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β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation a
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Evgeny N. Dobrov , Nikolai A. Nikitin , Ekaterina A. Trifonova , Evgenia Yu. Parshina , a
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Valentin V. Makarov , George V. Maksimov , Olga V. Karpova & Joseph G. Atabekov
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A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University , 1/40 Leninskie gory, Moscow , 119991 , Russia b
Department of Virology , Lomonosov Moscow State University , 1/12 Leninskie gory, Moscow , 119991 , Russia c
Department of Biophysics , Moscow State University , Moscow , 119991 , Russia Published online: 19 Jun 2013.
To cite this article: Evgeny N. Dobrov , Nikolai A. Nikitin , Ekaterina A. Trifonova , Evgenia Yu. Parshina , Valentin V. Makarov , George V. Maksimov , Olga V. Karpova & Joseph G. Atabekov (2013): β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation, Journal of Biomolecular Structure and Dynamics, DOI:10.1080/07391102.2013.788983 To link to this article: http://dx.doi.org/10.1080/07391102.2013.788983
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Journal of Biomolecular Structure and Dynamics, 2013 http://dx.doi.org/10.1080/07391102.2013.788983
β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation Evgeny N. Dobrova*, Nikolai A. Nikitinb, Ekaterina A. Trifonovab, Evgenia Yu. Parshinac, Valentin V. Makarova, George V. Maksimovc, Olga V. Karpovab and Joseph G. Atabekova,b a A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 1/40 Leninskie gory, Moscow 119991, Russia; bDepartment of Virology, Lomonosov Moscow State University, 1/12 Leninskie gory, Moscow 119991, Russia; cDepartment of Biophysics, Moscow State University, Moscow 119991, Russia
Communicated by Ramaswamy H. Sarma
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(Received 15 February 2013; final version received 20 March 2013) Conversion of the rod-like tobacco mosaic virus (TMV) virions into “ball-like particles” by thermal denaturation at 90–98 ° C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94–98 °C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53–800 nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures. Keywords: tobacco mosaic virus; thermal remodeling; spherical particles; α-and β-structures; amyloid structures.
Introduction Tobacco mosaic virus (TMV) particles are rods of 18 nm in diameter and 300 nm in modal length; they consist of 2130 identical 17.5 kDa protein subunits packed closely by hydrophobic bonds into a rigid tube. The subunits are helically arranged with a pitch of 23 Å around a cylindrical canal of 20 Å in radius. The RNA about 6400 nucleotides long is intercalated between the protein turns at a radius of 40 Å and follows the helix of protein subunits (Butler, 1999; Klug, 1999; Zaitlin & Israel, 1975). It has been known that TMV is very heat-stable: some infectivity is retained even after a 10 min exposure of the infectious sap to over 90 °C. Lauffer and Price (1940) found that heat inactivation of TMV is associated with CP denaturation. Hart (1956) had reported that TMV heating in the range of 80–98 °C resulted in a swelling of TMV particles at one or both ends and their conversion into “ball-like particles.” However, these studies have not been continued and the ball-like particles have not been characterized. Previously, we have described in detail the spherical nanoparticles (SPs) generated by thermal remodeling of the rigid helical particles of native TMV at 94–98 °C. *Corresponding author. Email: [email protected] Copyright Ó 2013 Taylor & Francis
The SPs were widely heterogeneous in size (53–800 nm) and uniform in shape. Contrary to TMV virions, the protein isolated from SPs could not be reassembled into any regular structure, indicating that the thermal denaturation was irreversible. It is noteworthy that no degradation of TMV CP occurred upon TMV heating. The size (but not the shape) of SPs depended on virus concentration and, therefore, could be controlled. The SPs are water insoluble and highly stable: they do not change their shape and size, do not fuse together and do not change the state of aggregation at reheating, freezing, or long storage. They could be readily revealed as black discs by transmission electron microscopy (TEM) with no need of negative staining. The TEM images obtained by this means were similar to those produced by traditional staining with 2% uranyl acetate (Atabekov, Nikitin, Arkhipenko, Chirkov, & Karpova, 2011). The SPs could be generated by different forms of TMV CP aggregates and individual protein subunits. The SPs consist of thermally denatured CP and contain no RNA; thus, they are biologically safe and biodegradable. It was suggested that, under the conditions of thermal denaturation, the TMV CP subunits acquire a specific
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conformation favorable for their assembly into SPs (Atabekov et al., 2011; Nikitin et al., 2011). A unique feature of the SPs is the capacity of their surface to bind various distinct proteins, structurally and functionally, of different origin and molecular weights (in the range of 16–66 kDa) including the green fluorescent protein, some plant virus coat proteins, bovine serum albumin, or Rubella virus and influenza virus A antigenic determinants (Karpova et al., 2012). The SPs proved significantly more immunogenic than native TMV. Specific antibody response to SPs was up to 20 times higher than that to native TMV. This analysis showed that native TMV and SPs were only distantly related antigenic (Atabekov et al., 2011). In a series of experiments on SPs immunogenic properties, separate groups of mice were immunized with SPs alone (from 500 μg to 1 mg) (Karpova et al., 2012). It was found that no toxic effect was caused by SPs. We demonstrated that immunogenic activity of foreign antigens bound to the surface of SP-nanoplatform increased significantly. It was suggested that SPs exhibit immunopotentiating ability of enhancing the humoral immune response to foreign proteins and the adjuvant ability. Therefore, the SP-based compositions could be regarded as candidate nanovaccines assembled in vitro (Atabekov et al., 2011; Karpova et al., 2012; Nikitin et al., 2011), and SPs represented a new type of biogenic particle nanoplatform that has no structural analogs. Here we describe some structural characteristics of SPs. Materials and methods Virus purification TMV U1 strain was isolated from systemically infected Nicotiana tabacum L. cv. Samsun plants as described by Novikov and Atabekov (1970). SPs generation SPs were obtained by TMV heating at the initial concentration of 0.1 or 5 mg/ml for 10 s as described by Atabekov et al. (2011). Nanoparticle tracking analysis Nanoparticle tracking analyses (NTA) were performed using a NanoSight NS500 system (NanoSight, United Kingdom) according to Nikitin, Trifonova, Karpova, and Atabekov (2013). Video images were analyzed by the NTA analytical software version 2.3. The measurements were made at room temperature and each video clip was captured for 60 s. Density analyses The comparative density analyses of TMV, TMV-like RNA-free aggregates assembled from TMV CP and the
SPs (300 nm) were carried out at 20 °C and the concentrations were of 1 mg/ml using DX45 Delta Range (Mettler Toledo). Raman spectroscopy The Raman spectra of SPs (c = 0.1 mg/ml), TMV virions (c = 0.1 mg/ml), or TMV RNA (c = 0.1 mg/ml) were obtained using a NTEGRA Spectra NanoLaboratory (NT-MDT, Russia) and Nova software (NT-MDT, Russia). Laser optical scheme is designed to produce a confocal picture of a sample using a laser light. An NTEGRA Spectra performs spectral measurement at certain point and acquires the spectral characteristics of a sample. An inverted optical microscope Olympus IX71 serves for positioning the laser point on the sample. The secondary radiation spectral composition is analyzed with the CCD-camera cooled with a thermoelectric Peltier element to 50 °С. The suspensions were deposited on mirror glass (30 μl), dried on air, and 15–20 spectra were recorded for 60 s each from the samples with a 532 nm laser (5.5 mW), 40 objective (NA = .65), grating of 600 lines/mm, and spectral resolution of 3.18 cm 1. The baseline was corrected, and the spectra were normalized to the 1004 cm 1 band. Spectra measurements Unless otherwise indicated, the phosphate buffer was used in all cases as the most suitable buffer for far-UV CD measurements. The absorption spectra in 240 to 340 nm region were measured in cells with an optical path of 1 cm in a Hitachi UV-2600 spectrophotometer. CD spectra in the 185–250 nm region were recorded in 1–2 mm cells at 25°C in a Chiroscan CD spectrometer (Applied Photophysics, UK). The spectra were measured in 1 mM phosphate buffer of pH 7.5. Sample concentrations were of 50–100 μg/ml. The spectra were recorded at a speed of .5–1.0 nm/s with baseline subtraction. The measured spectra were smoothed with the instrument software. The [h] values were calculated taking the mean molecular weight of amino acid residues to 110. The intrinsic fluorescence spectra were recorded in 1 cm cells using a FluoroMax spectrofluorometer (HORIBA Jobin Yvon, USA) at 25 °C. Fluorescence of TMV virions and SPs at the concentration of 0.03 mg/ml was excited in 1 mM phosphate buffer of pH 7.5 at 280 nm, and the emission spectra were recorded in the 300–400 nm range. ThT fluorescence assays ThT (Sigma) binding was assayed by adding freshly prepared ThT to TMV and spherical particles solutions to the 10 mM phosphate buffer of pH 7.0 (the molar ratio of TMV: ThT was 5:1). The final ThT concentration was 10 mM. The fluorescence emission spectra were
Structural changes in viral CP upon TMV to SP transition
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measured between 450 and 600 nm with excitation at 445 nm using a FluoroMax spectrofluorometer (HORIBA Jobin Yvon, USA). The background ThT fluorescence was measured using ThT buffer only. Results and discussion Two preparations of native TMV with the initial concentrations of 0.1 mg/ml and 5 mg/ml were used to generate the SPs with diameter of 50 and 300 nm, respectively. The SPs were characterized by NTA, which permits to measure the size of spherical particles, their state of aggregation, and concentration directly in liquid (Nikitin et al., 2013). The average size of the SPs generated by thermal denaturation of 0.1 mg/ml TMV was 52 ± 6 nm (Figure 1(a)), whereas the SPs with an average size of 305 ± 24 nm were produced by thermal treatment of 1 mg/ml TMV (Figure 1(b)). Similar results regarding the SPs’ size distribution were obtained by the methods of dynamic light scattering (data not shown) and transmission electron microscopy (Atabekov et al., 2011). A monomodal size distribution revealed by NTA pointed to the absence of SP aggregation in liquid. The concentration of SPs measured by NTA was 2.4 1016 and 4.0 1013 particles/ml for the preparations with the average size of 52 and 305 nm, respectively. It has long been known that buoyant density of native TMV (type strain) was about 1.3 g/cm3 (Sehgal, Jean, Bhalla, Soong, & Krause, 1959; Siegal & Hudson, 1959). We compared the SPs density with that of RNA-free helical virus-like CP aggregates assembled in vitro (Atabekov et al., 2011) and with that of native TMV using DX45 Delta Range (Mettler Toledo). It was found that the density of SPs (1.43 g/cm3) differed
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significantly from that of TMV-like CP aggregates (1.19 g/cm3) and native TMV (1.31 g/cm3). These data provided evidence that considerable changes were caused by the thermal denaturation of TMV CP subunits to provide them the conformation favorable for the assembly of highly dense and stable SPs. It should be noted that the SPs density exceeds the density of other helical plant viruses (Sehgal et al., 1959). Thereafter, we measured Raman spectra (Figure 2) of two abovementioned SP preparations of different size. To this end, a recently developed confocal Raman spectrometer NTEGRASpectra (Ashton, Lau, Winder, & Goodacre, 2011; Semenova et al., 2012) was employed. This instrument described in detail the materials and methods allowed us to obtain a resolution similar to that achieved on conventional Raman spectrometers but with much lower amounts of material and in 15 to 20 min. Conformationally sensitive peaks of the native TMV CP and SP proteins were clearly resolved by this instrument and demonstrated that the transformation of TMV virions (about 50% of α-helices in TMV CP (Namba, Pattanayek, & Stubbs, 1989)) into SPs is accompanied by an almost complete loss of α-helical structures and the emergence of a large fraction of β-structure: the very strong virion peak at 1656 cm 1 was shifted to 1668 cm 1 in SPs; a β-structure peak at 1235 cm 1 appeared; whereas the α-helix peak at 935 cm 1 disappeared entirely. No reliable differences between 50 and 300 nm SPs could be revealed by Raman spectrometry (Figure 2). Taking into account the structural stability of SPs and the fact that the size measurements of SPs by TEM (after drying) and by NTA (in liquid) agreed closely with each other (Nikitin et al., 2013), we propose that no SP distortion occurred during air drying of the specimens for Raman spectroscopy analyses.
Figure 1. Nanoparticle tracking analysis (the size distribution) of SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (52 ± 6 nm) and (b) 5 mg/ml (305 ± 24 nm).
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Figure 2. Raman spectra of TMV-virions (c = 0.1 mg ml 1) (1), SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm) (2), and 5 mg/ml (300 nm) (3) (c = 0.1 mg/ml) dried on air, 532 nm 5.5 mW laser, 40 objective with NA .65, and registration time of 60 s. The spectra presented were averaged for 15–20 registered spectra.
Many viruses (including large phages and helical plant viruses) possess considerable light scattering that is displayed as turbid appearance of suspensions. The presence of turbidity may significantly distort the results of such measurements because of false absorption, caused by light-scattering is added to a sample true absorption. To determine this contribution, the extrapolation method was devised. The main point of this method amounts to measuring UV absorption in the interval in which measured components possess no absorption, and therefore all measured values correspond to turbidity determined by light-scattering. We used the computerized extrapolation version described in Ksenofontov et al. (2006). The UV absorption spectra of native TMV (Figure 3 (a)) and of small (50 nm) TMV SPs samples (Figure 3 (b)) demonstrated that, despite significant light scattering, these particles had normal protein spectra and their concentrations determined from the true absorption values at 280 nm coincided with those measured by other methods. However, the absorption spectra of large (300 nm) SPs yielded negligible true absorption values (Figure 3(c)). This phenomenon was unexpected and has never occurred in our experience with the extrapolation procedure (Tikchonenko, Dobrov, Velikodvorskaya, & Kisseleva, 1966). Further work is required to elucidate this effect. To our knowledge, the first measurement of far UV CD spectrum of intact TMV virions was done by our group (Arutyunyan, Rafikova, Drachev, & Dobrov, 2001), whereas isolated TMV CP preparation was one of the first proteins (Schubert & Krafczyk, 1969), for which far UV CD spectrum was measured by CD spectrum of intact TMV measured only up to 200 nm was typical for β-structured proteins (Arutyunyan et al., 2001). Based on
the X-ray fiber diffraction data, Namba et al. reported that intravirus TMV CP contains 45 to 50% of α-helices and no more than 5% of β-structure (Namba et al., 1989). Therefore, we suggested that this type of TMV far UV CD spectrum may be due to some kind of strong intersubunit interactions (Arutyunyan et al., 2001). As an alternative, the β-type spectrum of TMV may be determined by some artifacts related to the particulate nature of virus suspensions (Homer & Goodman, 1975). Figure 4 presents far UV CD spectra of intact TMV and of the 50 nm SPs measured up to 188 nm using a new CD spectrometer Chiroscan. It can be seen that TMV virions had a highly reproducible spectrum with a very strong positive maximum ([h197] = 43,000 grad). The quantitation of the α-helix content from this peak is not yet possible, but its height and position undoubtedly correspond to an α-helix-rich protein. Furthermore, this suggests that the negative peak at longer wavelengths can hardly be an artifact. The spectrum of TMV 50 nm SPs (Figure 4) shows that the negative peak region practically coincides with the virion spectrum, but its positive peak at 194 nm has almost four times lower intensity than the virion peak. The difference spectrum in Figure 4 testifies to a complete (or almost complete) loss of α-helical structure in the SPs, which agrees with the Raman spectroscopy data in Figure 2. The shape of the virion spectrum suggests the absence of a significant proportion of unordered structure, but a strong negative peak at 198 nm in the difference spectrum suggests the appearance of a large fraction of unordered structure (including possible β-sheets) in SPs. It is noteworthy that the 300 nm SPs gave zero CD signal in the 185–250 nm region (data not shown). It is hard to explain the precise role, if any, of structural similarity between the negative region spectra for the SPs and TMV virions in Figure 4. We used the popular program K2D2 to predict the secondary structure in SPs based on their 188–250 nm CD spectra. The program predicted that CP subunits in SPs contain 14% of α-helices, 32% of β-structure, and 54% of unordered sequences. This agrees well with the Raman spectroscopy results indicating that conformational changes occurred upon TMV-SP transition. Next, we compared the intrinsic fluorescence spectra of SPs and TMV virions (Figure 5). TMV CP intrinsic fluorescence spectrum (Guttenplan & Calvin, 1973) represents a classical case of protein with a stable tertiary structure: high fluorescence intensity and location of aromatic amino acid residues (3 W and 4 Y) in fully hydrophobic environment (Figure 5, curve 1). In the cases of 50 and 300 nm SPs, the fluorescence intensity remained practically unchanged as compared to the virions but the maximum position shifted substantially (from 326 to 338–340 nm); thus, indicating the transition of Trp and Tyr residues to a much more
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Structural changes in viral CP upon TMV to SP transition
Figure 3. UV absorption spectrum of SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (b) and 5 mg/ml (c), control – native TMV virions (a). The spectra were measured in 1 mM phosphate buffer pH 7.5.
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Figure 4. Far UV CD spectra of native TMV, SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm), and their subtractive spectrum. The spectra were measured at 25°C in 1 mM phosphate buffer pH 7.5.
Figure 5. Intrinsic fluorescence spectra of native TMV as well as SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm) and 5 mg/ml (300 nm). The spectra were recorded at 25°C in 1 mM phosphate buffer pH 7.5.
hydrophilic environment. These results suggest that TMV-to-SPs transition is accompanied by changes in the nature of amino acid residues exposed on the particles’ surface. The fluorescence data agree with the Raman and CD spectroscopy (Figures 2 and 4) and further confirm that SPs possess a highly stable structure (as followed from their properties described above). However, this stability
is determined by the interactions between β-sheets rather than between α-helices (as in TMV virions). Diaz-Avalos and Caspar (2000) studied the structure of the “stackdisc,” water-soluble intermediate aggregates of native TMV CP assembled in vivo and in vitro (reviewed by Butler, 1999; Klug, 1999). The aggregation into the stack-discs is accompanied by the conformational changes of TMV CP. Diaz-Avalos and Caspar (2000)
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Structural changes in viral CP upon TMV to SP transition
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Figure 6. Thioflavin fluorescence spectra of TMV virions as well as SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm) and 5 mg/ml (300 nm). Spectra were recorded at 25°C in 10 mM phosphate buffer pH 7.5. The molar ratio of TMV: ThT was 5:1.
noted that TMV protein subunits in the stack-discs contained structural elements “characteristic of the amyloid fibers.” Finally, recent studies demonstrated cross β-structure in aggregates of Aβ peptide, prion, and β-lactalbumin (El Moustaine, Perrier, Smeller, Lange, & Torrent, 2009; Krebs, Devlin, & Donald, 2009; Sureshbabu, Kirubagaran, & Jayakumar, 2009). To further elucidate the possibility of cross β-structure in SPs, we applied the thioflavin T fluorescence test widely used in recent years to assess the presence of amyloid-like structures (LeVine III, 1995; Nilsson, 2004). Figure 6 demonstrates the results of this test for TMV virions and SPs of two sizes (50 and 300 nm). In the case of the virions (20 μg/ml, 1:5 protein:ThyoT molar ratio), only a small peak at 490 nm was observed, which may be an artifact or a real effect of β-structure formation in the terminal CP subunits. However, a very strong fluorescence peak at 490 nm was observed in the
SP spectra. The shape of this peak was almost identical for both of the 50 and 300 nm SPs. This fact and the fast drop in fluorescence intensity on both sides of the peak testify that light scattering plays no role in the observed effect. The presence of cross β-structure in SPs may explain their high stability. Out data indicate that in the course of SPs formation the CP subunits change their secondary structure from mostly α-helical to largely β –structural one. However, we cannot say at the moment in which segments of the protein sequence the changes occur during TMV-SP transformation. Collectively, our data are summarized in Table 1. In the subsequent experiments the effect of SPs in different animal amyloidosis development systems and analysis of amino acid residues of TMV CP subunit involved in the formation of cross β-structure in SPs that will be examined.
Table 1. Comparative structural characteristics of native TMV and SPs. (⁄) according to Namba et al. (1989). Structural parameters
TMV virions
SPs (50 nm)
SPs (300 nm)
Density (g/cm3) Conformational sensitive Raman peaks (cm 1)
1.31 1656 (α); 935 (α)
Secondary structure of CP subunits from CD spectra Intrinsic fluorescence maximum position (nm) and intensity (%)
α – 50%, β – 5%, unordered – 45%⁄ 326 (100%)
– 1668 (β); 1235 (β), no peak at 935 α – 14%, β – 32%, unordered – 54% 338 (105%)
1.43 1668 (β); 1235 (β), no peak at 935 – 340 (111%)
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Abbreviations TMV CP SPs TEM NTA
tobacco mosaic virus coat protein spherical particles transmission electron microscopy nanoparticle tracking analysis
Acknowledgments This work was supported in part by the Ministry of Education and Sciences of Russia (agreement no. 8564) and the Russian Foundation for Basic Research (Grant No. 12-04-01472-a) and M.V. Lomonosov Moscow State University Program of Development.
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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation a
b
b
c
Evgeny N. Dobrov , Nikolai A. Nikitin , Ekaterina A. Trifonova , Evgenia Yu. Parshina , a
c
b
Valentin V. Makarov , George V. Maksimov , Olga V. Karpova & Joseph G. Atabekov
a b
a
A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University , 1/40 Leninskie gory, Moscow , 119991 , Russia b
Department of Virology , Lomonosov Moscow State University , 1/12 Leninskie gory, Moscow , 119991 , Russia c
Department of Biophysics , Moscow State University , Moscow , 119991 , Russia Published online: 19 Jun 2013.
To cite this article: Evgeny N. Dobrov , Nikolai A. Nikitin , Ekaterina A. Trifonova , Evgenia Yu. Parshina , Valentin V. Makarov , George V. Maksimov , Olga V. Karpova & Joseph G. Atabekov (2013): β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation, Journal of Biomolecular Structure and Dynamics, DOI:10.1080/07391102.2013.788983 To link to this article: http://dx.doi.org/10.1080/07391102.2013.788983
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Journal of Biomolecular Structure and Dynamics, 2013 http://dx.doi.org/10.1080/07391102.2013.788983
β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation Evgeny N. Dobrova*, Nikolai A. Nikitinb, Ekaterina A. Trifonovab, Evgenia Yu. Parshinac, Valentin V. Makarova, George V. Maksimovc, Olga V. Karpovab and Joseph G. Atabekova,b a A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 1/40 Leninskie gory, Moscow 119991, Russia; bDepartment of Virology, Lomonosov Moscow State University, 1/12 Leninskie gory, Moscow 119991, Russia; cDepartment of Biophysics, Moscow State University, Moscow 119991, Russia
Communicated by Ramaswamy H. Sarma
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(Received 15 February 2013; final version received 20 March 2013) Conversion of the rod-like tobacco mosaic virus (TMV) virions into “ball-like particles” by thermal denaturation at 90–98 ° C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94–98 °C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53–800 nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures. Keywords: tobacco mosaic virus; thermal remodeling; spherical particles; α-and β-structures; amyloid structures.
Introduction Tobacco mosaic virus (TMV) particles are rods of 18 nm in diameter and 300 nm in modal length; they consist of 2130 identical 17.5 kDa protein subunits packed closely by hydrophobic bonds into a rigid tube. The subunits are helically arranged with a pitch of 23 Å around a cylindrical canal of 20 Å in radius. The RNA about 6400 nucleotides long is intercalated between the protein turns at a radius of 40 Å and follows the helix of protein subunits (Butler, 1999; Klug, 1999; Zaitlin & Israel, 1975). It has been known that TMV is very heat-stable: some infectivity is retained even after a 10 min exposure of the infectious sap to over 90 °C. Lauffer and Price (1940) found that heat inactivation of TMV is associated with CP denaturation. Hart (1956) had reported that TMV heating in the range of 80–98 °C resulted in a swelling of TMV particles at one or both ends and their conversion into “ball-like particles.” However, these studies have not been continued and the ball-like particles have not been characterized. Previously, we have described in detail the spherical nanoparticles (SPs) generated by thermal remodeling of the rigid helical particles of native TMV at 94–98 °C. *Corresponding author. Email: [email protected] Copyright Ó 2013 Taylor & Francis
The SPs were widely heterogeneous in size (53–800 nm) and uniform in shape. Contrary to TMV virions, the protein isolated from SPs could not be reassembled into any regular structure, indicating that the thermal denaturation was irreversible. It is noteworthy that no degradation of TMV CP occurred upon TMV heating. The size (but not the shape) of SPs depended on virus concentration and, therefore, could be controlled. The SPs are water insoluble and highly stable: they do not change their shape and size, do not fuse together and do not change the state of aggregation at reheating, freezing, or long storage. They could be readily revealed as black discs by transmission electron microscopy (TEM) with no need of negative staining. The TEM images obtained by this means were similar to those produced by traditional staining with 2% uranyl acetate (Atabekov, Nikitin, Arkhipenko, Chirkov, & Karpova, 2011). The SPs could be generated by different forms of TMV CP aggregates and individual protein subunits. The SPs consist of thermally denatured CP and contain no RNA; thus, they are biologically safe and biodegradable. It was suggested that, under the conditions of thermal denaturation, the TMV CP subunits acquire a specific
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conformation favorable for their assembly into SPs (Atabekov et al., 2011; Nikitin et al., 2011). A unique feature of the SPs is the capacity of their surface to bind various distinct proteins, structurally and functionally, of different origin and molecular weights (in the range of 16–66 kDa) including the green fluorescent protein, some plant virus coat proteins, bovine serum albumin, or Rubella virus and influenza virus A antigenic determinants (Karpova et al., 2012). The SPs proved significantly more immunogenic than native TMV. Specific antibody response to SPs was up to 20 times higher than that to native TMV. This analysis showed that native TMV and SPs were only distantly related antigenic (Atabekov et al., 2011). In a series of experiments on SPs immunogenic properties, separate groups of mice were immunized with SPs alone (from 500 μg to 1 mg) (Karpova et al., 2012). It was found that no toxic effect was caused by SPs. We demonstrated that immunogenic activity of foreign antigens bound to the surface of SP-nanoplatform increased significantly. It was suggested that SPs exhibit immunopotentiating ability of enhancing the humoral immune response to foreign proteins and the adjuvant ability. Therefore, the SP-based compositions could be regarded as candidate nanovaccines assembled in vitro (Atabekov et al., 2011; Karpova et al., 2012; Nikitin et al., 2011), and SPs represented a new type of biogenic particle nanoplatform that has no structural analogs. Here we describe some structural characteristics of SPs. Materials and methods Virus purification TMV U1 strain was isolated from systemically infected Nicotiana tabacum L. cv. Samsun plants as described by Novikov and Atabekov (1970). SPs generation SPs were obtained by TMV heating at the initial concentration of 0.1 or 5 mg/ml for 10 s as described by Atabekov et al. (2011). Nanoparticle tracking analysis Nanoparticle tracking analyses (NTA) were performed using a NanoSight NS500 system (NanoSight, United Kingdom) according to Nikitin, Trifonova, Karpova, and Atabekov (2013). Video images were analyzed by the NTA analytical software version 2.3. The measurements were made at room temperature and each video clip was captured for 60 s. Density analyses The comparative density analyses of TMV, TMV-like RNA-free aggregates assembled from TMV CP and the
SPs (300 nm) were carried out at 20 °C and the concentrations were of 1 mg/ml using DX45 Delta Range (Mettler Toledo). Raman spectroscopy The Raman spectra of SPs (c = 0.1 mg/ml), TMV virions (c = 0.1 mg/ml), or TMV RNA (c = 0.1 mg/ml) were obtained using a NTEGRA Spectra NanoLaboratory (NT-MDT, Russia) and Nova software (NT-MDT, Russia). Laser optical scheme is designed to produce a confocal picture of a sample using a laser light. An NTEGRA Spectra performs spectral measurement at certain point and acquires the spectral characteristics of a sample. An inverted optical microscope Olympus IX71 serves for positioning the laser point on the sample. The secondary radiation spectral composition is analyzed with the CCD-camera cooled with a thermoelectric Peltier element to 50 °С. The suspensions were deposited on mirror glass (30 μl), dried on air, and 15–20 spectra were recorded for 60 s each from the samples with a 532 nm laser (5.5 mW), 40 objective (NA = .65), grating of 600 lines/mm, and spectral resolution of 3.18 cm 1. The baseline was corrected, and the spectra were normalized to the 1004 cm 1 band. Spectra measurements Unless otherwise indicated, the phosphate buffer was used in all cases as the most suitable buffer for far-UV CD measurements. The absorption spectra in 240 to 340 nm region were measured in cells with an optical path of 1 cm in a Hitachi UV-2600 spectrophotometer. CD spectra in the 185–250 nm region were recorded in 1–2 mm cells at 25°C in a Chiroscan CD spectrometer (Applied Photophysics, UK). The spectra were measured in 1 mM phosphate buffer of pH 7.5. Sample concentrations were of 50–100 μg/ml. The spectra were recorded at a speed of .5–1.0 nm/s with baseline subtraction. The measured spectra were smoothed with the instrument software. The [h] values were calculated taking the mean molecular weight of amino acid residues to 110. The intrinsic fluorescence spectra were recorded in 1 cm cells using a FluoroMax spectrofluorometer (HORIBA Jobin Yvon, USA) at 25 °C. Fluorescence of TMV virions and SPs at the concentration of 0.03 mg/ml was excited in 1 mM phosphate buffer of pH 7.5 at 280 nm, and the emission spectra were recorded in the 300–400 nm range. ThT fluorescence assays ThT (Sigma) binding was assayed by adding freshly prepared ThT to TMV and spherical particles solutions to the 10 mM phosphate buffer of pH 7.0 (the molar ratio of TMV: ThT was 5:1). The final ThT concentration was 10 mM. The fluorescence emission spectra were
Structural changes in viral CP upon TMV to SP transition
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measured between 450 and 600 nm with excitation at 445 nm using a FluoroMax spectrofluorometer (HORIBA Jobin Yvon, USA). The background ThT fluorescence was measured using ThT buffer only. Results and discussion Two preparations of native TMV with the initial concentrations of 0.1 mg/ml and 5 mg/ml were used to generate the SPs with diameter of 50 and 300 nm, respectively. The SPs were characterized by NTA, which permits to measure the size of spherical particles, their state of aggregation, and concentration directly in liquid (Nikitin et al., 2013). The average size of the SPs generated by thermal denaturation of 0.1 mg/ml TMV was 52 ± 6 nm (Figure 1(a)), whereas the SPs with an average size of 305 ± 24 nm were produced by thermal treatment of 1 mg/ml TMV (Figure 1(b)). Similar results regarding the SPs’ size distribution were obtained by the methods of dynamic light scattering (data not shown) and transmission electron microscopy (Atabekov et al., 2011). A monomodal size distribution revealed by NTA pointed to the absence of SP aggregation in liquid. The concentration of SPs measured by NTA was 2.4 1016 and 4.0 1013 particles/ml for the preparations with the average size of 52 and 305 nm, respectively. It has long been known that buoyant density of native TMV (type strain) was about 1.3 g/cm3 (Sehgal, Jean, Bhalla, Soong, & Krause, 1959; Siegal & Hudson, 1959). We compared the SPs density with that of RNA-free helical virus-like CP aggregates assembled in vitro (Atabekov et al., 2011) and with that of native TMV using DX45 Delta Range (Mettler Toledo). It was found that the density of SPs (1.43 g/cm3) differed
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significantly from that of TMV-like CP aggregates (1.19 g/cm3) and native TMV (1.31 g/cm3). These data provided evidence that considerable changes were caused by the thermal denaturation of TMV CP subunits to provide them the conformation favorable for the assembly of highly dense and stable SPs. It should be noted that the SPs density exceeds the density of other helical plant viruses (Sehgal et al., 1959). Thereafter, we measured Raman spectra (Figure 2) of two abovementioned SP preparations of different size. To this end, a recently developed confocal Raman spectrometer NTEGRASpectra (Ashton, Lau, Winder, & Goodacre, 2011; Semenova et al., 2012) was employed. This instrument described in detail the materials and methods allowed us to obtain a resolution similar to that achieved on conventional Raman spectrometers but with much lower amounts of material and in 15 to 20 min. Conformationally sensitive peaks of the native TMV CP and SP proteins were clearly resolved by this instrument and demonstrated that the transformation of TMV virions (about 50% of α-helices in TMV CP (Namba, Pattanayek, & Stubbs, 1989)) into SPs is accompanied by an almost complete loss of α-helical structures and the emergence of a large fraction of β-structure: the very strong virion peak at 1656 cm 1 was shifted to 1668 cm 1 in SPs; a β-structure peak at 1235 cm 1 appeared; whereas the α-helix peak at 935 cm 1 disappeared entirely. No reliable differences between 50 and 300 nm SPs could be revealed by Raman spectrometry (Figure 2). Taking into account the structural stability of SPs and the fact that the size measurements of SPs by TEM (after drying) and by NTA (in liquid) agreed closely with each other (Nikitin et al., 2013), we propose that no SP distortion occurred during air drying of the specimens for Raman spectroscopy analyses.
Figure 1. Nanoparticle tracking analysis (the size distribution) of SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (52 ± 6 nm) and (b) 5 mg/ml (305 ± 24 nm).
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Figure 2. Raman spectra of TMV-virions (c = 0.1 mg ml 1) (1), SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm) (2), and 5 mg/ml (300 nm) (3) (c = 0.1 mg/ml) dried on air, 532 nm 5.5 mW laser, 40 objective with NA .65, and registration time of 60 s. The spectra presented were averaged for 15–20 registered spectra.
Many viruses (including large phages and helical plant viruses) possess considerable light scattering that is displayed as turbid appearance of suspensions. The presence of turbidity may significantly distort the results of such measurements because of false absorption, caused by light-scattering is added to a sample true absorption. To determine this contribution, the extrapolation method was devised. The main point of this method amounts to measuring UV absorption in the interval in which measured components possess no absorption, and therefore all measured values correspond to turbidity determined by light-scattering. We used the computerized extrapolation version described in Ksenofontov et al. (2006). The UV absorption spectra of native TMV (Figure 3 (a)) and of small (50 nm) TMV SPs samples (Figure 3 (b)) demonstrated that, despite significant light scattering, these particles had normal protein spectra and their concentrations determined from the true absorption values at 280 nm coincided with those measured by other methods. However, the absorption spectra of large (300 nm) SPs yielded negligible true absorption values (Figure 3(c)). This phenomenon was unexpected and has never occurred in our experience with the extrapolation procedure (Tikchonenko, Dobrov, Velikodvorskaya, & Kisseleva, 1966). Further work is required to elucidate this effect. To our knowledge, the first measurement of far UV CD spectrum of intact TMV virions was done by our group (Arutyunyan, Rafikova, Drachev, & Dobrov, 2001), whereas isolated TMV CP preparation was one of the first proteins (Schubert & Krafczyk, 1969), for which far UV CD spectrum was measured by CD spectrum of intact TMV measured only up to 200 nm was typical for β-structured proteins (Arutyunyan et al., 2001). Based on
the X-ray fiber diffraction data, Namba et al. reported that intravirus TMV CP contains 45 to 50% of α-helices and no more than 5% of β-structure (Namba et al., 1989). Therefore, we suggested that this type of TMV far UV CD spectrum may be due to some kind of strong intersubunit interactions (Arutyunyan et al., 2001). As an alternative, the β-type spectrum of TMV may be determined by some artifacts related to the particulate nature of virus suspensions (Homer & Goodman, 1975). Figure 4 presents far UV CD spectra of intact TMV and of the 50 nm SPs measured up to 188 nm using a new CD spectrometer Chiroscan. It can be seen that TMV virions had a highly reproducible spectrum with a very strong positive maximum ([h197] = 43,000 grad). The quantitation of the α-helix content from this peak is not yet possible, but its height and position undoubtedly correspond to an α-helix-rich protein. Furthermore, this suggests that the negative peak at longer wavelengths can hardly be an artifact. The spectrum of TMV 50 nm SPs (Figure 4) shows that the negative peak region practically coincides with the virion spectrum, but its positive peak at 194 nm has almost four times lower intensity than the virion peak. The difference spectrum in Figure 4 testifies to a complete (or almost complete) loss of α-helical structure in the SPs, which agrees with the Raman spectroscopy data in Figure 2. The shape of the virion spectrum suggests the absence of a significant proportion of unordered structure, but a strong negative peak at 198 nm in the difference spectrum suggests the appearance of a large fraction of unordered structure (including possible β-sheets) in SPs. It is noteworthy that the 300 nm SPs gave zero CD signal in the 185–250 nm region (data not shown). It is hard to explain the precise role, if any, of structural similarity between the negative region spectra for the SPs and TMV virions in Figure 4. We used the popular program K2D2 to predict the secondary structure in SPs based on their 188–250 nm CD spectra. The program predicted that CP subunits in SPs contain 14% of α-helices, 32% of β-structure, and 54% of unordered sequences. This agrees well with the Raman spectroscopy results indicating that conformational changes occurred upon TMV-SP transition. Next, we compared the intrinsic fluorescence spectra of SPs and TMV virions (Figure 5). TMV CP intrinsic fluorescence spectrum (Guttenplan & Calvin, 1973) represents a classical case of protein with a stable tertiary structure: high fluorescence intensity and location of aromatic amino acid residues (3 W and 4 Y) in fully hydrophobic environment (Figure 5, curve 1). In the cases of 50 and 300 nm SPs, the fluorescence intensity remained practically unchanged as compared to the virions but the maximum position shifted substantially (from 326 to 338–340 nm); thus, indicating the transition of Trp and Tyr residues to a much more
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Structural changes in viral CP upon TMV to SP transition
Figure 3. UV absorption spectrum of SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (b) and 5 mg/ml (c), control – native TMV virions (a). The spectra were measured in 1 mM phosphate buffer pH 7.5.
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Figure 4. Far UV CD spectra of native TMV, SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm), and their subtractive spectrum. The spectra were measured at 25°C in 1 mM phosphate buffer pH 7.5.
Figure 5. Intrinsic fluorescence spectra of native TMV as well as SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm) and 5 mg/ml (300 nm). The spectra were recorded at 25°C in 1 mM phosphate buffer pH 7.5.
hydrophilic environment. These results suggest that TMV-to-SPs transition is accompanied by changes in the nature of amino acid residues exposed on the particles’ surface. The fluorescence data agree with the Raman and CD spectroscopy (Figures 2 and 4) and further confirm that SPs possess a highly stable structure (as followed from their properties described above). However, this stability
is determined by the interactions between β-sheets rather than between α-helices (as in TMV virions). Diaz-Avalos and Caspar (2000) studied the structure of the “stackdisc,” water-soluble intermediate aggregates of native TMV CP assembled in vivo and in vitro (reviewed by Butler, 1999; Klug, 1999). The aggregation into the stack-discs is accompanied by the conformational changes of TMV CP. Diaz-Avalos and Caspar (2000)
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Structural changes in viral CP upon TMV to SP transition
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Figure 6. Thioflavin fluorescence spectra of TMV virions as well as SPs generated by thermal denaturation of TMV with an initial concentration of 0.1 mg/ml (50 nm) and 5 mg/ml (300 nm). Spectra were recorded at 25°C in 10 mM phosphate buffer pH 7.5. The molar ratio of TMV: ThT was 5:1.
noted that TMV protein subunits in the stack-discs contained structural elements “characteristic of the amyloid fibers.” Finally, recent studies demonstrated cross β-structure in aggregates of Aβ peptide, prion, and β-lactalbumin (El Moustaine, Perrier, Smeller, Lange, & Torrent, 2009; Krebs, Devlin, & Donald, 2009; Sureshbabu, Kirubagaran, & Jayakumar, 2009). To further elucidate the possibility of cross β-structure in SPs, we applied the thioflavin T fluorescence test widely used in recent years to assess the presence of amyloid-like structures (LeVine III, 1995; Nilsson, 2004). Figure 6 demonstrates the results of this test for TMV virions and SPs of two sizes (50 and 300 nm). In the case of the virions (20 μg/ml, 1:5 protein:ThyoT molar ratio), only a small peak at 490 nm was observed, which may be an artifact or a real effect of β-structure formation in the terminal CP subunits. However, a very strong fluorescence peak at 490 nm was observed in the
SP spectra. The shape of this peak was almost identical for both of the 50 and 300 nm SPs. This fact and the fast drop in fluorescence intensity on both sides of the peak testify that light scattering plays no role in the observed effect. The presence of cross β-structure in SPs may explain their high stability. Out data indicate that in the course of SPs formation the CP subunits change their secondary structure from mostly α-helical to largely β –structural one. However, we cannot say at the moment in which segments of the protein sequence the changes occur during TMV-SP transformation. Collectively, our data are summarized in Table 1. In the subsequent experiments the effect of SPs in different animal amyloidosis development systems and analysis of amino acid residues of TMV CP subunit involved in the formation of cross β-structure in SPs that will be examined.
Table 1. Comparative structural characteristics of native TMV and SPs. (⁄) according to Namba et al. (1989). Structural parameters
TMV virions
SPs (50 nm)
SPs (300 nm)
Density (g/cm3) Conformational sensitive Raman peaks (cm 1)
1.31 1656 (α); 935 (α)
Secondary structure of CP subunits from CD spectra Intrinsic fluorescence maximum position (nm) and intensity (%)
α – 50%, β – 5%, unordered – 45%⁄ 326 (100%)
– 1668 (β); 1235 (β), no peak at 935 α – 14%, β – 32%, unordered – 54% 338 (105%)
1.43 1668 (β); 1235 (β), no peak at 935 – 340 (111%)
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Abbreviations TMV CP SPs TEM NTA
tobacco mosaic virus coat protein spherical particles transmission electron microscopy nanoparticle tracking analysis
Acknowledgments This work was supported in part by the Ministry of Education and Sciences of Russia (agreement no. 8564) and the Russian Foundation for Basic Research (Grant No. 12-04-01472-a) and M.V. Lomonosov Moscow State University Program of Development.
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