Betty Prescott

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.

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.

Subunit Secondary Structure in Filamentous Viruses: Predictions and Observations

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

Raman spectrum and secondary structure of the phage λ operator site OL1: evidence for multiple nucleoside conformations in an aqueous B-form DNA

Abstract The laser Raman spectrum of OL1 has been obtained in the region 600–850 cm−1 and the data have been interpreted in terms of different nucleoside conformations within the 17 base pair operator site. The OL1 sequence, which is one of the tightest binding sites for the cI and Cro repressors of bacteriophage λ, displays several Raman conformation markers indicative of more than one backbone geometry for the same double-stranded DNA helix. Specific assignments for the Raman conformation markers are suggested by analogy with spectra of DNA single crystals and DNA fibers of known structure. Two Raman bands diagnostic of B-DNA backbone geometry are observed at 825 ± 3 and 838 ± 3 cm−1, and may be due, respectively, to inequivalent conformations of GC and AT pairs. In addition, a weak band at 706 cm−1 and a shoulder near 807 cm−1 are consistent with a minor contribution from residues which assume the A-DNA backbone geometry or a structurally related configuration. The complex bandshape in the 650–700 cm−1 interval, which is resolved into four peaks by Fourier deconvolution, is also consistent with the presence of multiple nucleoside conformers in OL1 in physiological conditions.

Evidence for conformational differences in aqueous and crystalline structures of α-bungarotoxin and cobratoxin

Laser Raman spectroscopy has been employed to investigate the structures of α-bungarotoxin (Bungarus multicinctus) and cobratoxin (Naja naja siamensis) in H2O and D2O solutions. Structures of the aqueous neurotoxins are compared with one another and with the X-ray crystal structures. The results indicate that the solution and crystal molecular structures of cobratoxin are in substantial agreement with one another, but those of α-bungarotoxin are not. Raman data provide no evidence for strained disulfides in aqueous α-bungarotoxin, although strained CSSC dihedral angles are indicated for the X-ray crystal structure. The data are interpreted as evidence for a strained molecular conformation of α-bungarotoxin in the crystal, which converts to a relaxed, more energetically favorable conformation in aqueous solution. Raman spectra also suggest more β-strand secondary structure in aqueous α-bungarotoxin (47 ± 5%) than in the crystalline form ( < 10%). The high β-strand content measured by Raman spectroscopy could be due to either a secondary structure in solution that is appreciably different than that of the crystal, or to the imprecision of the Raman method in distinguishing peptide configurations that are vibrationally equivalent but conformationally inequivalent. Aqueous α-bungarotoxin and cobratoxin also differ from one another in amino acid side chain orientations and interactions, though not in main chain conformations. Different geometries are indicated for cystine CCSS dihedral angles, and different hydrogen bonding states are indicated for internal tyrosines. Tyrosine-24 of α-bungarotoxin is shown to donate a strong hydrogen bond to a negative acceptor, deduced to be glutamate-41, whereas the equivalently positioned residue of cobratoxin is apparently hydrogen bonded to solvent molecules.