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.     

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)     

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.     

Structural characterization of the filamentous bacteriophage PH75 from Thermus thermophilus by Raman and UV-resonance Raman spectroscopy

The filamentous bacteriophage PH75, which infects the thermophile T. thermophilus, assembles in vivo at 70 degrees C and is stable to at least 90 degrees C. Although a high-resolution structure of PH75 is not available, the virion is known to comprise a closed single-stranded (ss) DNA circle of 6500 nucleotides sheathed by a capsid comprising 2700 copies of a 46-residue subunit (pVIII). Here, we employ Raman and UV-resonance Raman (UVRR) spectroscopy to identify structural details of the pVIII and DNA constituents of PH75 that may be related to the high thermostability of the native virion assembly. Analysis of the Raman amide I and amide III signatures reveals that the capsid subunit secondary structure is predominantly (87%) alpha-helical but contains a significant number of residues (6 +/- 1 or 13 +/- 3%) differing from the canonical alpha-helix. This minor structural component is not apparent in capsid subunits of the mesophilic filamentous phages, fd, Pf1, and Pf3, previously examined at similar spectral resolution. The Raman signature of PH75 also differs from those of fd, Pf1, and Pf3 by virtue of an unusual alanine marker (898 cm(-)(1) band), which is attributed to C(alpha)-H hydrogen-bond donation by subunit Ala residues. Because alanines of the PH75 subunit occur primarily within sXXXs motifs (where s is a small side chain, e.g. Gly, Ala, Ser), and because the occurrence of such motifs in alpha-helices is believed to thermostabilize interhelix associations via C(alpha)-H...O interactions [G. Kleiger et al. (2002) Biochemistry 41, 5990-5997], we propose that such hydrogen bonding may explain both the alanyl and amide I/III markers of PH75 capsid subunits and that C(alpha)-H...O interactions may serve as a significant source of virion thermostabilization. Raman and UVRR signatures of PH75 are also distinguished from those of fd, Pf1, and Pf3 by several marker bands that are indicative of hydrophilic Trp and Tyr environments, including hydrogen bonding interactions of aromatic ring substituents. These interactions are likewise proposed as contributors to the high thermostability of PH75 vis-a-vis fd, Pf1, and Pf3. Finally, PH75 is the only filamentous phage exhibiting UVRR markers diagnostic of a highly base-stacked ssDNA genome incorporating the low energy C2'-endo/anti deoxynucleoside conformation. The present results suggest that both intersubunit interactions and genome organization contribute to the enhanced thermostability of PH75 relative to mesophilic filamentous bacteriophages.     

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.     

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.     

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.     

Raman markers of nonaromatic side chains in an alpha-helix assembly: Ala, Asp, Glu, Gly, Ile, Leu, Lys, Ser, and Val residues of phage fd subunits

The study of filamentous virus structure by Raman spectroscopy requires accurate band assignments. In previous work, site- and residue-specific isotope substitutions were implemented to elucidate definitive assignments for Raman bands arising from vibrational modes of the alpha-helical coat protein main chain and aromatic side chains in the class I filamentous phage, fd [Overman, S. A., and Thomas, G. J., Jr. (1995) Biochemistry 34, 5440-5451; Overman, S. A., and Thomas, G. J., Jr. (1998) Biochemistry 37, 5654-5665]. Here, we extend the previous methods and expand the assignment scheme to identify Raman markers of nonaromatic side chains of the coat protein in the native fd assembly. This has been accomplished by Raman analysis of 11 different fd isotopomers selectively incorporating deuterium at specific sites in either alanine, aspartic acid, glutamic acid, glycine, isoleucine, leucine, lysine, serine, or valine residues of the coat protein. Raman markers are also identified for the corresponding deuterated side chains. In combination with previous assignments, the results provide a comprehensive understanding of coat protein contributions to the Raman signature of the fd virion and validate Raman markers assigned to the packaged single-stranded DNA genome. The findings described here show that nonaromatic side chains contribute prolifically to the fd Raman signature, that marker bands for specific nonaromatics differ in general from those observed in corresponding polypeptides and amino acids, and that the frequencies and intensities of many nonaromatic markers are sensitive to secondary and higher-order structures. Nonaromatic markers within the 1200-1400 cm-1 interval also interfere seriously with the diagnostic Raman amide III band that is normally exploited in secondary structure analysis. Implications of these findings for the assessment of protein conformation by Raman spectroscopy are considered.     

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.     

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.     

Secondary structure and interactions of the packaged dsDNA genome of bacteriophage P22 investigated by Raman difference spectroscopy.

Vibrational spectra of the double-stranded DNA genome of bacteriophage P22 in packaged and unpackaged states are compared by digital difference Raman spectroscopy. The difference Raman spectrum, which is sensitive to structural changes at the level of < 2% of a given nucleotide type, reveals the effects of packaging upon sugar pucker, glycosyl orientation, phosphodiester geometry, base pairing, base stacking, and the electrostatic environment of DNA phosphate groups. For both packaged and unpackaged states, the experiments were performed on aqueous solutions at 25 degrees C containing effective P22 DNA concentrations of 30-50 mg/mL in 200 mM NaCl + 10 mM MgCl2 + 10 mM Tris at pH 7.5. At the experimental conditions employed, the B-form secondary structure of unpackaged P22 DNA is minimally perturbed by packaging the viral genome in the virion capsid. However, the electrostatic environment of DNA phosphates is dramatically altered with packaging. Specifically, we find the following: (1) C2'-endo sugar pucker and anti glycosyl orientations are conserved for all nucleosides. (2) Watson-Crick base pairing is essentially completely retained. (3) Alternative secondary structures, whether right- (A or C form) or left-handed (Z form), are not evident in either the packaged or unpackaged viral genome. (4) Small Raman hyperchromic effects (< 10%) observed for certain marker bands of dG, dA, and dT in the packaged state of P22 DNA suggest slightly reduced base-stacking interactions with packaging. These are consistent with previously reported UV hyperchromic effects, but the Raman spectrum shows that they are not associated with either base unpairing or strand separation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural study of the lipid-containing bacteriophage PRD1 and its capsid and DNA components by laser Raman spectroscopy

The Raman spectrum of a virus contains the structural signature of each of its molecular components (Thomas, 1987). We report the first Raman spectrum obtained from an intact, lipid-containing virus--the icosahedral bacteriophage PRD1--and show that this spectrum contains characteristic structure markers for the major capsid protein, the packaged double-stranded DNA genome, and the viral membrane which resides between the capsid and DNA. We find that the packaged genome of PRD1 exhibits Raman markers typical of the B-DNA secondary structure. Comparison of the Raman spectrum of the packaged DNA with that of protein-free DNA extracted from the virion shows further that the B-form secondary structure is not significantly perturbed by packaging in the virion. The Raman signature of the PRD1 membrane, monitored within the virion at 4 degrees C, is that of a phospholipid liquid-crystalline phase. The PRD1 capsid, which comprises several hundred copies of the major coat protein P3 (product of viral gene III) and a few copies of minor proteins, incorporates P3 capsomers predominantly in the beta-sheet conformation. The beta-sheet structure of P3 is maintained in the fully assembled PRD1 virion, as well as in the empty capsid. The present results demonstrate the feasibility of obtaining structural information from the three different classes of biomolecules--nucleic acid, protein, and lipid--which constitute a membrane-lined virus particle. Our results also demonstrate that the coat protein and double-stranded DNA components of a lipid-containing bacteriophage share many structural features in common with bacteriophage lacking a lipid membrane.     

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.     

Raman spectroscopy of Z-form poly[d(A-T)].poly[d(A-T)

Helical structures of double-stranded poly[d(A-T)] in solution have been studied by Raman spectroscopy. While the classical right-handed conformation B-type spectra are obtained in the case of sodium chloride solutions, a Z-form Raman spectrum is observed by addition of nickel ions at high sodium concentration, conditions in which the inversion of the circular dichroic spectrum of poly[d(A-T)] is detected, similar to that observed for high-salt poly[d(G-C)] solutions [Bourtayre, P., Liquier, J., Pizzorni, L., & Taillandier, E. (1987) J. Biomol. Struct. Dyn. 5, 97-104]. The characterization of the Z-form spectrum of poly[d(A-T)] is proposed by comparison with previously obtained characteristic Raman lines of Z-form poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)] solutions and of d(CG)3 and d(CGCATGCG) crystals [Thamann, T. J., Lord, R. C., Wang, A. H.-J., & Rich, A. (1981) Nucleic Acids Res. 9, 5443-5457; Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., Rich, A., & Thomas, G. J., Jr. (1984) Nucleic Acids Res. 14, 5913-5925]. Detailed spectroscopic data are presented reflecting the reorientation of the purine-deoxyribose entities (C2'-endo/anti----C3'-endo/syn), the modification of the phosphodiester chain, and the adenosine lines in the 1300-cm-1 region. The role played by the hydrated nickel ions in the B----Z transition is discussed.     

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 approximately 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 degrees from the virion axis and that the indolyl pseudo-twofold axis is inclined at 46 degrees to the virion axis. Using the presently determined orientation of the indolyl ring and side-chain torsion angles, chi(1) (N-C(alpha)-C(beta)-C(gamma)) and chi(2,1) (C(alpha)-C(beta)-C(gamma)-C(delta1)), 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-pi 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.     

Demonstration of Z-d(5BrCGAT5BrCG) and B-d(CGCGATCGCG) form crystal structures in DNA-cobalt hexammine complexes by Kr 647.1 nm excitation of Raman spectra.

Cobalt hexammine [Co(NH3)6(3+)] is an efficient DNA complexing agent which significantly perturbs nucleic acid secondary structure. We have employed red excitation (647.1 nm) from a krypton laser to obtain Raman spectra of the highly colored complexes formed between cobalt hexammine and crystals of the DNA oligomers, d(5BrCGAT5BrCG) and d(CGCGATCGCG), both of which incorporate out-of-alternation pyrimidine/purine sequences. The Co(NH3)6(3+) complex of d(5BrCGAT5BrCG) exhibits a typical Z-form Raman signature, similar to that reported previously for the alternating d(CGCGCG) sequence. Comparison of the Raman bands of d(5BrCGAT5BrCG) with those of other oligonucleotide and polynucleotide structures suggests that C3'-endo/syn and C3'-endo/anti thymidines may exhibit distinctive nucleoside conformation markers, and tentative assignments are proposed. The Raman markers for C2'-endo/anti adenosine in this Z-DNA are consistent with those reported previously for B-DNA crystals containing C2'-endo/anti dA. Raman bands of the cobalt hexammine complex of d(CGCGATCGCG) are those of B-DNA, but with significant differences from the previously characterized B-DNA dodecamer, d(CGCAAATTTGCG). The observed differences suggest an unusual deoxyguanosine conformer, possibly related to a previously characterized structural intermediate in the B-->Z transition. The present results show that crystallization of d(CGCGATCGCG) in the presence of cobalt hexammine is not alone sufficient to induce the left-handed Z-DNA conformation. This investigation represents the first application of off-resonance Raman spectroscopy for characterization of highly chromophoric DNA and illustrates the feasibility of the Raman method for investigating other structurally perturbed states of DNA-cobalt hexammine complexes.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

Phage phi X174 probed by laser Raman spectroscopy: evidence for capsid-imposed constraint on DNA secondary structure

The Raman spectrum of the isometric bacteriophage phi X174 contains a number of well-resolved bands which have been assigned unambiguously to proteins of the capsid or to the single-stranded DNA (ssDNA) genome. Additional Raman bands of protein and DNA, which are partially overlapped in the spectrum of virus, have been resolution enhanced by Fourier deconvolution to permit improved semiquantitative measurement of spectral intensities and frequencies for structural conclusions. Raman conformation markers indicate that the ssDNA molecule within the capsid contains nucleosides of C2'-endo sugar pucker and anti-glycoside bond orientation, but the nucleic acid backbone lacks the geometry characteristic of B-form DNA. The Raman profile of encapsidated phi X DNA indicates a backbone more similar to heat-denatured DNA than to DNA containing hairpinlike secondary structure. This finding suggests limited interbase interactions in the packaged genome, which is presumably the result of constraints imposed by the viral capsid. Thus, the extensive pairing and stacking of bases indicated by Raman profiles from ssRNA viruses are not evident for the phi X174 chromosome. Overall, the proteins of the virion contain extensive beta-sheet and irregular secondary structures. Fourier deconvolution of the Raman amide I band provides an estimate of the percentage of total beta-sheet structure (approximately 60%) in all proteins of the virion. The amide III region of the spectrum confirms that beta-sheet and irregular domains are the predominant protein secondary structures. Samples of phi X174 concentrated for Raman spectroscopy by either ultracentrifugation or ultrafiltration exhibit nearly identical Raman spectra, indicating that either method can be employed to prepare intact virus without significant loss of DNA or protein components.     

The Raman spectra of left-handed DNA oligomers incorporating adenine-thymine base pairs+.

Raman spectra were obtained from single crystals of [d(CGCATGCG)]2 and [d(m5CGTAm5CG)]2, both of which incorporate A-T pairs into Z-DNA structures and contain C2'-endo/syn conformers of deoxyguanosine at the oligonucleotide ends. Correlation with x-ray results permits the following Raman assignments for nucleoside conformers: C3'-endo/syn G, 623 +/- 1; C2'-endo/syn G, 671 +/- 2; C2'-endo/anti C, 782 +/- 1; C2'endo/anti T, 650 +/- 5 and ca. 750; C3'-endo/syn A, 729 +/- 1 cm-1. These results show that (i) the 670 cm-1 line of syn G is highly sensitive to the change from C3'-endo to C2'-endo pucker, (ii) the 729 cm-1 line of A is affected neither by furanose pucker nor glycosidic bond orientation and (iii) the 1200-1500 cm-1 region of the Raman spectrum of the A-T double helix is greatly altered by the B-to-Z transition. Conformation sensitive Raman frequencies in the 850-1700 cm-1 region are identified for both octamer and hexamer, and the Z-to-B transition of each is monitored by spectral changes which occur upon dissolving the crystal in H2O solution.