated that tyrosyl groups are not involved in the protein-protein interactions of the monomer-dimer equilibrium, that tyrosyl groups stack with DNA bases in the complexes, and that each subunit of gene V protein in the intracellular complexes with fd-DNA is replaced by exactly two subunits of major coat protein during final assembly of the virus.     

Elongation of duplex DNA by recA protein

recA protein, which is essential for the recombination process in Escherichia coli, was incubated in the presence of 5′-γ-thiotriphosphate with circular plasmid pBR_βG containing small single-stranded gaps. Stable complexes were formed which appear in the electron microscope as fibres with a diameter about five times that of naked DNA. Complex formation appears to be a co-operative process whereby the average rise per base-pair with respect to the fibre axis increases from 3·39 ± 0·08 Å to 5·20 ± 0·18 Å. The elongation of DNA by about 50% is compatible with an unwinding of the double helix and an intercalating mode of binding of recA and/or 5′-γ-thiotriphosphate to DNA.