G. J. Thomas

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 studies of nucleic acids VIII estimation of RNA secondary structure from raman scattering by phosphate-group vibrations

Abstract Analysis of the Raman spectra of a large number of ribonucleotide monomers and polymers indicates that the frequencies and intensities characteristic of the P-O stretching vibrations provide a basis for the quantitative determination of RNA secondary structure. The intensity ratio of the Raman lines at 815 and 1100 cm−1 is shown to have a value of 1.64 ± 0.04 in completely ordered structures and a value of zero in disordered structures. Ordered structures in this context include double-helical configurations containing A · U or G · C pairs and ordered single-stranded configurations (such as occur in poly(rA) and poly(rC)) but exclude other type of base pairing as well as random-chain configurations. The percentages of RNA nucleotides in such ordered configurations, as computed from Raman intensity measurements, are 16-S RNA ( 95 ± 5 ), 23-S RNA ( 85 ± 5 ), tRNAfMet ( 84 ± 3 ), tRNAVal ( 84 ± 3 ), tRNAPhe ( 85 ± 3 ) and R17 RNA ( 87 ± 3 % ). Since the present method makes use of spectral transitions originating in the RNA backbone and not in the bases, no limitations are imposed by lack of knowledge of the RNA base sequence. In combination with data obtained from quantitative infrared spectroscopy the Raman results are also shown to yield information on the percentages of RNA nucleotides in ordered single-stranded configurations.

The dependence of raman scattering on the conformation of ribosomal RNA

Summary The intensities of Raman scattering from certain vibrations of nucleotide residues in 16S and 23S ribosomal RNA are sensitive to changes of RNA conformation with temperature. The intensity effects differ from those exhibited by polynucleotides and may be due to a specific RNA tertiary structure or to a sequence dependence of the Raman hypochromic effect.

Studies of virus structure by Laser-Raman spectroscopy: II. MS2 phage, MS2 capsids and MS2 RNA in aqueous solutions☆

Abstract Laser-Raman spectra of the bacteriophage MS2, and of its isolated coat-protein and RNA components, have been obtained as a function of temperature in both H2O and D2O (deuterium oxide) solutions. The prominent Raman lines in the spectra are assigned to the amino acid residues and polypeptide backbone of the viral coat protein and to the nucleotide residues and ribosyl-phosphate backbone of the viral RNA. The Raman frequencies and intensities, and their temperature dependence, indicate the following features of MS2 structure and stability. Coat-protein molecules in the native phage maintain a conformation determined largely by regions of β-sheet (~60%) and random-chain (~40%) structures. There are no disulfide bridges in the virion and all sulfhydryl groups are accessible to solvent molecules. Protein-protein interactions in the virion are stable up to 50 °C. Release of viral RNA from the virion does not affect either the conformation of the coat-protein molecules or the thermal stability of the capsid. MS2 RNA within the virion contains a highly ordered secondary structure in which most (~85%) of the bases are either paired or stacked or both paired and stacked and in which the RNA backbone assumes a geometry of the A-type. When RNA is partially or fully released from the virion its overall secondary structure at 32 °C is unchanged. However, the exposed RNA is more susceptible to changes in secondary structure promoted by increasing the temperature. Thus the viral capsid exerts a significant stabilizing effect on the secondary structure of MS2 RNA. This stabilization is ionic-strength dependent, being more pronounced in solutions containing high concentrations of KCl. Raman intensity profiles as a function of temperature reveal that disordering of the MS2 RNA backbone and rupture of hydrogen-bonding between complementary bases are gradual processes, the major portions of which occur above 40 °C. However, the unstacking of purine and pyrimidine bases is a more co-operative phenomenon occurring almost exclusively above 55 °C.

Studies of virus structure by Raman spectroscopy I. R17 virus and R17 RNA

Abstract The laser-excited Raman spectrum of the RNA virus, R17, is shown to contain a large number of Raman lines assignable to scattering by vibrations of the nucleotide residues of RNA and the amino-acid residues of protein capsomers. The Raman lines from specific nucleotide vibrations in the phage are compared with their counterparts in the spectrum of protein-free RNA to suggest many similarities of RNA structure in the phage and protein-free states. However, the average configuration of guanine residues in the phage is apparently very different from that of protein-free RNA, suggesting that guanine plays an important role in RNA-protein interactions.

Raman studies of nucleic acids X. Conformational structures of Escherichia coli transfer RNAs in aqueous solution

Abstract Laser-excited Raman spectra of aqueous solutions of purified samples of Escherichia coli tRNA Glu , tRNA Arg , tRNA Val , tRNA fMet and tRNA 2 Phe are compared. The characteristic Raman frequencies and intensities, from scattering by symmetrical stretching vibrations of the phosphodiester groups, reveal that the total secondary structure of each tRNA is qualitatively and quantitatively the same. The percentage of nucleotide residues in each tRNA which exist in a highly ordered configuration is 84 ± 4 %, consistent with a cloverleaf structure provided two-thirds of the unpaired bases in loops are also in ordered configurations. The remaining nucleotides (16 ± 4 %) must occur in randomly oriented or highly-strained configurations of the tRNA chain. The Raman spectra also reveal specific differences in the amount of stacking of A and G residues in different tRNAs. These results provide strong evidence for the presence of specific tertiary structure (folding) in aqueous tRNA. A study of the melting transition of tRNA Glu over the range 32–90 °C reveals that appreciable order persists in the tRNA backbone at 90 °C and is attributable to residual stacking of the purines. Gross changes in the conformation of tRNA Val are also detected when excess Mg 2+ is present. The improved spectral sensitivity in this work demonstrates that the Raman spectrum of tRNA may be used to detect the presence of the minor nucleoside, dihydrouridine, and that the frequency region 1150–1450 cm −1 is highly sensitive to the base composition, base sequence and base-stacked secondary structure of tRNA.

Raman studies of nucleic acids IV: Vibrational spectra and associative interactions of aqueous inosine derivatives

Abstract Laser-excited Raman spectra of H 2 O and 2 H 2 O solutions of inosine, inosine-5′-monophosphate and I-methylinosine are reported. The spectra are discussed in terms of the changes in molecular structure produced by changes in pH and p 2 H. The temperature dependence of Raman scattering intensities from aqueous inosine and inosine-5′-monophosphate are also reported. Raman hypochromism occurs in several lines in the spectra and is attributed to stacking of the hypoxanthine rings at low temperature. The association of the nucleotide is accompanied by enhanced Raman scattering near 820 cm −1 , similar to that observed previously for ordered polynucleotide structures and RNA. Effects of solute concentration and Mg 2+ concentration on spectra of the nucleotide are also discussed. The Raman data suggest that the nucleotide bases are stacked singly and that the phosphate substituents are largely unaffected in structure and environment by the base stacking.

Raman studies of nucleic acids VI. Conformational structures of tRNAfMet, tRNAVal and tRNAPhe2☆

Abstract Laser-excited Raman spectra of tRNA fMet , tRNA Val and tRNA Phe 2 from Escherichia coli indicate that each tRNA has a similar conformation in aqueous solution. The spectra of tRNA fMet and tRNA Val show further that their specific base-paired and base-stacked secondary structures are consistent with the proposed clover-leaf models. The onset of melting in tRNA fMet and tRNA Val occurs at about 40°C. At 70°C all base pairs are broken. Above 70°C and up to 90°C, significant order is retained in the tRNA backbones and appreciable stacking of G residues persists. In the presence of a 3-fold molar excess of Mg 2+ , the onset of melting occurs at about 60°C. Comparison of Raman spectra of tRNA and rRNA reveals that the latter molecules have a more ordered ribose-phosphate backbone and contain more A but less G residues in stacked configurations at 32°C.

Raman Spectroscopy and Virus Research

A survey is given of developments leading to the application of laser-Raman spectroscopy in structural studies of viruses and model nucleoproteins. The major constituents of viruses — nucleic acid and protein molecules — exhibit Raman spectra which differ greatly from one another, both in the spectral ranges that contain vibrational frequencies of conformational interest and in the relative intensities of Raman scattering of their respective subgroups. These features, not common to the infrared spectra, allow laser-Raman spectroscopy to be exploited for the study of viral assembly and nucleoprotein interactions. Examples considered here are the RNA-containing virus MS2, the DNA-containing viruses Pfl and fd, and the complex of polylysine with DNA.

On the tautomeric structure of inosine

Abstract The suggestion that 6-keto and 6-enol tautomers of inosine may be of similar stability in neutral aqueous solution has been investigated by means of Raman spectroscopy. Spectra of aqueous solutions of inosine and its methylated analogs were obtained in the region 2000-200 cm−1 using argon laser excitation, and indicate that inosine exists predominantly in the keto form.