Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK1
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK2
Department of Chemistry, DTU 207, Technical University of Denmark, DK-2800 Lyngby, Denmark3
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 lack of information about the molecular structure of potexviruses highlights the lack of physical techniques applicable to the determination of virus structure and function at the molecular level. A novel spectroscopic technique called Raman optical activity (ROA) appears to be promising for such studies and has recently been applied to intact viruses; specifically filamentous bacteriophages (Blanch et al., 1999 ), and tobacco rattle virus (TRV) and tobacco mosaic virus (TMV) (Blanch et al., 2001a
). Here we report an ROA study of the DX strain of PVX (Jones, 1982
) and the MC strain of NMV (Wilson et al., 1991
) from which new information about the folds of the coat proteins is deduced, mainly from a comparison with the ROA spectrum of the U1 strain of TMV.
A brief review of Raman spectroscopy and ROA
Since the ROA technique is unfamiliar to most virologists, a brief description may be helpful. Raman spectroscopy itself is a form of vibrational spectroscopy, complementary to infrared absorption, which provides vibrational spectra of molecules by means of inelastic scattering of visible or ultraviolet laser light. During the so-called Stokes Raman scattering event, the interaction of the molecule with the incident laser photon of energy , where
is its angular frequency, can leave the molecule in an excited vibrational state of energy h/
v, with a corresponding energy loss, and hence a shift to lower angular frequency
-
v, of the scattered photon. Therefore, by analysing the spectrum of the scattered light with a visible or ultraviolet spectrometer, a complete vibrational spectrum of the molecule may be obtained. Conventional Raman spectroscopy has a number of favourable characteristics, including the fact that water is an excellent solvent for Raman studies, which have led to many applications in biochemistry (Carey, 1982
; Miura & Thomas, 1995
). It has proved especially valuable in structural virology on account of its ability to provide information about both protein and nucleic acid constituents of intact virions (Thomas, 1987
, 1999
).
ROA is a novel form of Raman spectroscopy that is sensitive to chirality, meaning handedness, in molecular structure. The two distinguishable mirror-image forms of a chiral molecule are called enantiomers. Biomolecules such as proteins and nucleic acids are chiral on account of the intrinsic chirality of their constituent amino acids (almost exclusively the L-enantiomers) and sugars (almost exclusively the D-enantiomers), respectively. Raman spectroscopy becomes sensitive to chirality by utilizing circularly polarized light: thus ROA measures small differences in the Raman spectra of chiral molecules acquired using right- and left-circularly polarized incident laser light. ROA bears the same relation to conventional Raman spectroscopy as does the widely used biochemical technique of UVCD to conventional visible and ultraviolet absorption spectroscopy. The advantage of ROA is that it provides vibrational optical activity spectra which contain much more stereochemical information than the electronic optical activity spectra provided by UVCD (just as conventional vibrational spectra, Raman scattering or infrared absorption, contain much more information about molecular structure than conventional visible and ultraviolet absorption spectra). Recent general reviews of ROA include Nafie (1997) and Barron & Hecht (2000)
.
ROA has recently been applied to biomolecules such as proteins, carbohydrates, nucleic acids and viruses. This work has been reviewed by Barron et al. (2000) . On account of its sensitivity to chirality, ROA is proving to be an incisive probe of biomolecular structure and dynamics. One reason for this is that the largest ROA signals are often associated with the most rigid 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 peptide backbone conformation, unlike the parent Raman spectra which are often dominated by bands from the amino acid side-chains, which often obscure the peptide backbone bands. As well as bands arising from secondary structure, protein ROA spectra also contain bands from loops and turns and so can provide information about the tertiary fold. A few ROA bands from side-chains also appear, including one for tryptophan from which the absolute stereochemistry can be deduced, even in the coat proteins of intact viruses (Blanch et al., 2001b
). Carbohydrate ROA spectra provide information about anomeric preference, the pattern of OH substituents and the nature of the glycosidic link, and can detect extended secondary structure in polysaccharides. Nucleic acid ROA spectra provide information about base stacking, the mutual orientations of sugar and base rings, and the sugarphosphate backbone conformations.
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Methods |
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Both viruses were studied as solutions at 25 mg/ml in 15 mM phosphate buffer at pH 7·4 held in small quartz microfluorescence cells at ambient temperature (
20 °C). The ROA measurements were performed using an instrument described previously (Hecht et al., 1999
). It has a backscattering configuration and employs a single-grating spectrograph fitted with a backthinned charge coupled device (CCD) camera as detector and an edge filter to block the Rayleigh line. The small ROA signals are accumulated by synchronizing the Raman spectral acquisition with an electrooptic modulator that switches the polarization of the argon-ion laser beam between right- and left-circular at a suitable rate. The spectra are displayed in analogue-to-digital counter units as a function of the Stokes Raman wavenumber shift with respect to the exciting laser wavelength. The ROA spectra, which have undergone minimal smoothing (2 point FFT), are presented as circular intensity differences IR-IL and the parent Raman spectra as circular intensity sums IR+IL, where IR and IL are the Raman-scattered intensities in right- and left-circularly polarized incident light, respectively. The experimental conditions were as follows: laser wavelength 514·5 nm; laser power at the sample
700 mW; spectral resolution
10 cm-1; acquisition time
48 h.
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Results and Discussion |
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The protein ROA data are consistent with the UVCD results, which predicted large amounts of -helix with little
-sheet (Homer & Goodman, 1975
; Wilson et al., 1991
). Furthermore, the overall appearances of the ROA spectra of PVX and NMV are very similar to each other and to that of TMV reported recently (Blanch et al., 2001a
) for which the coat protein subunits are based on four-helix bundles containing both water-exposed residues and residues at hydrophobic helixhelix interfaces (Namba et al., 1989
; Stubbs, 1999
). One difference, however, is that the positive
1337 cm-1 ROA bands in PVX and NMV are significantly more intense than the corresponding band in TMV, which suggests that PVX and NMV contain more hydrated
-helix than TMV. For convenience, a re-measured ROA spectrum of TMV (strain U1) of better quality than that recorded in the earlier study is shown at the bottom of Fig. 1
. Our results therefore suggest that the coat protein folds of PVX and NMV are similar to that of TMV, being based on a helix bundle, but with differences of detail resulting from differences in the appended loops and turns and from the extra sequences present in PVX and NMV (the coat proteins of the DX strain of PVX, the MC strain of NMV and the U1 strain of TMV contain 237, 240 and 159 amino acids, respectively). Our results do not support the model of Baratova et al. (1992a)
for the PVX coat proteins, which may contain too much well-defined
-sheet (which is predicted at both the N- and C-terminal ends). However, unlike the N-terminal regions of PVX which tritium planigraphy shows are exposed, the C-terminal regions of PVX are virtually inaccessible to tritium labelling in the intact virion (Baratova et al., 1992b
), but become accessible after
20 N-terminal amino acids are proteolyticaly removed (Baratova et al., 1992a
). For TMV, on the other hand, tritium planigraphy shows that both N- and C-terminal regions are exposed (Goldanskii et al., 1988
), as expected from the X-ray structure (Stubbs, 1999
). Some of the additional
80 residues in PVX and NMV compared with TMV might constitute an N-terminal region which tends to bury the C-terminal end, but the ROA spectra suggest that this region is not organized into a
-sheet as proposed by Baratova et al. (1992a)
. In this connection it is worth noting that there is no similarity in primary sequence of PVX and TMV coat proteins since no alignment of the potexvirus and tobamovirus conserved primary sequences is possible and also that, according to a prediction of Sawyer et al. (1987)
, the N-terminal 50 residues of PVX have zero probability of being
-helix. This last prediction is unfortunate since it is tempting to assign the greater hydrated
-helix content of PVX and NMV relative to TMV to the extra sequences.
TRV exhibits a strong positive ROA band at 1315 cm-1 assigned to polyproline II (PPII)-helical structure in the additional central and C-terminal sequences which the coat proteins of this virus contain compared with TMV (Blanch et al., 2001a
). However, since there is no additional positive intensity in this region of the ROA spectra of PVX and NMV compared with TMV, the additional sequences of PVX and NMV do not appear to contain much PPII structure. (All three virus ROA spectra in Fig. 1
show similar small but clear positive ROA bands at
1316 cm-1 assigned to PPII structure.)
A few ROA bands from side-chains can also be identified. ROA bands in the range 14201480 cm-1 originate in both aliphatic and aromatic side-chains. Proteins often show a tryptophan ROA band in the
1550 cm-1 region, assigned to a W3 type vibration of the indole ring, which reflects the sign and magnitude of the torsion angle
2,1 (Blanch et al., 2001b
). The PVX and NMV strains used here contain five and four tryptophans, respectively. The absence of significant ROA in this spectral region of both viruses therefore suggests conformational heterogeneity among these tryptophans (Blanch et al., 2001b
). On the other hand, the two and six tyrosines present in PVX and NMV, respectively, could be responsible for the small but significant negative ROA bands at
1604 cm-1 associated with Y8a,b type modes of the aromatic ring (Miura & Thomas, 1995
), suggesting these side-chains have mainly fixed conformations generating ROA signals which tend to reinforce. A similar negative band is present at
1608 cm-1 in the ROA spectrum of TMV, which contains four tyrosines. These negative Y8 ROA bands may originate in the participation of some tyrosine side-chains in subunit binding, as observed in the TMV X-ray fibre structure of Namba et al. (1989)
.
Principal component analysis of coat protein folds
We are developing a pattern recognition program, based on the multivariate statistical approach of principal component analysis (PCA), to identify protein folds from ROA spectral band patterns. The method is similar to one developed for the analysis of conventional Raman spectra of parchment (Nielsen et al., 1999 ) and is related to methods used for the determination of the structure of proteins from infrared vibrational circular dichroism (Pancoska et al., 1991
) and UVCD (Venyaminov & Yang, 1996
) spectra. From the ROA spectral data, the PCA algorithm calculates a set of sub-spectra that serve as basis functions, the algebraic combination of which with appropriate expansion coefficients can be used to reconstruct any member of the original set of experimental ROA spectra. The level of accuracy is determined by the number of basis functions used; for example, a set of ten basis functions is sufficient to reproduce adequately any member of our current set of 56 polypeptide, protein and virus ROA spectra. This set contains the ROA spectra of poly(L-lysine) and poly(L-glutamic acid) in model
-helical and unordered (random coil) states, 32 proteins with well-defined tertiary folds known mostly from X-ray crystallography with a few known from multidimensional solution nuclear magnetic resonance, 11 denatured or natively unfolded proteins with structures known to be mostly unordered mainly from spectroscopic techniques such as UVCD, six viruses (including TMV) with coat protein folds known from X-ray crystallography or fibre diffraction and three viruses (PVX, NMV and TRV) with unknown coat protein folds. Since the initial results are promising, we show in Fig. 2
a plot of the coefficients for the whole set of ROA spectra for the two most important basis functions. This provides a two-dimensional representation of the structural relationships among the members of the set. The proteins are colour-coded with respect to the seven different structural types listed on the figure and defined more fully in the caption. These structural types provide a useful initial classification that will be refined and enlarged in later work. Typical examples of these structural types are: alpha, human serum albumin; mainly alpha, hen lysozyme; alpha beta, hen ovalbumin; mainly beta, bovine
-lactoglobulin; beta, jack bean concanavalin A; mainly unordered, bovine
-casein; unordered, hen phosvitin. This provides a starting point for the pattern recognition method since it reveals an initial separation of the spectra into clusters corresponding to different dominant types of protein structural elements, with increasing
-helix content to the left, increasing
-sheet content to the right, and increasing disorder from bottom to top. The way in which PVX, NMV and TMV cluster together suggests that the folds of their coat proteins may be similar, but with those of PVX and NMV more similar to each other (since their positions are almost identical) than to that of TMV (which is shifted a little from the other two). This cluster lies within the mainly alpha region and so reinforces our conclusion above that the model of Baratova et al. (1992a
) for the PVX coat proteins contains too much
-sheet. Plots of other coefficients, which will be given in a later paper, provide further discrimination between different structural types. We have shown these preliminary results here because this unbiased mathematical analysis of the ROA spectral data reinforces the visual impression of a close similarity between the coat protein folds of PVX, NMV and TMV, and because pattern recognition methods are expected to become increasingly important in future applications of ROA to structural virology.
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Acknowledgments |
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References |
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Received 29 June 2001;
accepted 13 September 2001.