1 Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA
2 Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA
Correspondence
Prasad L. Polavarapu
Prasad.L.Polavarapu{at}Vanderbilt.Edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
VCD is a measure of the difference in absorption for left and right circularly polarized light in the infrared (IR) region (Keiderling, 1981; Nafie, 1984
; Polavarapu, 1998
; Barron, 2004
). It is a useful method for studying the conformation of biomolecules in different environments and has therefore been used to investigate the structures of peptides, polypeptides and proteins in solution (Baumruk & Keiderling, 1993
; Yoder et al., 1995
; Kocak et al., 1998
; Zhao & Polavarapu, 1999
; Schweitzer-Stenner et al., 2003
; Shanmugam & Polavarapu, 2004a
, b
, c
). The predominant secondary structure of a peptide or protein, such as
-helix,
-sheet or random coil, is reflected in the unique signs and/or patterns of the VCD bands that appear for the amide I (mainly due to peptide C=O stretching) and the amide II (mainly due to peptide NH deformation) vibrations. The amide I vibrations appear in the 17501600 cm1 region while amide II vibrations appear in the 16001500 cm1 region. Several reports have discussed in detail the characteristic VCD features for these secondary structural components (Pancoska et al., 1991
; Shanmugam & Polavarapu, 2004a
). Briefly, a right-handed
-helical structure exhibits a positive VCD couplet (a positive VCD band on the lower wave number side and a negative VCD band on the higher wave number side) in the amide I region and a small negative VCD band in the amide II region. The random coil VCD spectrum has a negative VCD couplet in the amide I region, where a stronger negative band appears at a lower wave number and a positive band appears at a higher wave number (opposite to that seen in a right-handed
-helix). A
-sheet structure exhibits a small negative VCD band near 1620 cm1 (amide I) and a negative VCD couplet in the amide II region. Characteristic VCD patterns in the amide I region for the 310-helix,
-turn and polyproline II structures have also been reported (Zhao et al., 2000
; Silva et al., 2002
).
Since water absorbs strongly in the infrared region, it is not a favourable solvent for IR absorption and VCD spectroscopy. One approach to overcoming this limitation is to use higher sample concentrations and minimize the interference from solvent absorption. An alternative is to use other solvents, such as D2O or DMSO, although these solvents may not reflect physiological environments. Using one or both of these options, several VCD reports have been published for peptides, proteins, nucleic acids and carbohydrates in solution (Annamalai & Keiderling, 1987; Tummalapalli et al., 1988
; Bose & Polavarapu, 1999a
, b
; Zhao et al., 2000
; Shanmugam & Polavarapu, 2004b
, c
). The need for higher concentrations when aqueous solutions are used has been a limiting factor for applications of VCD to biological systems. Another limitation for measurements in D2O solvent is that only the protons on exposed amide groups may exchange for deuterium, while the buried amide protons may not. A different approach to overcoming these limitations is to deposit aqueous solutions as films and undertake studies on films. As most of the solvent is dried out in preparing the films, solvent absorption interference is no longer a problem. In addition, dilute stock solutions can be used to prepare the films for investigation, so concentration limitations are not as troubling. The film VCD spectra generally have better signal-to-noise ratios than the corresponding spectra for solutions because of increased light transmission. VCD measurements performed on films of proteins (Shanmugam & Polavarapu, 2004a
) and carbohydrates (Petrovic et al., 2004
) have been reported recently. Also, thermally induced structural changes in bovine serum albumin have been reported using the film method (Shanmugam & Polavarapu, 2004b
). The ability to record VCD spectra for films opens up opportunities for applying this method to many biological systems, including viruses.
VCD spectral studies on viruses have not been reported before now. In this paper, we report the first measurement of VCD spectra in the amide I and II regions for viruses in the form of films derived from dilute aqueous buffer solutions. For comparative studies, we also measured VCD spectra of viruses in solution. Four filamentous plant viruses, Tobacco mosaic virus (TMV), Papaya mosaic virus (PMV), Potato virus X (PVX) and Narcissus mosaic virus (NMV), were investigated. In addition, we investigated a deletion mutant of PVX (PVX-21). TMV belongs to the genus Tobamovirus comprising rigid, rod-shaped, helical plant viruses (Lewandowski & Dawson, 1999
), approximately 3000 Å (300 nm) long and 180 Å (18 nm) in diameter. Tobamoviruses contain single-stranded positive-sense RNA. The detailed structure of TMV has been determined by X-ray fibre diffraction (Namba et al., 1989
). PVX, PMV and NMV are members of the genus Potexvirus (AbouHaidar & Gellatly, 1999
), comprising flexuous filamentous viruses about 5000 Å (500 nm) long and 140 Å (14 nm) in diameter, also containing single-stranded positive-sense RNA. Previous studies using X-ray fibre diffraction, ROA and ECD have shown that the protein structures in these viruses are dominated by
-helices (Homer & Goodman, 1975
; Erickson et al., 1981
; Namba et al., 1989
; Wilson et al., 1991
; Orlov et al., 2001
; Blanch et al., 2002
).
Our VCD results also suggested that the coat proteins of these viruses contain -helices, both in film and in solution. The structures of PVX and NMV were found to be similar to each other, as expected, but different from TMV. Although the viral RNA could not be detected by VCD measurements in solution, the film VCD spectra showed characteristics of both nucleic acid and protein. Our results demonstrate the usefulness of VCD in the structural analysis of viruses and provide new structural information. The VCD spectra of PVX and NMV suggest that both the coat protein and RNA structures are extremely similar for these two viruses, based on the VCD band positions and sign patterns. Furthermore, the presence of small amounts of
-sheet conformation in PVX and NMV was clearly demonstrated; previous reports of
structure in these viruses have been ambiguous. The secondary structure of the coat protein was similar in solution and dried (film) states for the viruses studied here, except for the mutant virus PVX-
21.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
For solution VCD spectra, viruses were centrifuged and resuspended twice in D2O buffers. The first centrifugation was for 1 h at 100 000 g; the second for 1 h at 150 000 g.
VCD study.
All VCD spectra were recorded on a commercial Chiralir spectrometer (Bomem-Bio Tools) modified to minimize artefacts using the double polarization modulation method (Nafie, 2000). Details of the modification of the VCD instrument have been published previously (Shanmugam & Polavarapu, 2004a
). Film and solution VCD spectra were collected for 1 and 3 h, respectively, at a resolution of 8 cm1. Virus concentrations and buffer solutions used for solution and film state measurements are given in Table 1
.
|
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Our VCD data are consistent with previous results from proteins containing -helical structures. The coat protein subunits were seen to have a large proportion of
-helical structure; in the case of TMV, this structure is known to be in the form of a four-helix bundle containing both water-exposed residues and residues at hydrophilic and hydrophobic helixhelix interfaces (Namba et al., 1989
). Furthermore, far-UV CD and ROA results predicted a high proportion of helical structure in aqueous solution (Orlov et al., 2001
; Blanch et al., 2002
).
The VCD and vibrational absorption spectra of PVX and NMV films are shown in Fig. 1(a, top two spectra). The overall appearances of the VCD spectra of PVX and NMV were very similar, particularly between 1700 and 1600 cm1, indicating that the structures of these two viruses are similar. The VCD spectrum of PVX showed a positive couplet (a negative band at 1661 cm1 followed by a positive band at 1650 cm1) characteristic of an
-helical structure. This interpretation was confirmed by the negative amide II band at 1505 cm1, also characteristic of a helical structure. The presence of an additional negative band at 1633 cm1 indicated that
-sheet structure was also present in the film state. The presence of another positive couplet on the higher energy side (a negative band at 1694 cm1 and a positive band at 1679 cm1) is characteristic of nucleic acid bases, particularly guanine. The VCD spectrum of NMV showed a positive VCD couplet (negative at 1660 cm1 and positive at 1647 cm1) and a weak negative band at 1632 cm1, all similar to those of PVX, except for small shifts in band positions. The VCD spectra also showed a W pattern between 1600 and 1500 cm1 (a negative band at 15051518 cm1 and a positive band at 1536 cm1 followed by a negative band at 15451551 cm1), which was similar for both PVX and NMV, except for changes in the relative intensities of the individual components. The VCD bands due to nucleic acid bases were also seen for NMV at 1670 (positive) and 1682 (negative) cm1, similar to those for PVX. Thus, unlike TMV and PMV, the spectra of PVX and NMV in the film state clearly showed bands that were characteristic of both coat protein and nucleic acid bases. It is premature to suggest structures for the nucleic acid from the limited number of VCD observations reported here, but with more VCD investigations on viruses in the future it might be possible to infer the structures of nucleic acids in viruses.
ROA data indicate that PVX and NMV in aqueous solution contain -helical and hydrated
structures (Blanch et al., 2002
). However, UV CD data predict large amounts of
-helix but little
-sheet structure (Homer & Goodman, 1975
; Wilson et al., 1991
). Our results indicated the presence of
structures in PVX and NMV. For the PVX coat protein, Baratova et al. (1992)
predicted from the amino acid sequence a coat protein structure that included a well-defined
structure at both the N- and C-terminal ends of the protein. Sawyer et al. (1987)
suggested that the N-terminal 50 residues of PVX have zero probability of having an
-helical structure. Our VCD results confirm the presence of both
-helical and
-sheet structures in the PVX and NMV coat proteins.
There is no similarity in the amino acid sequences of TMV and the potexvirus coat proteins. Our results clearly suggest that there is some difference between the structures of TMV and the potexviruses. VCD has provided evidence for helical structures in all of the viruses and some -sheet structures in PVX, NMV and PMV, but not in TMV. It has been suggested (Sawyer et al., 1987
; Shukla et al., 1988
; Blanch et al., 2002
) that the coat protein folds of PVX and NMV may be similar to that of TMV, being based on the helix bundle, but with differences in detail resulting from differences in the appended loops and turns and from the extra sequences present in the potexviruses. These differences may be responsible for the observed VCD differences.
For comparative studies, we also measured the VCD and vibrational absorption spectra of TMV, PMV, PVX and NMV in D2O buffer solution at pH 8. It should be noted that, unlike the situation in film measurements, the absorption background from D2O precludes observations in the amide II region. Furthermore, the signal-to-noise ratio is lower in solution than in film, owing to the lower amount of light transmitted through D2O solutions. The solution spectra are shown in Fig. 1(b). Both TMV and PMV in Tris buffer at pH 8 (Table 1
) showed a positive VCD couplet (a negative band at 1666 cm1 and a positive band at 1650 cm1) characteristic of a helical structure. The negative band in this couplet was slightly shifted relative to the corresponding couplet in the film VCD. A weak negative VCD band at 1624 cm1 for PMV was associated with a
-sheet structure, as in the film VCD. This
-sheet VCD band was not seen for TMV. These data suggest that the virus coat protein structures did not change significantly between the solution and film states for PMV and TMV. The vibrational absorption spectra in Fig. 1(b)
also showed the characteristic helical amide I band at 1651 cm1 for TMV and PMV.
The VCD spectrum of PVX in Tris/EDTA buffer at pH 8 (Fig. 1b) showed diagnostic bands for an
-helical structure (a positive couplet between 1640 and 1670 cm1). Furthermore, the spectrum had a weak negative VCD band in the
-sheet region (1636 cm1), as observed for PVX film (Fig. 1a
). A weak positive couplet in the higher frequency region (not marked in the spectrum) could be due to nucleic acid bases. For NMV, the low concentration of the virus sample resulted in a lower signal quality. Nevertheless, the solution VCD spectrum of NMV (Fig. 1b
) revealed features similar to those of PVX. The positive VCD band at
1640 cm1 was quite weak and was not seen clearly above the noise. The vibrational absorption spectra of PVX and NMV clearly showed the characteristic helical band centred at
1651 cm1 (Fig. 1b
, bottom). These results suggested that there is no significant change in the protein structure of PVX and NMV between aqueous buffer solution and films derived from the same buffer solution, although the quality of the VCD spectra was better in film than in solution.
The VCD spectra of the viruses in solution were dominated by protein bands, unlike the film VCD spectra in which bands due to RNA were also observed. The VCD bands from the RNA were expected to be much weaker than those from the protein because of the low RNA content (6 %). Even though the concentrations of virus samples used for film and solution studies were approximately the same, VCD clearly showed characteristic nucleic acid bands in the film state but not in solution. This result indirectly confirmed our conclusion that the positive couplet observed for different viruses between 1670 and 1635 cm1 in the film VCD spectra was from the peptide backbone carbonyl stretching and not from the nucleic acid bases.
We also measured the VCD spectra of a deletion mutant of PVX (PVX-21), from which residues 222 of the wild-type coat protein had been removed. Spectra were measured both in solution and in the film state to investigate the conformational differences (if any) between PVX-
21 and PVX. The film VCD spectrum of PVX-
21 was significantly different from that of PVX (Fig. 2a
). The VCD spectrum of PVX-
21 film showed a single negative band centred at 1651 cm1 in the amide I region, indicative of an
-helical structure. However, for an
-helical structure there should also be a positive VCD band on the lower frequency side of the 1651 cm1 negative VCD. This positive band did not appear in the film VCD spectrum. The presence of a negative VCD band at 1521 cm1 (amide II) in the film VCD of PVX-
21 also suggested a helical structure. The film VCD spectrum of PVX-
21 did not show any characteristic band for the nucleic acid bases, unlike the film VCD of PVX. In order to eliminate the possibility of artefacts in the film VCD of mutant PVX, we repeated the VCD measurements with films prepared from stock solutions of three different concentrations (see Table 1
); in all cases the same VCD features were obtained. In addition, the film VCD was found to be independent of film orientation when the film was rotated around the light beam axis. These tests led us to believe that the film VCD spectra for mutant PVX did not contain artefacts. Fig. 2(b)
compares the VCD (top) and vibrational absorption (bottom) spectra of PVX-
21 and PVX in D2O buffer, pH 8. For comparison, the corresponding noise spectrum is presented above the VCD spectrum. Only those VCD bands that had magnitudes >0·5x105 are discussed here, since the noise level in the VCD spectra was
0·5x105. The observed VCD spectrum of PVX-
21 clearly showed the positive couplet (negative band at 1661 cm1 and positive band at 1647 cm1) and the absorption spectrum showed an absorption maximum at 1651 cm1, indicating an
-helical structure similar to that of PVX. This result suggested that in solution the coat protein structure of PVX-
21 is not significantly different from that of wild-type PVX.
|
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Annamalai, A. & Keiderling, T. A. (1987). Vibrational circular dichroism of polyribonucleic acids. A comparative study in aqueous solution. J Am Chem Soc 109, 31253132.[CrossRef]
Baratova, L. A., Grebenshchikov, N. I., Dobrov, E. N. & 7 other authors (1992). The organization of potato virus X coat proteins in virus particles studied by tritium planigraphy and model building. Virology 188, 175180.[CrossRef][Medline]
Barron, L. D. (2004). Molecular Light Scattering and Optical Activity, 2nd edn. London: Cambridge University Press.
Baumruk, V. & Keiderling, T. A. (1993). Vibrational circular dichroism of proteins in H2O solution. J Am Chem Soc 115, 69396942.[CrossRef]
Blanch, E. W., Robinson, D. J., Hecht, L., Syme, C. D., Nielsen, K. & Barron, L. D. (2002). Solution structures of potato virus X and narcissus mosaic virus from Raman optical activity. J Gen Virol 83, 241246.
Boedtker, H. & Simmons, N. S. (1958). The preparation and characterization of essentially uniform tobacco mosaic virus particles. J Am Chem Soc 80, 25502556.[CrossRef]
Bose, P. K. & Polavarapu, P. L. (1999a). Vibrational circular dichroism of monosaccharides. Carbohyd Res 319, 172183.[CrossRef]
Bose, P. K. & Polavarapu, P. L. (1999b). Acetate groups as probes of the stereochemistry of carbohydrates: a vibrational circular dichroism study. Carbohyd Res 322, 135141.[CrossRef]
Chapman, S., Hills, G., Watts, J. & Baulcombe, D. (1992). Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity. Virology 191, 223230.[CrossRef][Medline]
Erickson, J. W. & Bancroft, J. B. (1978). The self-assembly of papaya mosaic virus. Virology 90, 3646.[CrossRef][Medline]
Erickson, J. W., Bancroft, J. B. & Stillman, M. J. (1981). Circular dichroism studies of papaya mosaic virus coat protein and its polymers. J Mol Biol 147, 337349.[CrossRef][Medline]
Goodman, R. M. (1975). Reconstitution of potato virus X in vitro. I. Properties of the dissociated protein structural subunits. Virology 68, 287298.[CrossRef][Medline]
Gupta, V. P. & Keiderling, T. A. (1992). Vibrational CD of the amide II band in some model polypeptides and proteins. Biopolymers 32, 239248.[CrossRef][Medline]
Homer, R. B. & Goodman, R. M. (1975). Circular dichroism and fluorescence studies on potato virus X and its structural components. Biochim Biophys Acta 378, 296304.[Medline]
Keiderling, T. A. (1981). Vibrational circular dichroism. Appl Spectrosc Rev 17, 189225.
Kocak, A., Luque, R. & Diem, M. (1998). The solution structure of small peptides: an infrared CD study of aqueous solutions of (L-Ala)n [n=3, 4, 5, 6] at different temperatures and ionic strengths. Biopolymers 46, 455463.[CrossRef]
Lewandowski, D. J. & Dawson, W. O. (1999). Tobamoviruses. In Encyclopedia of Virology, 2nd edn, pp. 17801783. Edited by A. Granoff & R. G. Webster. London: Academic Press.
Nafie, L. A. (1984). Experimental and theoretical advances in vibrational optical activity. In Advances in Infrared and Raman Spectroscopy, vol. 11, pp. 4993. Edited by R. J. H. Clark & R. E. Hester. London: Heyden & Sons.
Nafie, L. A. (2000). Dual polarization modulation: a real-time, spectral-multiplex separation of circular dichroism from linear birefringence spectral intensities. Appl Spectrosc 54, 16341645.[CrossRef]
Namba, K., Pattanayek, R. & Stubbs, G. (1989). Visualization of proteinnucleic acid interactions in a virus; refinement of intact tobacco mosaic virus structure at 2·9Å resolution by fiber diffraction. J Mol Biol 208, 307325.[CrossRef][Medline]
Orlov, V. N., Arutyunyan, A. M., Kust, S. V., Litmanovich, E. A., Drachev, V. A. & Dobrov, E. N. (2001). Macroscopic aggregation of tobacco mosaic virus coat protein. Biochemistry 66, 154162.[Medline]
Pancoska, P., Yasui, S. C. & Keiderling, T. A. (1991). Statistical analyses of the vibrational circular dichroism of selected proteins and relationship to secondary structures. Biochemistry 30, 50895103.[CrossRef][Medline]
Parker, L., Kendall, A. & Stubbs, G. (2002). Surface features of potato virus X from fiber diffraction. Virology 300, 291295.[CrossRef][Medline]
Petrovic, A. G., Bose, P. K. & Polavarapu, P. L. (2004). Vibrational circular dichroism of carbohydrate films formed from aqueous solutions. Carbohydr Res 339, 27132720.[Medline]
Polavarapu, P. L. (1998). Vibrational Spectra: Principles and Applications with Emphasis on Optical Activity. New York: Elsevier Publications.
Sawyer, L., Tollin, P. & Wilson, H. R. (1987). A comparison between the predicted secondary structures of potato virus X and papaya mosaic virus coat proteins. J Gen Virol 68, 12291232.
Schweitzer-Stenner, R., Eker, F., Perez, A., Griebenow, K., Cao, X. & Nafie, L. A. (2003). The structure of tri-proline in water probed by polarized Raman, Fourier transform infrared, vibrational circular dichroism, and electric ultraviolet circular dichroism spectroscopy. Biopolymers 71, 558568.[CrossRef][Medline]
Shanmugam, G. & Polavarapu, P. L. (2004a). Vibrational circular dichroism of protein films. J Am Chem Soc 126, 1029210295.[CrossRef][Medline]
Shanmugam, G. & Polavarapu, P. L. (2004b). Structure of A(25-35) peptide in different environments. Biophys J 87, 622630.[CrossRef][Medline]
Shanmugam, G. & Polavarapu, P. L. (2004c). Vibrational circular dichroism spectra of protein films: thermal denaturation of bovine serum albumin. Biophys Chem 111, 7377.[CrossRef][Medline]
Shukla, D. D., Strike, P. M., Tracy, S. L., Gough, K. H. & Ward, C. W. (1988). The N and C termini of the coat proteins of potyviruses are surface-located and the N terminus contains the major virus specific epitopes. J Gen Virol 69, 14971508.
Silva, R. A., Yasui, S. C., Kubelka, J., Formaggio, F., Crisma, M., Toniolo, C. & Keiderling, T. A. (2002). Discriminating 310- from -helices: vibrational and electronic CD and IR absorption study of related Aib-containing oligopeptides. Biopolymers 65, 229243.[CrossRef][Medline]
Tummalapalli, C. M., Back, D. M. & Polavarapu, P. L. (1988). Fourier-transform infrared vibrational circular dichroism of simple carbohydrates. J Chem Soc Faraday Trans 1 84, 25852594.[CrossRef]
Wilson, H. R., Tollin, P., Sawyer, L., Robinson, D. J., Price, N. C. & Kelly, S. M. (1991). Secondary structures of narcissus mosaic virus coat protein. J Gen Virol 72, 14791480.[Abstract]
Yoder, G., Keiderling, T. A., Formaggio, F., Crisma, M. & Toniolo, C. (1995). Characterization of -bend ribbon spiral forming peptides using electronic and vibrational CD. Biopolymers 35, 103111.[CrossRef][Medline]
Zhao, C. & Polavarapu, P. L. (1999). Vibrational circular dichroism is an incisive structural probe: ion-induced structural changes in gramicidin D. J Am Chem Soc 121, 1125911260.[CrossRef]
Zhao, C., Polavarapu, P. L., Das, C. & Balaram, P. (2000). Vibrational circular dichroism of -hairpin peptides. J Am Chem Soc 122, 82288231.[CrossRef]
Received 22 March 2005;
accepted 10 May 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |