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.     

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.     

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.     

Structure similarity, difference and variability in the filamentous viruses fd, If1, IKe, Pf1 and Xf. Investigation by laser Raman spectroscopy

The filamentous bacteriophages fd, If1, IKe, Pf1, Xf and Pf3 in aqueous solutions of low, moderate and high ionic strength have been investigated as a function of temperature by laser Raman difference spectroscopy. By analogy with Raman spectra of model compounds and viruses of known structure, the data reveal the following structural features: the predominant secondary structure of the coat protein subunit in each virus is the alpha-helix, but the amount of alpha-helix differs from one virus to another, ranging from an estimated high of 100% in Pf1 to a low of approximately 50% in Xf. The molecular environment and intermolecular interactions of tyrosine, tryptophan and phenylalanine residues differ among the different viruses, as do the conformations of aliphatic amino acid side-chains. The foregoing features of coat protein structure are highly sensitive to changes in Na+ concentration, temperature or both. The backbones of A-DNA and B-DNA structures do not occur in any of the viruses, and unusual DNA structures are indicated for all six viruses. The alpha-helical protein subunits of Pf1, like those of Pf3 and Xf, can undergo reversible transitions to beta-sheet structures while retaining their association with DNA; yet fd, IKe and If1 do not undergo such transitions. Raman intensity changes with ionic strength or temperature suggest that transgauche rotations of aliphatic amino acid side-chains and stacking of aromatic side-chains are important structural variables in each virus.     

Mass,length, composition and structure of the filamentous bacterial virus fd

Determinations by three independent methods gave an average of (14.6 ± 0.6) × 10⁶ daltons for the anhydrous mass of the filamentous bacterial virus fd; a determination of the mass per unit length by light scattering of the virus in solution gave 1560 ±60 daltons/Å; and three independent methods show that 12.0±O.2% of the virus mass is from the single-stranded circular DNA molecule. The data give an average axial distance of 3.82 ±0.15 Å between major coat protein subunits (5240 daltons each) for virus in solution. The DNA has an up strand and a down strand within the filament, and an average axial distance of 3.29 ± 0.14 Å between neighbouring nucleotides in a given strand is obtained from the data. There are 2.32 ±0.07 nucleotides per major coat protein subunit and hence each of the nucleotides cannot interact in the same way with subunits of coat protein. The results provide a basis for the interpretation of X-ray diffraction patterns of oriented fibers of the virus. The uncertainties cited above are 95% confidence limits.     

A theory of the symmetries of filamentous bacteriophages.

A mathematical model is presented which explains the symmetries observed for the protein coats of filamentous bacterial viruses. Three viruses (Ff, IKe, and If1) all have five-start helices with rotation angles of 36 degrees and axial translations of 16 A (Type I symmetry), and three other viruses (Pf1, Xf, and Pf3) all have one-start helices with rotation angles of approximately equal to 67 degrees and translations of approximately 3 A (Type II symmetry). The coat protein subunits in each group diverge from each other in amino acid sequence, and Type II viruses differ dramatically in DNA structure. Regardless of the differences, both Type I and Type II symmetry can be understood as direct, natural consequences of the close-packing of alpha-helical protein subunits. In our treatment, an alpha-helical subunit is modeled as consisting of two interconnected, flexible tubular segments that follow helical paths around the DNA, one in an inner layer and the other in an outer layer. The mathematical model is a set of algebraic equations describing the disposition of the flexible segments. Solutions are described by newly introduced symmetry indices and other parameters. An exhaustive survey over the range of indices has produced a library of all structures that are geometrically feasible within our modeling scheme. Solutions which correspond in their rotation angles to Type I and Type II viruses occur over large ranges of the parameter space. A few solutions with other symmetries are also allowed, and viruses with these symmetries may exist in nature. One solution to the set of equations, obtained without any recourse to the x-ray data, yields a calculated x-ray diffraction pattern for Pf1 which compares reasonably with experimental patterns. The close-packing geometry we have used helps explain the near constant linear mass density of known filamentous phages. Helicoid, rigid cylinder, and maximum entropy structure models proposed by others for Pf1 are reconciled with the flexible tube models and with one another.     

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.     

Subunit secondary structure in filamentous viruses: predictions and observations

The algorithm of Garnier, Osguthorpe and Robson (J. Mol. Biol. 120, 97-120, 1978) for prediction of protein secondary structure has been applied to the coat protein sequences of six filamentous bacteriophages: fd, If1, IKe, Pf1, Xf and Pf3. For subunits of Class I virions (fd, If1, IKe), the algorithm predicts a very high percentage of helix in comparison to other structure types, which is in accord with the results of laser Raman and circular dichroism measurements. For subunits of the Class II virions (Pf1, Xf, Pf3), the algorithm consistently predicts a predominance of beta structure, which is compatible with the demonstrated facility for conversion of Class II subunits from alpha-helix to beta-strand under appropriate experimental conditions (Thomas, Prescott and Day, J. Mol. Biol. 165, 321-356, 1983). Even when the algorithm is biased to favor helix, the Class II virion subunits are predicted to contain considerably more strand than helix. Qualitatively similar results are obtained using the algorithm of Chou and Fasman (Adv. Enzym. 47, 45-148, 45-148). Therefore, both predictive and experimental methods indicate a distinction between Class I and II subunits, which is reflected in a greater tendency of the latter to adopt other than uniform alpha-helical conformation. The results suggest a possible model for the disassembly of filamentous viruses which may involve the unraveling of coat protein helices at the N terminus.     

Quantitative analysis of nucleic acids, proteins, and viruses by Raman band deconvolution

A constrained, iterative Fourier deconvolution method is employed to enhance the resolution of Raman spectra of biological molecules for quantitative assessment of macromolecular secondary structures and hydrogen isotope exchange kinetics. In an application to the Pf1 filamentous bacterial virus, it is shown that the Raman amide I band contains no component other than that due to alpha-helix, indicating the virtual 100% helicity of coat proteins in the native virion. Comparative analysis of the amide I band of six filamentous phages (fd, If1, IKe, Pf1, Xf, and Pf3), all at the same experimental conditions, indicates that the subunit helix-percentage ranges from a high of 100% in Pf1 to a low of 71% in Xf. Deconvolution of amide I of Pf3 at elevated temperatures, for which an alpha-to-beta transition was previously reported (Thomas, G. J., Jr., and L. A. Day, 1981, Proc. Natl. Acad. Sci. USA., 78:2962-2966), allows quantitative evaluation of the contributions of both alpha-helix and beta-strand conformations to the structure of the thermally perturbed viral coat protein. Weak Raman lines of viral DNA bases and coat protein side chains, which are poorly resolved instrumentally, are also distinguished for all viruses by the deconvolution procedure. Application to the carbon-8 hydrogen isotope exchange reaction of a purine constituent of transfer RNA permits accurate determination of the exchange rate constant, which is in agreement with calculations based upon curve-fitting methods.     

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.     

Silver and mercury probing of deoxyribonucleic acid structures in the filamentous viruses fd, If1, IKe, Xf, Pf1, and Pf3

Ag+ binding and Hg2+ binding to both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) have been examined in some detail, and the results have been applied to study the structures of circular ssDNA in several filamentous viruses. It has been known for some time that Ag+ and Hg2+ bind to the bases of DNA producing characteristic large changes in absorbance and circular dichroism (CD) spectra, as well as changes in sedimentation rates. In the case of Ag+, it is known that there are three modes of binding to isolated dsDNA, referred to as types I, II, and III. Type III binding, by definition, occurs when Ag+ binds to Ag-dsDNA complexes having sites for binding types I and II extensively occupied, if not saturated. It produces CD spectra, assigned in this study, and absorbance spectra that are isosbestic with those of the Ag-dsDNA complexes present prior to its onset. In phosphate buffers binding is restricted to types I and II, whereas in borate buffers weaker type III binding can occur. Characteristics of types I, II, and III were observed for the DNAs in fd, If1, IKe, and Xf, but not for those in Pf1 and Pf3. Similarly, many of the spectral changes seen when Hg2+ binds to isolated double-stranded DNA are mimicked by Hg2+ binding to the DNAs within fd, IKe, If1, and Xf, but not for those in Pf1 and Pf3. The Ag+ and Hg2+ results indicate the presence of right-handed DNA helices in fd, If1, IKe, and Xf, with the two antiparallel strands of the covalently closed single-stranded DNAs having the bases directed toward the virion axes. For Pf1 and Pf3, Ag+ and Hg2+ binding cause large absorbance changes but only small CD changes. The very different results for Pf1 and Pf3 are consistent with the presence of inverted DNA structures (I-DNA) with the bases directed away from the structure axes, but the two structures differ from one another. Sedimentation velocity changes with Ag+ and Hg2+ binding strongly suggest structural linkages between the DNA and the surrounding protein sheath in each of the viruses.     

Structural details of the thermophilic filamentous bacteriophage PH75 determined by polarized Raman microspectroscopy

The filamentous virus PH75, which infects the thermophile Thermus thermophilus, consists of a closed DNA strand of 6500 nucleotides encapsidated by 2700 copies of a 46-residue coat subunit (pVIII). The PH75 virion is similar in composition to filamentous viruses infecting mesophilic bacteria but is distinguished by in vivo assembly at 70 degrees C and thermostability to at least 90 degrees C. Structural details of the PH75 assembly are not known, although a fiber X-ray diffraction based model suggests that capsid subunits are highly alpha-helical and organized with the same symmetry (class II) as in the mesophilic filamentous phages Pf1 and Pf3 [Pederson et al. (2001) J. Mol. Biol. 309, 401-421]. This is distinct from the symmetry (class I) of phages fd and M13. We have employed polarized Raman microspectroscopy to obtain further details of PH75 architecture. The spectra are interpreted in combination with known Raman tensors for modes of the pVIII main chain (amide I) and Trp and Tyr side chains to reveal the following structural features of PH75: (i) The average pVIII peptide group is oriented with greater displacement from the virion axis than peptide groups of fd, Pf1, or Pf3. The data correspond to an average helix tilt angle of 25 degrees in PH75 vs 16 degrees in fd, Pf1, and Pf3. (ii) The indolyl ring of Trp 37 in PH75 projects nearly equatorially from the subunit alpha-helix axis, in contrast to the more axial orientations for Trp 26 of fd and Trp 38 of Pf3. (iii) The phenolic rings of Tyr 15 and Tyr 39 project along the subunit helix axis, and one phenoxyl engages in hydrogen-bonding interaction that has no counterpart in either fd or Pf1 tyrosines. Also, in contrast to fd, Pf1, and Pf3, the packaged DNA genome of PH75 exhibits no Raman anisotropy, suggesting that DNA bases are not oriented unidirectionally within the nucleocapsid assembly. The structural findings are discussed in relation to intrasubunit and intersubunit interactions that may confer hyperthermostability to the PH75 virion. A refined molecular model is proposed for the PH75 capsid subunit.     

Kinetics of microtubule formation after cold disaggregation of the mitotic apparatus

The rate of spindle-fiber reformation following cold treatment of the giant amoeba, Chaos carolinensis, has been determined and used to test a single growth point, subunit incorporation model of microtubule assembly. Mitotic apparatuses isolated at one-minute intervals after rewarming contain progressively longer spindle fibers; re-assembly begins at the kinetochore region, proceeds at a rate of 1·5 μm per minute, then slows as the normal length of 5 μm is approached. From information on microtubule ultrastructure, the total number of 40-Å subunits in mitotic apparatuses per amoeba, and hence the concentration released during disassembly, was calculated to be 1·0 × 10¹⁵ molecules per cm³. Calculation of diffusion and assembly kinetics indicates that this concentration of microtubule subunits is equal to the concentration required to produce a growth rate of 1·5 μm per minute by diffusion of single subunits to one assembly point per microtubule.     

DNA packing in the filamentous viruses fd, Xf, Pf1 and Pf3.

Spectral data for filamentous viruses in the presence and absence of Ag+, together with other parameters, indicate that the DNA structures in two of the viruses, fd and Xf, are similar to each other but that these differ from two quite unusual and different DNA structures in Pf1 and Pf3.     

Electron microscopy of the stacked disk aggregate of tobacco mosaic virus protein. I. Three-dimensional image reconstruction

The three-dimensional structure of the stacked disk aggregate of tobacco mosaic virus protein has been determined from “phase plate” electron micrographs to an effective resolution of about 12 Å. It is a long rod comprised of paired rings of protein (disks), the subunits of which have different conformations according to which ring they belong. The two subunit conformations are such that the rings come close together within a disk near the outer surface of the particle, but between disks on the inside. This property, interpreted on the basis of a polar packing of the subunits, was established from an earlier, lower resolution, study by Finch &; Klug (1971). The present study shows, in addition, that the pairing is contributed mainly by axial distortions of the subunits in one of the rings, the axial distortions of the subunits in the other being largely replaced at lower radii by a tilt or twist and, at higher radii, by a slew. The subunits in the latter ring appear to have a conformation similar to that of the protein molecules in the virus.     

Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.     

Estimation of tyrosine-40-DNA distance in the filamentous phage Pf1 by analysis of its intrinsic fluorescence properties

Iodination of the exposed Tyr-25 in the coat protein decreases the fluorescence intensity of the filamentous phage Pf1 to less than 3% of its original fluorescence. If one assumes that the total residual fluorescence originates from the non-iodinated, buried Tyr-40, one can estimate the distance between Tyr-40 and the DNA bases in Pf1 to be less than 7 A, making use of the Foerster law for fluorescence energy transfer. The result is consistent with the idea that Tyr-40-DNA interaction is responsible for the unusually large axial base separation in Pf1-DNA.     

Structure of the filamentous bacteriophage,Pf3, by X-ray fiber diffraction

Fiber diffraction data have been obtained for the filamentous bacteriophage Pf3. The virus crystallizes on a hexagonal net with lattice constants a = b = 5.61 nm and c = 7.50 nm at 0% relative humidity and a = 6 = 6.20 nm and c = 7.99 nm at 98% relative humidity. The X-ray diffraction pattern resembles those of two other bacteriophages, Pf 1 and Xf, and therefore belongs to class II. The data are consistent with a 27₅ helix symmetry with an axial rise of 0.277 (dry) to 0.296 (wet) nm.     

Subunit Secondary Structure in Filamentous Viruses: Predictions and Observations

Abstract

The algorithm of Gamier, Osguthorpe and Robson (J. Mol. Biol. 120, 97–120, 1978) for prediction of protein secondary structure has been applied to the coat protein sequences of six filamentous bacteriophages: fd, Ifl, IKe, Pfl, Xf and Pf3. For subunits of Class I virions (fd, Ifl, IKe), the algorithm predicts a very high percentage of helix in comparison to other structure types, which is in accord with the results of laser Raman and circular dichroism measurements. For subunits of the Class II virions (Pfl, Xf, Pf3), the algorithm consistently predicts a predominance of β structure, which is compatible with the demonstrated facility for conversion of Class II subunits from α-helix to β-strand under appropriate experimental conditions (Thomas, Prescott and Day, J. Mol. Biol. 165, 321–356, 1983). Even when the algorithm is biased to favor helix, the Class II virion subunits are predicted to contain considerably more strand than helix. Qualitatively similar results are obtained using the algorithm of Chou and Fasman {Adv. Enzym. 47, 45–148,45-148). Therefore, both predictive and experimental methods indicate a distinction between Gass I and II subunits, which is reflected in a greater tendency of the latter to adopt other than uniform β-helical conformation. The results suggest a possible model for the disassembly of filamentous viruses which may involve the unraveling of coat protein helices at the N terminus.     

Crystal structure of rubredoxin from Pyrococcus furiosus at 0.95 Å resolution, and the structures of N-terminal methionine and formylmethionine variants of Pf Rd. Contributions of N-terminal interactions to thermostability

The high-resolution crystal structure of the small iron-sulfur protein rubredoxin (Rd) from the hyperthermophilic archeon Pyrococcus furiosus (Pf) is reported in this paper, together with those of its methionine ([_0M]Pf Rd) and formylmethionine (f[_0M]Pf Rd) variants. These studies were conducted to assess the consequences of the presence or absence of a salt bridge between the amino terminal nitrogen of Ala1 and the side chain of Glu14 to the structure and stability of this rubredoxin. The structure of wild-type Pf Rd was solved to a resolution of 0.95?Å and refined by full-matrix least-squares techniques to a crystallographic agreement factor of 12.8% [F>2σ(F) data, 25?617 reflections], while those of the [_0M]Pf and f[_0M]Pf Rd variants were solved at slightly lower resolutions (1.1?Å, R=11.5%, 17?213 reflections; 1.2?Å, R=13.7%, 12?478 reflections, respectively). The quality of the data was such that about half of the hydrogen atoms of the protein were clearly visible. All three structures were ultimately refined using the program SHELXL-93 with anisotropic atomic displacement parameters for all non-hydrogen protein atoms, and calculated hydrogen positions included but not refined. In this paper we also report thermostability data for all three forms of Pf Rd, and show that they follow the sequence wild-type >[_0M]Pf>formyl[_0M]Pf. Comparison of the three Pf Rd structures in the N-terminal region show that the structures of wild-type Pf Rd and f[_0M]Pf are rather similar, while that of [_0M]Pf Rd shows a number of additional hydrogen bonds involving the extra methionine group. While the salt bridge between the Ala1 amino group and the Glu14 carboxylate is not the primary determinant of the thermostability of Pf Rd, alterations to the amino terminus do have a moderate influence on the thermostability of this protein.