Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK1
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK2
Author for correspondence: Laurence Barron. Fax +44 141 330 4888. e-mail laurence{at}chem.gla.ac.uk
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Abstract |
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Introduction |
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The coat protein sequences of the tobraviruses contain 196223 residues, compared with 156161 for the tobamoviruses. Proteins of both genera possess at their C termini a region of variable sequence, which consists of 1538 residues in tobraviruses, whereas the corresponding region in tobamoviruses contains only 39 residues. Another region of variable sequence, at the N terminus of tobravirus proteins, has no counterpart in tobamoviruses. In addition, tobraviruses have a central insertion of 2633 residues of variable sequence relative to tobamoviruses. Unlike TMV particles, which give no proton nuclear magnetic resonance (NMR) signals, those of TRV give an NMR signal that has been attributed to segmental mobility of the extra C-terminal sequence (Mayo et al., 1993 ). Furthermore, the C-terminal region of TRV was found to be strongly antigenic and a central region (residues 110121) to be more weakly antigenic, suggesting that these regions are exposed externally in the intact virus particle and could be associated with its transmission by nematodes (Legorburu et al., 1996
).
The lack of detailed information about the molecular structure of tobraviruses highlights the dearth of physical techniques generally applicable to the determination of virus structure and function. Of the few techniques available, Raman spectroscopy is particularly useful on account of its potential ability to provide structural information about both protein and nucleic acid constituents of intact virions and viral precursors over a broad range of sampling conditions (Thomas, 1999 ). A novel form of Raman spectroscopy called Raman optical activity (ROA), which measures small differences in the Raman spectra of chiral molecules acquired using right- and left-circularly polarized incident light, has recently been applied to biomolecules and provides new information on their solution structure and dynamics (Barron et al., 2000
). ROA bears the same relation to conventional Raman spectroscopy as does ultraviolet circular dichroism (UVCD) to conventional UV absorption spectroscopy. The largest ROA signals are often associated with vibrational coordinates which sample the most rigid and chiral parts of the structure. In proteins these are usually within the peptide backbone and often give rise to ROA band patterns characteristic of the backbone conformation, unlike the parent Raman spectra in which many bands from the amino acid side-chains often obscure the peptide backbone bands. As well as bands arising from secondary structure (together with a few bands from side-chains), protein ROA spectra also contain bands from loops and turns and so can provide information about the tertiary fold of the peptide backbone. Nucleic acid ROA spectra contain information on base stacking, the mutual orientations of sugar and base rings, and the sugar-phosphate backbone conformations. The first observations of ROA on intact viruses, specifically filamentous bacteriophages, were published recently (Blanch et al., 1999
). Here we report ROA data on the PRN strain of TRV and the U1 strain of TMV from which new information is deduced about the coat protein structure of TRV.
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Methods |
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Results and Discussion |
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Positive ROA intensity in the range 13151325 cm-1 appears to be characteristic of PPII helix, or at least of loop structure containing residues clustering in the PPII region of the Ramachandran surface (Smyth et al., 2001
; Blanch et al., 2000b
). The positive ROA intensity in this range shown by TMV could therefore arise from some of the loops within the coat protein fold (Klug, 1999
; Stubbs, 1999
). However, the ROA spectrum of TRV contains much more intensity in this region in the form of a strong sharp positive ROA band at
1315 cm-1, which indicates that the coat protein of this virus contains much more PPII structure than the TMV protein. The prominent negative extended amide III ROA band of TRV peaking at
1236 cm-1 is characteristic of
-strand, which suggests that TRV contains more of this conformational element than TMV for which the negative ROA intensity in this region is weaker and less well-defined. Since PPII structure is often hydrated, flexible and found mainly in loop regions on protein surfaces (Adzhubei & Sternberg, 1993
; Stapley & Creamer, 1999
), it is a likely conformational element to be found in the additional C-terminal sequence which the earlier NMR antigenicity work has suggested is exposed externally (Mayo et al., 1993
; Legorburu et al., 1996
). It therefore seems likely that PPII helix is a significant conformational element in the additional central and C-terminal sequences that are present in the TRV coat protein compared with the TMV protein. There may also be some
-strand in these sequences but little, if any,
-helix.
Goulden et al. (1992) identified amino acid residues, conserved in the coat protein sequences of four tobraviruses, that were counterparts of residues with important functions in the hydrophobic core structure of the TMV coat protein subunit. Inspection of the 13 tobravirus coat protein sequences now available confirms the conserved status of almost all of these residues. Thus, the idea that tobravirus and tobamovirus coat proteins have similar overall folding patterns, reflecting a common evolutionary origin, and in particular that both structures are based on a four-helix bundle, still seems valid, even though the four-helix bundle of TRV may be more hydrated and open than that of TMV. Mainly due to the larger radius of the cylindrical tobravirus particles, the volume occupied by a tobravirus coat protein subunit is significantly larger than that occupied by a tobamovirus subunit.
The ROA data suggest that the additional volume of TRV may be associated with large amounts of open hydrated PPII structure together with increased hydration of the -helical regions. This could be consistent with the structural model of the complete TRV particle proposed by Goulden et al. (1992)
in which corresponding structural elements of the subunits are displaced radially by 4 nm relative to their positions in TMV, the number of subunits per turn is increased from 16
to 25
, and the subunits acquire an increased bulk. Exposed hydrated PPII structure (plus some
-strand) from the additional central sequence might occupy the vacated volume around the axial hole, that from the additional C-terminal sequences could be present on the outer surface, and additional hydrated
-helix could provide increased bulk either within the helix bundles by increasing the distances between some helices or externally by increasing the separations between adjacent helix bundles. The present ROA study therefore provides partial experimental support of the model of Goulden et al. (1992)
, but with some additional details.
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Acknowledgments |
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References |
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Received 10 January 2001;
accepted 5 March 2001.