On the structures of filamentous bacteriophage Ff (fd,f1, M13)

The filamentous bacteriophage (Inovirus) strain Ff (fd, f1, M13) is widely used in molecular biophysics as a simple model system. A low resolution molecular model of the fd protein coat has been reported, derived from iterative helical real space reconstruction of cryo-electron micrographs (cryoEM). This model is significantly different from the model previously derived from X-ray fibre diffraction and solid-state NMR. We show that the cryoEM model agrees neither with solid-state NMR data nor with X-ray fibre diffraction data of fd, and has some puzzling structural features, for instance nanometre holes through the protein coat. We refine the cryoEM model against the X-ray data, and find that the model after refinement closely approximates the model derived directly from X-ray fibre diffraction and solid-state NMR data. We suggest possible reasons for the differences between the models derived from cryoEM and X-ray diffraction.     

Raman spectroscopy of filamentous bacteriophage Ff (fd, M13, f1) incorporating specifically-deuterated alanine and tryptophan side chains. Assignments and structural interpretation.

Structural interpretation of the Raman spectra of filamentous bacteriophages is dependent upon reliable assignments for the numerous Raman vibrational bands contributed from coat protein and packaged DNA of the virion. To establish unambiguous assignments and facilitate structural conclusions derived from them, we have initiated a systematic study of filamentous bacteriophage Ff (fd, f1, M13) incorporating protein subunits with specifically deuterated amino-acid side chains. Here, we report and interpret the Raman spectra of fd virions which incorporate: (a) a single deuterio-tryptophan residue per coat protomer [fd(Wd5)], (b) ten deuterio-alanines per protomer [fd(10Ad3)], and (c) both deuterio-tryptophan and deuterio-alanine [fd(Wd5 + 10Ad3)]. The unambiguous assignment of coat protein Raman bands in normal and deuterated isotopomers of fd establishes the validity of earlier empirical assignments of many key Raman markers, including those of packaged ssDNA (Thomas et al., 1988). Present results confirm that deoxyguanosine residues of the packaged ssDNA molecule depart from the usual C2'-endo/anti conformation characteristic of protein-free DNA in aqueous solution, although C2'-endo/anti conformers of thymidine are not excluded by the data. The combined results obtained here on normal fd, and on fd incorporating deuterio-tryptophan [fd(Wd5) and fd(Wd5 + 10Ad3)], show also that the microenvironment of the single tryptophan residue per coat protomer (W26) can be clearly deduced as follows: (a) The indole 1-NH donor group of each protomer in fd forms a moderately strong hydrogen bond, most likely to a hydroxyl oxygen acceptor. (b) The planar indole ring exists in a hydrophilic environment. (c) The torsion angle describing the orientation of the indole ring (C3-C2 linkage) with respect to the side-chain (C alpha-C beta bond) is unusually large, i.e., magnitude of X2,1 approximately 120 degrees. With respect to alanine isotopomers, the present results show that alanine residues, and possibly other methyl-containing side chains, are significant contributors to the fd Raman spectrum. The present study provides new information on protomer side chains of fd and demonstrates a Raman methodology which should be generally useful for investigating single-site interactions and macromolecular conformations in other nucleoprotein assemblies.     

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.     

The gene II proteins of the filamentous phages IKe and Ff (M13, fd and f1) are not functionally interchangeable during viral strand replication.

Gene II protein is the only phage-encoded protein required for the double-stranded DNA replication of the distantly related filamentous phages IKe and Ff (M13, fd and f1). Complementation studies have demonstrated that, despite a significant degree of homology between the nucleotide sequences of the gene II's of IKe and Ff and the core's (domains A) of their viral strand replication origins, the biological functions of the gene II proteins are not interchangeable. The specificity of these proteins is not determined by the nucleotide sequence (domain B) which is required for efficient initiation of viral strand replication of Ff. In fact, our data indicate that a sequence with a similar function as domain B in Ff does not form part of the viral strand replication origin of IKe.     

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.     

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.     

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.     

Metal ion-induced lateral aggregation of filamentous viruses fd and M13

We report a detailed comparison between calculations of inter-filament interactions based on Monte-Carlo simulations and experimental features of lateral aggregation of bacteriophages fd and M13 induced by a number of divalent metal ions. The general findings are consistent with the polyelectrolyte nature of the virus filaments and confirm that the solution electrostatics account for most of the experimental features observed. One particularly interesting discovery is resolubilization for bundles of either fd or M13 viruses when the concentration of the bundle-inducing metal ion Mg(2+) or Ca(2+) is increased to large (>100 mM) values. In the range of Mg(2+) or Ca(2+) concentrations where large bundles of the virus filaments are formed, the optimal attractive interaction energy between the virus filaments is estimated to be on the order of 0.01 kT per net charge on the virus surface when a recent analytical prediction to the experimentally defined conditions of resolubilization is applied. We also observed qualitatively distinct behavior between the alkali-earth metal ions and the divalent transition metal ions in their action on the charged viruses. The understanding of metal ions-induced reversible aggregation based on solution electrostatics may lead to potential applications in molecular biology and medicine.     

Effects of virion and salt concentrations on the Raman signatures of filamentous phages fd, Pf1, Pf3, and PH75

Filamentous phages consist of a single-stranded DNA genome encapsidated by several thousand copies of a small alpha-helical coat protein subunit plus several copies of four minor proteins at the filament ends. The filamentous phages are important as cloning vectors, vehicles for peptide display, and substrates for macromolecular alignment. Effective use of a filamentous phage in such applications requires an understanding of experimental factors that may influence the propensity of viral filaments to laterally aggregate in solution. Because the Raman spectrum of a filamentous phage is strongly dependent on the relative orientation of the virion with respect to the polarization direction of the electromagnetic radiation employed to excite the spectrum, we have applied Raman spectroscopy to investigate lateral aggregation of phages fd, Pf1, Pf3, and PH75 in solution. The results show that lateral aggregation of the virions and anisotropic orientation of the aggregates are both disfavored by high concentrations of salt (>200 mM NaCl) in solutions containing a relatively low virion concentration (<10 mg/mL). Conversely, the formation of lateral aggregates and their anisotropic orientation are strongly favored by a low salt concentration (<0.1 mM NaCl), irrespective of the virion concentration over a wide range. The use of Raman polarization effects to distinguish isotropic and anisotropic solutions of filamentous phages is consistent with previously reported Raman analyses of virion structures in both solutions and fibers. The Raman data are supported by electron micrographs of negatively stained specimens of phage fd, which permit an independent assessment of salt effects on lateral aggregation. The present results also identify new Raman bands that serve as potential markers of subunit side-chain orientations in filamentous virus assemblies.     

Raman spectroscopy and deuterium exchange of the filamentous phage fd

The filamentous phage fd has been investigated using the techniques of Raman spectroscopy and deuterium exchange. Despite the rather uniform secondary structure of the fd phage coat protein, which is predominantly alpha-helix, the deuterium exchange is complex. A substantial fraction of the helical peptides exchange deuterium by 8 h at room temperature, yet another substantial fraction does not exchange following an additional 5 months at 4 degrees C. Heating the phage to 70 degrees C for several hours leads to additional deuterium exchange compared to samples soaked for 5 months in heavy water. We suggest that the wide variation in peptide exchange rates may be related to the phage protein quaternary structure, which has been shown to be a double layer of tightly packed helices. The accomplishment of enhanced exchange by reaction at high temperature combined with digital difference spectroscopic methods has enabled us to define the structure of the amide III and III' bands. The complexity of these bands is unexpected for a simple helical protein, but we suggest that the complexity arises at least in part from end-effects that become important in short alpha-helices.     

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.     

Sugar pucker and phosphodiester conformations in viral genomes of filamentous bacteriophages: fd, If1, IKe, Pf1, Xf, and Pf3

The laser Raman spectra of filamentous viruses contain discrete bands which are assignable to molecular vibrations of the encapsidated, single-stranded DNA genomes and which are informative of their molecular conformations. Discrimination between Raman bands of the DNA and those of the coat proteins is facilitated by analysis of viruses containing deuterium-labeled amino acids. Specific DNA vibrational assignments are based upon previous studies of A-, B-, and Z-DNA oligonucleotide crystals of known structure [Thomas, G.J., Jr., & Wang, A.H.-J. (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., & Lilley, D.M.J., Eds.) Vol. 2, Springer-Verlag, Berlin]. The present results show that canonical DNA structures are absent from six filamentous viruses: fd, If1, IKe, Pfl, Xf, and Pf3. The DNAs in three viruses of symmetry class I (fd, If1, IKe) contain very similar nucleoside sugar puckers and glycosyl torsions, deduced to be C3'-endo/anti. However, nucleoside conformations are not the same among the three class II viruses examined: Pf1 and Xf DNAs contain similar conformers, deduced to be C2'-endo/anti, whereas Pf3 DNA exhibits bands usually associated with C3'-endo/anti conformers. Conformation-sensitive Raman bands of the DNA 3'-C-O-P-O-C-5' groups show that in all class I viruses and in Pf1 the ssDNA backbones do not contain regularly ordered phosphodiester group geometries, like those found in ordered single- and double-stranded nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal difference circular dichroism of Pf1 filamentous virus and effects of mercury(II), silver(I), and copper(II)

The circular dichroism (CD) of Pfl filamentous virus has been examined over the temperature range 0-40 degrees C, in the absence and presence of Hg(II), Ag(I), and Cu(II). Thermal difference CD spectra were obtained by subtraction of spectra recorded above and below a thermally induced structure transition near 12 degrees C. The thermal difference spectra look like they arise from shifts in two exciton bands, one centered at 230 nm and the other at 290 nm. The amplitudes on either side of a crossover at 230 nm are 10 times those of a crossover at 290 nm. It is proposed that the difference spectra result from thermally induced shifts in coupled oscillator interactions between Tyr40 residues of the coat protein and the guanine and cytosine bases of the DNA. Metal ions can reduce or block these shifts. The changes in ellipticities at 220, 237, and 270 nm induced by changing the temperature have inflections near 12 degrees C. Ag(I) and Hg(II), which are known to bind to the DNA bases in Pfl, reduce or eliminate the inflections in the thermal profiles, depending on the metal ion type and concentration. Cu(II) ions do not affect the profiles. The spectral changes and the effects of the metal ions indicate intimate contact between the DNA bases and the protein subunits in the virion.     

A CD determination of the alpha-helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3

The CD spectra of four filamentous bacteriophages--fd, IKe, Pf1, and Pf3--were analyzed to determine the alpha-helix contents of their major coat proteins. Measured spectra included the 192-nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins [J.T. Yang, C.S.C. Wu, and H.M. Martinez (1986) Methods in Enzymology 130, 208-269] and (b) from poly(L-lysine) [N. Greenfield and G.D. Fasman (1960) Biochemistry 8, 4108-4116]. Extended analyses were also performed with the addition of the spectrum of a model beta-turn to the Greenfield and Fasman reference set, with the spectrum of a short alpha-helix in the Yang et al. reference set, and with an estimate of the spectrum of Trp added to both reference sets. The reference set based on the simple poly(L-lysine) polypeptide, plus a spectrum of a model beta-turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of alpha-helix. The class I phages--fd and IKe--had large percentages of alpha-helix of 98 +/- 2 and 97 +/- 5%, respectively, while the two class II phages--Pf1 and Pf3--had similar but smaller alpha-helix contents of 83 +/- 6 and 84 +/- 2, respectively. While these alpha-helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.     

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.     

Nucleotide sequence and genetic organization of the genome of the N-specific filamentous bacteriophage IKe. Comparison with the genome of the F-specific filamentous phages M13, fd and f1

The nucleotide sequence and genetic organization of the genome of the N-specific filamentous single-stranded DNA phage IKe has been established and compared with that of the F-specific filamentous phages M13, fd and f1 (Ff). The IKe DNA sequence comprises 6883 nucleotides, which is 476 (475) nucleotides more than the nucleotide sequence of the Ff genome. The data indicate that IKe and Ff have evolved from a common ancestor (overall homology approx. 55%) and that their genomes contain ten homologous genes, the order of which is identical. Similar to Ff, the major coat protein and the gene III-encoded pilot protein of IKe are synthesized via precursor molecules. The extent of homology between the genes of IKe and Ff differs significantly from one gene to another. Genes that code for viral capsid proteins are less homologous than genes whose products are involved in the processes of DNA replication and phage morphogenesis. During evolution, large nucleotide sequence rearrangements have occurred in the gene (gene III) whose product is needed for the attachment of the virion to the conjugative pili of the host cell, suggesting that these rearrangements have led to phages with different host specificities. Extensive nucleotide sequence homology was noted between the structural elements involved in DNA replication and phage morphogenesis, indicating that the mechanisms involved in DNA replication and morphogenesis are highly conserved.     

Tyrosine Raman signatures of the filamentous virus Ff are diagnostic of non-hydrogen-bonded phenoxyls: demonstration by Raman and infrared spectroscopy of p-cresol vapor

p-Cresol is a simple molecular model for the para phenolic side chain of tyrosine. Previously, Siamwiza and co-workers [(1975) Biochemistry 14, 4870-4876] investigated p-cresol solutions to identify Raman spectroscopic signatures for different hydrogen-bonding states of the tyrosine phenoxyl group in proteins. They found that the phenolic moiety exhibits an intense Raman doublet in the spectral interval 820-860 cm(-1) and that the doublet intensity ratio (I2/I1, where I2 and I1 are Raman peak intensities of the higher- and lower-wavenumber members of the doublet) is diagnostic of specific donor and acceptor roles of the phenoxyl OH group. The range of the doublet intensity ratio in proteins (0.30 < I2/I1 < 2.5) was shown to be governed by Fermi coupling between the phenolic ring-stretching fundamental nu1 and the first overtone of the phenolic ring-deformation mode nu(16a), such that when the tyrosine phenoxyl proton is a strong hydrogen-bond donor, I2/I1 = 0.30, and when the tyrosine phenoxyl oxygen is a strong hydrogen-bond acceptor, I2/I1 = 2.5. Here, we interpret the Raman and infrared spectra of p-cresol vapor and extend the previous correlation to the non-hydrogen-bonded state of the tyrosine phenoxyl group. In the absence of hydrogen bonding, the Raman intensity of the higher-wavenumber component of the canonical Fermi doublet is greatly enhanced such that I2/I1 = 6.7. Thus, for the non-hydrogen-bonded phenoxyl, the lower-wavenumber member of the Fermi doublet loses most of its Raman intensity. This finding provides a basis for understanding the anomalous Raman singlet signature (approximately 854 cm(-1)) observed for tyrosine in coat protein subunits of filamentous viruses Ff and Pf1 [Overman, S. A., et al. (1994) Biochemistry 33, 1037-1042; Wen, Z. Q., et al. (1999) Biochemistry 38, 3148-3156]. The implications of the present results for Raman analysis of tyrosine hydrogen-bonding states in other proteins are considered.     

Molecular structure of fd (f1, M13) filamentous bacteriophage refined with respect to X-ray fibre diffraction and solid-state NMR data supports specific models of phage assembly at the bacterial membrane

Filamentous bacteriophage (Inovirus) is a simple and well-characterized model system. The phage particle, or virion, is about 60 angstroms in diameter and several thousand angstrom units long. The virions are assembled at the bacterial membrane as they extrude out of the host without killing it, an example of specific transport of nucleoprotein assemblages across membranes. The Ff group (fd, f1 and M13) has been especially widely studied. Models of virion assembly have been proposed based on a molecular model of the fd virion derived by X-ray fibre diffraction. A somewhat different model of the fd virion using solid-state NMR data has been proposed, not consistent with these models of assembly nor with the X-ray diffraction data. Here we show that reinterpreted NMR data are also consistent with the model derived from X-ray fibre diffraction studies, and discuss models of 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.