Virion Axis Research Articles

Pf1 bacteriophage hydration by magic angle spinning solid-state NMR.

High resolution two- and three-dimensional heteronuclear correlation spectroscopy ((1)H-(13)C, (1)H-(15)N, and (1)H-(13)C-(13)C HETCOR) has provided a detailed characterization of the internal and external hydration water of the Pf1 virion. This long and slender virion (2000 nm × 7 nm) contains highly stretched DNA within a capsid of small protein subunits, each only 46 amino acid residues. HETCOR cross-peaks have been unambiguously assigned to 25 amino acids, including most external residues 1-21 as well as residues 39-40 and 43-46 deep inside the virion. In addition, the deoxyribose rings of the DNA near the virion axis are in contact with water. The sets of cross-peaks to the DNA and to all 25 amino acid residues were from the same hydration water (1)H resonance; some of the assigned residues do not have exchangeable side-chain protons. A mapping of the contacts onto structural models indicates the presence of water "tunnels" through a highly hydrophobic region of the capsid. The present results significantly extend and modify results from a lower resolution study, and yield a comprehensive hydration surface map of Pf1. In addition, the internal water could be distinguished from external hydration water by means of paramagnetic relaxation enhancement. The internal water population may serve as a conveniently localized magnetization reservoir for structural studies.

A structural model for the single-stranded DNA genome of filamentous bacteriophage Pf1.

The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament ( approximately 2000 x 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue alpha-helical subunit. The subunit alpha-helices, which are inclined at a small average angle ( approximately 16 degrees ) from the virion axis, are arranged compactly around the DNA core. Orientations of the Pf1 DNA nucleotides with respect to the filament axis are not known. In this work we report and interpret the polarized Raman spectra of oriented Pf1 filaments. We demonstrate that the polarizations of DNA Raman band intensities establish that the nucleotide bases of packaged Pf1 DNA are well ordered within the virion and that the base planes are positioned close to parallel to the filament axis. The present results are combined with a previously proposed projection of the intraviral path of Pf1 DNA [Liu, D. J., and Day, L. A. (1994) Science 265, 671-674] to develop a novel molecular model for the Pf1 assembly.

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.

Orientation and Interactions of an Essential Tryptophan (Trp-38) in the Capsid Subunit of Pf3 Filamentous Virus

The filamentous bacteriophage Pf3 consists of a covalently closed DNA single strand of 5833 nucleotides sheathed by ∼2500 copies of a 44-residue capsid subunit. The capsid subunit contains a single tryptophan residue (Trp-38), which is located within the basic C-terminal sequence (–RWIKAQFF) and is essential for virion assembly in vivo. Polarized Raman microspectroscopy has been employed to determine the orientation of the Trp-38 side chain in the native virus structure. The polarized Raman measurements show that the plane of the indolyl ring is tilted by 17° from the virion axis and that the indolyl pseudo-twofold axis is inclined at 46° to the virion axis. Using the presently determined orientation of the indolyl ring and side-chain torsion angles, χ 1 (N–C α –C β –C γ ) and χ 2,1 (C α –C β –C γ –C δ1 ), we propose a detailed molecular model for the local structure of Trp-38 in the Pf3 virion. The present Pf3 model is consistent with previously reported Raman, ultraviolet-resonance Raman and fluorescence results suggesting an unusual environment for Trp-38 in the virion assembly, probably involving an intrasubunit cation- π interaction between the guanidinium moiety of Arg-37 and the indolyl moiety of Trp-38. Such a C-terminal Trp-38/Arg-37 interaction may be important for the stabilization of a subunit conformation that is required for binding to the single-stranded DNA genome during virion assembly.

Protein and DNA residue orientations in the filamentous virus Pf1 determined by polarized Raman and polarized FTIR spectroscopy.

The Pseudomonas bacteriophage Pf1 is a long ( approximately 2000 nm) and thin ( approximately 6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue alpha-helical subunit. The coat subunits are arranged as a superhelix of C(1)()S(5.4)() symmetry (class II). Polarized Raman and polarized FTIR spectroscopy of oriented Pf1 fibers show that the packaged single-stranded DNA genome is ordered specifically with respect to the capsid superhelix. Bases are nonrandomly arranged along the capsid interior, deoxynucleosides are uniformly in the C2'-endo/anti conformation, and the average DNA phosphodioxy group (PO(2)(-)) is oriented so that the line connecting the oxygen atoms (O.O) forms an angle of 71 degrees +/- 5 degrees with the virion axis. Raman and infrared amide band polarizations show that the subunit alpha-helix axis is inclined at an average angle of 16 degrees +/- 4 degrees with respect to the virion axis. The alpha-helical symmetry of the capsid subunit is remarkably rigorous, resulting in splitting of Raman-active helix vibrational modes at 351, 445 and 1026 cm(-)(1) into apparent A-type and E(2)()-type symmetry pairs. The subunit tyrosines (Tyr 25 and Tyr 40) are oriented with phenoxyl rings packed relatively close to parallel to the virion axis. The Tyr 25 and Tyr 40 orientations of Pf1 are surprisingly close to those observed for Tyr 21 and Tyr 24 of the Ff virion (C(5)()S(2)() symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed alpha-helical subunits, irrespective of capsid symmetry. The polarized Raman spectra also provide information on the orientations of subunit alanine, valine, leucine and isoleucine side chains of the Pf1 virion.

Intensity of the polarized Raman band at 1340-1345 cm-1 as an indicator of protein alpha-helix orientation: application to Pf1 filamentous virus.

Raman spectra of oriented alpha-helical protein molecules exhibit a prominent band near 1340-1345 cm(-)(1), the intensity of which is highly sensitive to molecular orientation. Polarization of the 1340-1345 cm(-)(1) marker is evident in Raman spectra of alpha-helical poly-L-alanine (alphaPLA) and alpha-helical poly-gamma-benzyl-L-glutamate (alphaPBLG). Corresponding polarization is also observed in Raman spectra of the filamentous virus Pf1, which is an assembly of alpha-helical coat protein molecules. In alphaPLA and alphaPBLG, we assign the band to a normal mode of symmetry type E(2) and specifically to a vibration localized in the (O=C)-C(alpha)-H linkages of the main chain peptide group. Although strict helical symmetry does not apply to coat subunits of filamentous viruses, an approximate E(2)-type mode may be presumed to account for a corresponding Raman band of Pf1 and fd filamentous viruses. Spectroscopic studies of N-methylacetamide and isotopically-edited fd viruses support the present assignment of the 1340-1345 cm(-)(1) band. Polarization anisotropy indicates that this band may be exploited as a novel indicator of protein alpha-helix orientation. Application of this approach to the polarized Raman spectrum of Pf1 suggests that, on average, the axis of the alpha-helical coat protein subunit in the native virion structure forms an angle of 20 +/- 10 degrees with respect to the virion axis.

Orientations of Tyrosines 21 and 24 in Coat Subunits of Ff Filamentous Virus: Determination by Raman Linear Intensity Difference Spectroscopy and Implications for Subunit Packing

Virions of the Ff group of bacteriophages ( fd, f1, M13) are morphologically identical filaments (∼6-nm diameter × ∼880-nm length) in which a covalently closed, single-stranded DNA genome is sheathed by ∼2700 copies of a 50-residue α-helical subunit (pVIII). Orientations of pVIII tyrosines (Tyr 21 and Tyr 24) with respect to the filament axis have been determined by Raman linear intensity difference (RLID) spectroscopy of flow-oriented mutant virions in which the tyrosines were independently mutated to methionine. The results show that the twofold axis of the phenolic ring (C1–C4 line) of Tyr 21 is inclined at 39.5 ± 1.4° from the virion axis, and that of Tyr 24 is inclined at 43.7 ± 0.6°. The orientation determined for the Tyr 21 phenol ring is close to that of a structural model previously proposed on the basis of fiber x-ray diffraction results (Protein Data Bank, identification code 1IFJ). On the other hand, the orientation determined for the Tyr 24 phenol ring differs from the diffraction-based model by a 40° rotation about the C α-C β bond. The RLID results also indicate that each tyrosine mutation does not greatly affect the orientation of either the remaining tyrosine or single tryptophan (Trp 26) of pVIII. On the basis of these results, a refined model is proposed for the coat protein structure in Ff.

Properties of the coat protein of a new tobacco mosaic virus coat protein ts-mutant.

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 A from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21=>T and D66=>G, both in the 70-A region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21-66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-A region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.

Evidence for Tilted Smectic Liquid Crystalline Packing of fd Inovirus from X-ray Fiber Diffraction

Fibers of the fd strain of Inovirus (filamentous bacteriophage) prepared above its isoelectric point (about pH 4) give X-ray diffraction patterns, higher pH patterns, that differ from the patterns of fd fibers below the isoelectric point, lower pH patterns. The overall distribution of intensity on higher and lower pH patterns is substantially the same, indicating that the virion structure is substantially the same, but the crystalline reflections that define the packing of the virions in crystallites are different. For the lower pH patterns, the crystalline reflections can be indexed on a conventional hexagonal lattice. However, for the higher pH patterns, the crystalline reflections on the equator and first layer line are spread out in a broad, angular, but specific fashion (layer-line fanning), unlike disorientation spreading. We interpret this observation to mean that the crystallites in the higher pH fiber are tilted with respect to the fiber axis. This interpretation is supported by simulated fiber diffraction patterns calculated from models of the packing of tilted virions. The diffraction patterns from fibers of some mutants of fd, and from other wild-type strains of Inovirus (M13, IKe, If1), do not show layer-line fanning. We propose that the tilting of crystallites that gives rise to layer-line fanning in fd fibers is due both to a specific pattern of charge on the surface of the fd virion at higher pH and to the presence of a specific inter-subunit hydrogen bond. Apparently these features of the fd virion structure require a strict 2-fold screw axis along the virion axis that affects the packing between neighboring virions, resulting in the formation of a tilted smectic liquid crystal phase.

Orientation of tryptophan-26 in coat protein subunits of the filamentous virus Ff by polarized Raman microspectroscopy.

The Ff filamentous virus, which includes the closely related strains fd, fl and M13, serves as a model for membrane protein assembly and is employed extensively as a cloning vector and vehicle for peptide display. The threadlike virion (approximately 6 x 880 nm) comprises a single-stranded DNA genome sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit, the product of viral gene VIII. The pVIII subunit contains a single tryptophan residue (tryptophan-26) which is essential for assembly. We have employed polarized Raman microspectroscopy to determine the orientation of tryptophan-26 in pVIII subunits of oriented fd fibers. The present application is based upon the transfer of tryptophan Raman tensors from a recent study of N-acetyl-L-tryptophan single crystals [Tsuboi et al. (1996) J. Mol. Struct. 379, 43-50]. The polarized Raman spectra of fd indicate that the plane of the indole ring in each pVIII subunit is close to parallel to the virion axis. In this orientation, the line connecting indole ring atoms N1 and C2 is nearly perpendicular to the virion axis, while the indole pseudo-2-fold axis (a line connecting atom C2 to the midpoint of the C5-C6 bond) is approximately 36 degrees from the virion axis. We have used the present results in combination with preferred tryptophan side-chain torsions [chi 1 (C3-C beta-C alpha-N) and chi 2.1 (C2-C3-C beta-C alpha)] in other proteins and a previously determined experimental value of chi 2.1 in fd [Aubrey. K. L., & Thomas, G. J., Jr. (1991) Biophys, J. 60, 1337-1349] to propose a detailed molecular model for the orientation of the tryptophan-26 side chain in the native virus.

Pf1 filamentous bacteriophage: refinement of a molecular model by simulated annealing using 3.3 A resolution X-ray fibre diffraction data.

The filamentous bacteriophage Pf1 is structurally similar to the well known Ff (fd, fl, M13) strains, but it gives much better X-ray diffraction patterns, enabling a more detailed analysis of the molecular structure. The 46-residue protein subunit can be closely approximated by a single gently curved stretch of alpha-helix. The axes of the subunits are at a small angle to the virion axis, and several thousand subunits form an overlapping inter-digitated helical array surrounding a DNA core. We have derived a detailed model of the virion based on X-ray data and stereochemical constraints. We have considered potential sources of error in the diffraction data, and used the improved data to study regions where the protein subunit of Pf1 may deviate from a continuous alpha-helix. We use simulated annealing to escape from local minima, and various kinds of electron-density maps to guide the model building. Refinement of the model shows that the first few residues at the N terminus are non-helical, and there is a slight discontinuity in the alpha-helix near the middle of the sequence. The model is consistent both with general structural principles derived from high-resolution analysis of other proteins, and with specific chemical and spectroscopic data about Pf1. We apply the same refinement techniques to an alternative model with a non-helical surface loop between residues 13 and 19. Comparative analysis of models with and without a loop shows that the loop model is not supported by 3.3 A resolution X-ray diffraction data.

DNA packing in filamentous bacteriophages.

in that the is assumed to have the smoothest possible distribution of electron density. Contributions from side chains of varying size and disposition are subsumed in the smoothness assumption. Model­ ing by this procedure with data to 3 A resolution axially and 4 A resolution radially produced a model that is a gently bent, continuous IX-helix of 1 3 turns. The axis of approximately the first five turns is approximately parallel to the main virion axis (Figure 10 of Reference 81) . The overall shapes of the three models are similar, as indicated by the lattice diagram of Figure 4. Ff Subunit Structure Determination Opella and his coworkers ( 108) have undertaken to determine the structure of the Ff subunits by measuring the nuclear magnetic spin interaction tensors for all 49 peptide backbone planes in the molecule. The spin interaction data, including dipole-dipole spin coupling, chemical shift anisotropy, and quadrupole interactions, yield a limited set of orientation angles for each peptide plane relative to its neighbors and to the main structure axis. The data also provide information on radial positioning due to the dependence of dipole-dipole interactions on distance. The set Table 4 Structural parameters for the protein coats Parameter Ff Xf Pfl PO References Symmetry of helix Rise per subunit (A) 16.0-16.2b 2.85 2.78-3.05 2.78-2.96 37,80,8�88, 101, 112 Rotation per subunitC _30.00b _66.7° -65.9--66.r -66.7° 77,85,88, 101, 112, 157 Pitch of helix' (A) 16.0-16.2b 15.4 15.0-16.6 15.0-15.8 37,80,85,88,101, 112 Symmetry designationd CSS2.0 CISS.4 CISS.4-- CISS.46 CISS.4 75 Symmetry designation (2/10; 3/15) (2/11; 3/17) (2/11; 3.17) (2/11; 3/17) 91 Subunit IX-helix content and orientation CD amplitudesf 18,33, 138 [111208 (cm2 deg dmol-') -27,000 -18,000 -35,000 -31,000 [111m (em2 deg dmol-') -41,000 -15,000 -32,000 -38,000 % IX-Helix 80-100 40--6 5 80-100 80-100 Raman 1650 cm -I amide I band 136, 137, 139 Deconvolved components 1648 (0.92) 1646 (0.71) 1652 (1.0) 1645 (0.82) 1667 (0.08) 1658 (0.29) 1666 (0.08) % IX-Helix 92 71 100 82 Infrared 1650 em-I amide I band 43,44 % IX-Helix 95-100 90-100 Average a-helix inclination to axis Birefringence 20-30° 145 Infrared dichroism 37±W 30± 10° 43, 44 NMR _10° -0, 20° 29, 108 • Helical parameters observed by fiber diffraction for various experimental conditions of temperature, humidity, and chemical modification. b Ff possesses fivefold rotational symmetry (76, 77, 104); the translation and rotation per pentamer of subunits are listed. cThe rotation per is limited to a narrow range afvalues, even far large changes in experimental conditions (91). d Symmetry notation of Klug et al (63a) (see text and Reference 75). An alternative notation gives the number af subunits and the number of helical turns in the apparent structural repeat, such as 27u/5t = 5.4 ult or 7Iu/13t = 5.46 u/t far Pfl (8\). 'N otation developed in a theoretical treatment of the symmetries of the filamentous phages (see text and Reference 91). f See Figure 6. Ul IV 0 > - Crl ..., > t' DNA PACKING IN FILAMENTOUS PHAGE 525

Pf1 Inovirus: Electron density distribution calculated by a maximum entropy algorithm from native fibre diffraction data to 3 Å resolution and single isomorphous replacement data to 5 Å resolution

We have calculated the electron density distribution of the Pf1 strain of filamentous bacteriophage by a maximum entropy method. In the calculation we included native X-ray fibre diffraction data extending to 3 Å resolution in the meridional direction on 60 layerlines that are resolved to 4 Å in the equatorial direction, and lower resolution data from a single isomorphous derivative iodinated on the Tyr25 residue. The electron density map indicates that the 46-residue protein subunit is a single, gently curved stretch of α-helix with its axis at an angle of about 20° to the axis of the virion. The α-helix subunit curves around the virion axis by about 1 6 turn, and decreases from about 27 Å radius to about 13 Å radius in the virion as the amino acid sequence of the subunit runs from the N terminus to the C terminus. Nearest-neighbour α-helical subunits are about 10 Å apart along their length, and the axis of each subunit makes an unexpected negative angle with its nearest neighbours in the virion. To confirm the validity of the maximum entropy calculation, we have varied the constraints on the calculation. All variations result in either a map that is close to the original map or a map that cannot be interpreted in terms of secondary structure: we find only one map that makes structural sense.

Maximum-entropy calculation of the electron density at 4 A resolution of Pf1 filamentous bacteriophage.

A 4 A electron-density map of Pf1 filamentous bacterial virus has been calculated from x-ray fiber diffraction data by using the maximum-entropy method. This method produces a map that is free of features due to noise in the data and enables incomplete isomorphous-derivative phase information to be supplemented by information about the nature of the solution. The map shows gently curved (banana-shaped) rods of density about 70 A long, oriented roughly parallel to the virion axis but slewing by about 1/6th turn while running from a radius of 28 A to one of 13 A. Within these rods, there is a helical periodicity with a pitch of 5 to 6 A. We interpret these rods to be the helical subunits of the virion. The position of strongly diffracted intensity on the x-ray fiber pattern shows that the basic helix of the virion is right handed and that neighboring nearly parallel protein helices cross one another in an unusual negative sense.

Electron microscopic observations on fd bacteriophage, its alkali denaturation products and its DNA

The length of fd bacteriophage, one of the filamentous viruses specific for male strains of Escherichia coli, has been found to be 8800 ± 150 Å by electron microscopy. From contour length measurements of its double-stranded forms it has been estimated that the single strand of fd DNA contains about 6700 nucleotides. From these data the base-base separation in the direction of the virion axis is estimated to be 2.6 Å. Alkaline degradation of the virion, monitored by changes in optical rotatory properties and infectivity and by the appearance of degradation products in electron micrographs, can be described as essentially an all-or-none process. Accordingly, a bacteriophage suspension subjected to alkali would contain, in addition to intact and fully denatured filaments, a very small fraction of filaments actually in the process of denaturation. In some of the micrographs presented, a few filaments can be seen which apparently have been caught in the act of denaturing. They are shorter than intact filaments and appear to be split longitudinally, as though native virions have two halves or strands.

F1, a Rod-shaped male-specific bacteriophage that contains DNA

Filamentous bacteriophages are interesting paradigms in structural molecular biology, in part because of the unusual mechanism of filamentous phage assembly. During assembly, several thousand copies of an intracellular DNA-binding protein bind to each copy of the replicating phage DNA, and are then displaced by membrane-spanning phage coat proteins as the nascent phage is extruded through the bacterial plasma membrane. This complicated process takes place without killing the host bacterium.The bacteriophage is a semi-flexible worm-like nucleoprotein filament. The virion comprises a tube of several thousand identical major coat protein subunits around a core of single-stranded circular DNA. Each protein subunit is a polymer of about 50 amino-acid residues, largely arranged in an α-helix. The subunits assemble into a helical sheath, with each subunit oriented at a small angle to the virion axis and interdigitated with neighbouring subunits. A few copies of “minor” phage proteins necessary for infection and/or extrusion of the virion are located at each end of the completed virion.Here we review both the structure of the virion and aspects of its function, such as the way the virion enters the host, multiplies, and exits to prey on further hosts. In particular we focus on our understanding of the way the components of the virion come together during assembly at the membrane. We try to follow a basic rule of empirical science, that one should chose the simplest theoretical explanation for experiments, but be prepared to modify or even abandon this explanation as new experiments add more detail.