J. Phys. Chem. B 2007, 111, 8619-8625
8619
Studies on the Conformation of a Polyelectrolyte in Solution: Local Conformation of Cucumber Green Mottle Mosaic Virus RNA Compared with Tobacco Mosaic Virus RNA† Yoshio Muroga,*,¶ Yoh Sano,‡ Hiroyuki Tagawa,§ and Shigeru Shimizu§ Graduate School of Engineering, Nagoya UniVersity, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan, Faculty of Pharmaceutical Sciences, Setsunan UniVersity, 45-1 Nagaotouge-machi, Hirakata, Osaka 573-0101, Japan, and College of Science and Technology, Nihon UniVersity, 1-8-14 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-8308, Japan ReceiVed: December 27, 2006; In Final Form: March 21, 2007
The thermal stability of the local structures of cucumber green mottle mosaic virus RNA, CGMMV-RNA, and tobacco mosaic virus RNA, TMV-RNA, was studied by circular dichroism (CD) and small-angle X-ray scattering (SAXS) and compared with each other in the temperature domain from 20 to 50 °C. The temperature dependence of the molar ellipticity and mean-square radius of the cross section of a chain shows that the structure of CGMMV-RNA is more vulnerable than that of TMV-RNA. Such a different thermal stability of their structures was also reflected in the temperature dependence of the length and number of the constituent rods when the structures of the two RNA chains were represented by a model which consisted of rods joined with freely hinged joints. From these results, a possibility was suggested that the structural stability of CGMMVRNA and TMV-RNA might be correlated with the infectivity of the corresponding virus, CGMMV and TMV, respectively.
1. Introduction Cucumber green mottle mosaic virus (CGMMV) is found in crops such as cucumber, watermelon, and melon. It is a rodshaped virus, 300 nm in length and 18 nm in diameter, and is almost identical in size, shape, and its chemical and immunological characteristics to tobacco mosaic virus (TMV). Both viruses consist1-3of a single-stranded RNA chain, CGMMV- and TMV-RNA, respectively, and coat proteins surrounding the RNA chain. The structure is constructed in a similar way by interaction of coat proteins with the RNA chain via a nucleation process followed by an elongation process, where the presence of a specified segment of a hairpin-loop form in the RNA chain is indispensable for the nucleation process to start. Despite such a remarkable resemblance between their structures and between the schemes of the constructions of the two viruses, they have fairly different biological activity. When the two constituents of the virus, proteins and RNA, are cultivated in a greenhouse at the temperature above 30 °C, the yield of TMV is much better than CGMMV, and moreover, the former is known to have more intensive infectivity than the latter. The question still remains open why the viruses have such different infectivity. The biological infectivity process involves attachment of an intact virus to the leaf, penetration of the virus into the cell, survival of the virus on a time scale long enough to shed its protein coat, avoidance of the cell’s defenses, and so on. Therefore, it would be certain that the intact structure of the virus is needed, at least, in the initial stage of infection. † Part of the special issue “International Symposium on Polyelectrolytes (2006)”. * To whom correspondence should be addressed. E-mail: ymuroga@ apchem.nagoya-u.ac.jp. ¶ Nagoya University. ‡ Setsunan University § Nihon University.
Population of intact viruses might depend on the structural stability of the free RNA chain if it is assumed that there is equilibrium in the cellular environment between the whole virus (RNA in its protein coat) and free RNA, although no evidence for existence of such an equilibrium in vivo has been presented. That is, if the structure of free RNA, especially the structure of the hairpin-loop segments, is destroyed by raising the temperature, the equilibrium will be driven away from the intact virus, and consequently, the infectivity will be reduced. A more fragile structure of CGMMV-RNA, compared with that of TMV-RNA, is suggested from the data that shows that the thermodynamic stability4 of the former, -32.6, is fairly lower than that of TMVRNA, -30.3 kcal mol-1, where the former contains significantly less adenine base and more urasil base than TMV-RNA.5 In our previous work,6 the effect of temperature on the local structure of TMV-RNA was shown to be negligibly small. The present work is devoted to the studies on the temperature dependence of the helix content of CGMMV-RNA and its local structure by circular dichroism (CD) and small-angle X-ray scattering (SAXS) in order to see whether there is a noticeable difference between the thermal stabilities of CGMMV-RNA and TMV-RNA. The scattering function, which is needed in the analysis of SAXS data, is analytically derived by extending the Muroga treatment.7-12 2. Experimental Section 2.1. Materials and Sample Preparation. Cucumber green mottle mosaic virus (watermelon strain) particles were purified from infected Cucumis satiVus L. Var. Hatsukari. The RNA was obtained13,14 by extracting from the virus with an aqueous phenol solution and bentonite and precipitating by adding 2 vol of cold ethanol to the extracted solution, followed by washing the precipitated RNA with ethanol three times. Special attention was paid to the careful sterilization of all solute and solvents in order to avoid degradation of RNA by RNAse.
10.1021/jp068944j CCC: $37.00 © 2007 American Chemical Society Published on Web 05/04/2007
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In general, the RNA chain in an aqueous solution tends to aggregate due to its high molecular weight, and therefore, filtration with a filter of pore size 0.45 µm (Millipore filter) was carried out15,16 right before CD or SAXS measurements so that the solution thus filtered would involve monomer RNA only. Then, all sample solutions were further dialyzed against 0.1 mol L-1 phosphate buffer (ionic strength S ) 0.2) at 4 °C. 2.2. CD Measurements. Circular dichroism (CD) spectra were recorded for TMV-RNA and CGMMV-RNA in 0.1 mol L-1 phosphate buffer solution (S ) 0.2) on a JASCO J-600 and J-700 apparatus with a 0.1 cm optical cell, where the concentrations of the solute, Cp, were adjusted to 0.032 and 0.127 mg mL-1, respectively, by using an absorptivity value of 20.0 mL cm-1 mg-1 at 258 nm. The molar ellipticity [θ] in deg cm2 mol-1 was computed from the CD data using the height of the peak at 261 nm and the average molecular weight of the nucleotide, 322 au. 2.3. Small-Angle X-ray Scattering Measurements. SAXS experiments were carried out using synchrotron orbital radiation as an X-ray source with the optics and detector system of SAXSES (small-angle X-ray scattering equipment for solution samples) in the Photon Factory of the High Energy Accelerator Organization at Tsukuba, Ibaragi, Japan. The scattered intensity I(q) was registered by a position-sensitive proportional counter (PSPC) with 512 channels over a scattered vector q ranging from 0.005 to 0.25 Å-1, where q is defined by (4π/λ)sin(θ/2), λ is the wavelength of the X-ray, and θ is a scattering angle. Since the size of the X-ray beam at the sample position was small enough compared with the camera length, the effect of slit length and slit width on the scattered data was neglected. The details of the instrumentation and the procedure are given in our previous papers.17-20 SAXS was measured for CGMMV-RNA in 0.1 mol L-1 phosphate buffer solution (S ) 0.2). Cp, 2.8 mg mL-1, was sufficiently lower6 than the critical concentration Cp*, ca. 20 mg mL-1, above which different chains begin to overlap each other. Therefore, I(q) was regarded to reflect the behavior of an isolated chain and allowed to be multiplied by a constant factor for comparison with the particle scattering function P(θ). 3. Computation of Scattering Function As is well established, the RNA chain forms the structure of a partially double-stranded helical chain1 consisting of unpaired bases and hydrogen-bonded paired bases by pairing some segments with other segments in a single RNA chain. Taking into account such a scheme of the structural construction and the nucleotide sequence of RNA,5,21-26 most of the unpaired bases existing along the partially helical chain would have a comparatively short sequence length and be forced into restricted conformations and hindered molecular motions since they intervene between the adjacent paired bases. Consequently, most unpaired bases have the feature of an inflexible chain and form an inflexible chain domain (Domain I), together with the adjacent paired bases. On the other hand, if unpaired bases happen to occur in a moderate sequence length, they would form flexible chain domains (Domain II) and play a role in the joint to connect sequences of Domain I. Thus, one simple model for the partially double-stranded helix might be given by sequences of Domain I joined with joint of Domain II in a branched way. The computer simulation26 predicts that an expected structure of the RNA chain would contain a maximum of three branches, at most, as the branch points. Use of this model might be justified, as long as each sequence of the unpaired bases is isolated so far apart from its
Figure 1. Structural Model for a partially double-stranded helical structure: (A) Model A for tobacco mosaic virus RNA, TMV-RNA, and (B) Model B for cucumber green mottle mosaic virus RNA, CGMMV-RNA, where the inflexible domain (Domain I) is designated by the solid lines and the flexible domain (Domain II) by the circles.
adjacent sequences along the partially helical chain and thus cannot be combined to form extended random-coiled regions even at a moderate population of unpaired bases. At a greater population of unpaired bases, extended random-coiled regions would appear in the partially double-stranded helical chain. The simple model stated above and its related ones were satisfactorily employed in some studies on the structure of the RNA chain; Osterberg et al.27 have found that SAXS data of Escherichia Coli 5S-RNA are consistently elucidated if the RNA chain takes the structure consisting of one large and two small double-stranded helices arranged in the form of the letter Y and joined with one flexible region. Min et al.,28 Noller et al.,29 Holley et al.,30 and Doty et al.31 proposed a similar model for a part of the structure of the RNA chain. In our previous work,6 the local structure of TMV-RNA was represented by the model, Model A, shown in Figure 1A, where 9 rods (Domain I) designated by solid lines are arranged in the
Local Conformation od CGMMV-RNA Compared with TMV-RNA form of combinations of the letter Y and joined with joints (Domain II) designated by small circles, where rotation about adjacent rods is free, and thus, the rods are joined with freely hinged joints. P(θ) computed for this model has successfully elucidated the SAXS data of TMV-RNA. In the present work, Model A was refined to Model B shown in Figure 1B so that it could be employed at a greater population of unpaired bases, where each rod in Model A was replaced by a sequence of rods alternatively joined with freely hinged joints. The length a of the rods in different sequences as well as the number of rods m are given identical values when the sequences are regarded as equivalent ones with respect to the locations in the molecular structure. That is, Sequence-1, -2, -4, -6, -8, and -9 consist of mx rods of length ax, Sequence-3 and -7 consist of my rods of length ay, and Sequence-5 consists of mz rods of length az. Model B is reduced to Model A if mx, my, and mz are equal to unity. In general, the scattering intensity I(q) is proportional to Rθ, given by the summation over all pairs of scattering points, j and k, in a molecule
Rθ )
∑j ∑k exp[iqb ‚rbjk]
(1)
Rθ (2)
∑j ∑k 1
In Model B, Rθ is given as a sum of Rθ,ss (s ) 1-9) and Rθ,st (s and t ) 1-9), where Rθ,ss means the scattering intensity obtained when two scatterers belong to the same Sequence-s, and Rθ,st means the scattering intensity obtained when one scatterer belongs to Sequence-s and the other to Sequence-t. For a broken rodlike chain where several straight rods are alternatively joined by random-coil chains and its related models, we previously computed7-12 Rθ averaged over all possible conformations of the chain, . By a straightforward application of the Muroga treatment and assuming homogeneous electron density along the rods, and for Model B are given below. (A) for s ) 1, 2, 4, 6, 8, and 9
a2x
{
) mx 2W(Rx) -
( ) ( )} Rx 4 sin2 2 2 Rx
2W2(Rx)
W(Rx) )
() 1 Rx
µx )
+
mx 1-µ 1 - µx (1 - µ )2
∫0R sint t dt
Rx ) ax‚q sin(Rx) Rx
a2x
mx x
}
) W2(Rx)
( ) 1 - µmx x 1 - µx
2
(7)
(C) for s ) 1, t ) 3; s ) 3, t ) 1; s ) 2, t ) 3; s ) 3, t ) 2; s ) 3, t ) 4; s ) 4, t ) 3; s ) 6, t ) 7; s)7, t ) 6; s ) 7, t ) 9; s ) 9, t ) 7; s ) 7, t ) 8; and s ) 8, t ) 7
( )( )
1 - µmx x 1 - µmy y ) W(Rx)W(Ry) axay 1 - µx 1 - µy
(8)
for s ) 3, t ) 5; s ) 5, t ) 3; s ) 5, t ) 7; and s ) 7, t ) 5 is obtained by replacing subscript x by y and subscript y with z in eq 8. for s ) 4, t ) 5; s ) 5, t ) 4; s ) 6, t ) 5; and s ) 5, t ) 6 is obtained by replacing subscript y with z in eq 8. (D) for s ) 1, t ) 4; s ) 4, t ) 1; s ) 2, t ) 4; s ) 4, t ) 2; s ) 6, t ) 9; s ) 9, t ) 6; s ) 6, t ) 8; and s ) 8, t ) 6
( )
) W2(Rx)
1 - µmx x 2 my µ 1 - µx y
(9)
for s ) 3, t ) 7 and s ) 7, t ) 3 is obtained by replacing subscript x by y and subscript y with z in eq 9. for s ) 4, t ) 6 and s ) 6, t ) 4 is obtained by replacing subscript y with z in eq 9. (E) for s ) 1, t ) 5; s ) 5, t ) 1; s ) 2, t ) 5; s ) 5, t ) 2; s ) 9, t ) 5; s ) 5, t ) 9; s ) 8, t ) 5; and s ) 5, t ) 8
( )( )
1 - µmx x 1 - µmz z my ) W(Rx)W(Rz) µ axaz 1 - µx 1 - µz y
(10)
for s ) 3, t ) 6; s ) 6, t ) 3; s ) 4, t ) 7; and s ) 7, t ) 4 is obtained by replacing subscript z with y and subscript y with z in eq 10. (F) for s ) 1, t ) 6; s ) 6, t ) 1; s ) 2, t ) 6; s ) 6, t ) 2; s ) 4, t ) 9; s ) 9, t ) 4; s ) 4, t ) 8; and s ) 8, t ) 4
a2x
{ x
a2x
P(θ) is defined
P(θ) )
J. Phys. Chem. B, Vol. 111, No. 29, 2007 8621
) W2(Rx)
( )
1 - µmx x 2 my mz µ µ 1 - µx y z
(11)
(3)
(G) for s ) 1, t ) 7; s ) 7, t ) 1; s ) 2, t ) 7; s ) 7, t ) 2; s ) 3, t ) 9; s ) 9, t ) 3; s ) 3, t ) 8; and s ) 8, t ) 3
(4)
1 - µmx x 1 - µmy y my mz ) W(Rx)W(Ry) µ µ axay 1 - µx 1 - µy y z
(5) (6)
In the cases of s ) 3 and 7, is obtained by replacing subscript x by y in eq 3 and, in the case of s ) 5, by replacing subscript x with z. (B) for s ) 1, t ) 2; s ) 2, t ) 1; s ) 8, t ) 9; and s ) 9, t ) 8
( )( )
(12)
(H) for s ) 1, t ) 8; s ) 8, t ) 1; s ) 2, t ) 8; s ) 8, t ) 2; s ) 1, t ) 9; s ) 9, t ) 1; s ) 2, t ) 9; and s ) 9, t ) 2
a2x
( )
1 - µmx x 2 2my mz ) W (Rx) µ µz 1 - µx y 2
(13)
Thus, is given by 9
)
9
∑ ∑ i)1 j)1
(14)
8622 J. Phys. Chem. B, Vol. 111, No. 29, 2007
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and P(θ) is given by
P(θ) )
(6mxax + 2myay + mzaz)2
(15)
4. Results and Discussion Figure 2 shows the temperature dependences of the arbitrarily normalized CD (NCD) for TMV-RNA (O) and CGMMVRNA (×), where NCD was derived by normalizing the molar ellipticity [θ] of 5.350 mdeg cm2 mol-1 for TMV-RNA at 15 °C and 2.530 mdeg cm2 mol-1 for CGMMV-RNA at 13.5 °C to unity and [θ] of 1.134 mdeg cm2 mol-1 for the former at 75 °C and 0.646 mdeg cm2 mol-1 for the latter at 76 °C to zero. The dependence clearly shows that NCD for CGMMV-RNA begins to decrease at a lower temperature, around 38 °C, compared with the corresponding temperature for TMV-RNA, around 50 °C. That is, it is shown that the local structure of CGMMV-RNA is more fragile than that of TMV-RNA and tends to be more easily modified or destroyed as the temperature is raised. As was reviewed by Porod,32 I(q) for a semiflexible chain is given as the product of the axial factor Ithin(q) and the crosssectional factor Ics(q)
I(q) ) Ithin(q)Ics(q) 1 Ics(q) ∝ exp - q2 2
(16)
(
)
(17)
where is the mean square radius of a cross section of a chain. For a rodlike molecule, Ithin(q) is expressed by
Ithin(q) ∝
1 q
(18)
In the scattering vector region of q2 < 1, the local structure of a semiflexible chain is regarded as a rod, and Ithin(q) is still expressed by eq 18. In that q2 range, therefore, when the scattering data of a semiflexible chain are plotted in the form of ln(I(q)q) versus q2, it forms a straight line with a slope of -1/2. Once is evaluated, Ithin(q) for the chain is given by I(q)exp{1/2q2} over a whole q range. Figure 3 shows the plot of ln(I(q)q) versus q2 for CGMMVRNA in a 0.1 mol L-1 phosphate buffer solution (S ) 0.2) at (1) 20 (O); (2) 30 (4); (3) 40 (+); and (4) 50 °C (×), where the data were shifted by a magnitude along the ordinate so that each data set could be easily discriminated. For the data sets 1 and 2, the observed points form a straight line in a wide range of q2, as is shown by the dotted lines, and the slope gives 1/2 values of 8.6 and 7.8 Å, respectively. It is noted that an arrow on each curve designates the value of q*2, where q*2 ) 1. It is seen that the linear relation between ln(I(q)q) and q2 holds well sufficiently far down from q*2 to obtain a value of . For the data sets 3 and 4, however, the data points upward deviate from the assumed dotted line in a range of q2 < 0.022 Å-2. Therefore, for the data sets 3 and 4 was evaluated by successive approximations of the assumed values in the comparison of the experimental data with P(θ) for Model B, as is discussed below. As a result, 1/2 ) 6.6 and 5.5 Å were obtained for the data sets 3 and 4, respectively. The solid curves in Figure 3 are drawn with P(θ) of Model B with 1/2 thus estimated.
Figure 2. Temperature dependence of the arbitrarily normalized CD (NCD) for TMV-RNA (O) and CGMMV-RNA (×).
The root-mean-square radius of a cross section of a chain, 1/2, for CGMMV-RNA thus estimated is listed in Table 1, together with our previous result6 for TMV-RNA. 1/2 for both RNA chains at 20 and 30 °C, ranging from 7.6 to 8.6 Å, are comparable to those for the Ehrlich ascites tumor cell H-RNA, 8.0 Å.33,34 It is seen that 1/2 for CGMMV-RNA tends to decrease from 8.6 to 5.5 Å and 1/2 for TMVRNA from 8.0 to 6.0 Å as the temperature is raised from 20 to 50 °C. It has been shown in our previous paper35 that, when a polymer chain consists of helical rods and random-coil chains, is an average of the value at each state, helix and coil, respectively
) ghelix + (1 - g)coil
(19)
where g is the helix content. Taking into consideration the result in Table 1 and the relation of eq 19 and the expectation that 1/2 of a free RNA chain in the random-coiled state should be much smaller than at double-stranded helical state, it is shown that the local structure of CGMMV-RNA is more fragile than that of TMV-RNA and tends to be more easily modified or destroyed as the temperature is raised. This result is very compatible with the temperature dependence of NCD in Figure 2. Figure 4A shows the Kratky plot, Ithin(q)q2 versus q for CGMMV-RNA, where the symbols have the same meanings as those in Figure 3 and the data were shifted by a magnitude along the ordinate so that each data set could be easily discriminated. In Figure 4B, our previous data6 of TMV-RNA in a 0.1 mol L-1 phosphate buffer solution is reproduced (S )
Local Conformation od CGMMV-RNA Compared with TMV-RNA
J. Phys. Chem. B, Vol. 111, No. 29, 2007 8623
Figure 3. Plot of ln(I(q)q) versus q2 for CGMMV-RNA in a 0.1 mol L-1 phosphate buffer solution (S ) 0.2) at (1) 20 (O); (2) 30 (4); (3) 40 (+); and (4) 50 °C (×). An arrow on each curve designates the value of q*2, where q*2 ) 1. The solid curves are theoretical ones computed for Model B using the molecular parameters listed in Table 1. The dotted straight lines are drawn through the data points in a sufficiently high q2 region.
TABLE 1: Root-Mean-Square Radius of Cross Section 1/2 and Molecular Parameters for Model B CGMMV-RNA
TMV-RNA
temp (°C)
1/2 (Å)
ax (Å)
mx
ay (Å)
my
az (Å)
mz
1/2 (Å)
ax (Å)
mx
ay (Å)
my
az (Å)
mz
20 30 40 50
8.6 7.8 6.6 5.5
140 100 15 7
1 1 5 25
140 120 70 50
1 1 2 3
140 140 140 140
1 1 1 1
8.0 7.6 7.4 6.0
100 100 100 100
1 1 1 1
120 120 120 120
1 1 1 1
140 140 140 140
1 1 1 1
0.2) at (1) 20 (O); (2) 30 (4); (3) 40 (+); and (4) 50 °C (×). It is clearly seen that the profile of CGMMV-RNA significantly depends on the temperature, while the profile of TMV-RNA is almost independent of the temperature. In fact, data sets 1-4 in Figure 4 can be overlapped by multiplying the scattering intensity by a suitable constant factor, as is shown by data set 0 in the figure. In Figure 4A, the Kratky plot of CGMMV-RNA is compared with the scattering function P(θ) of Model B, eq 15, to find out the theoretical curve which mimics the observed curve the most satisfactorily. Generally speaking, the least-square method is applied to the observed data over an entire q range. However, Model B represents the local structure of the RNA chain, and therefore, its P(θ) should be employed to analyze the scattering data in a relevant q range. Taking into account this respect, a suitable theoretical curve (solid curve) was visually decided with trial-and-error so that it could satisfactorily mimic the scattering data in a range of ca. 0.008 Å-1 < q < ca. 0.112 Å-1. The theoretical curve thus obtained also well reproduced all of the observed curves plotted in the form of ln(I(q)q) versus q2, as is shown by the solid curves in Figure 3. The molecular parameters thus obtained are listed in Table 1. The values of the rod lengths at 20 °C, ax ) 140, ay ) 140, and az ) 140 Å, are comparable to the length33,34 for the Ehrlich ascites tumor cell H-RNA in the medium of pH 7 and an ionic strength of 0.15, 100 Å. As the temperature is raised, the rod lengths ax and ay are shortened, and the rod numbers mx and my are simultaneously increased, showing that the
partially double-stranded helical structure for CGMMV-RNA would be gradually frayed as the temperature is raised from 20 to 50 °C. On the other hand, as is shown in the data set 0 in Figure 4B, all Kratky plots of TMV-RNA can be well overlapped to each other into a single curve, showing that the partially doublestranded helical structure for TMV-RNA would be substantially preserved as stable over the temperature ranging from 20 to 50 °C. That is, as is shown by solid curves in Figures 4B 1-4, all of the observed scattering curves could be well reproduced by a theoretical curve whose molecular parameters are ax ) 100 Å and mx ) 1, ay ) 120 Å and my ) 1, and az ) 140 Å and mz ) 1. Here, a possibility is not excluded that there might be a slight variation in the length or number of constituent rods if more elaborate scattering data is obtained and more detailed analysis is carried out with the data. Such temperature insensitivity of the local structure for TMVRNA apparently seems to contradict the CD data (Figure 2) and the mean-square radius of the chain cross section (Table 1) showing the partial collapse of the local structure. However, the result would be obtained if the sequence length of the unpaired bases in TMV-RNA would be sufficiently short, and they exist so far apart from their adjacent unpaired bases along the partially helical chain that they cannot be combined to form extended random-coiled regions. In this case, the feature of the local structure or the chain flexibility is preserved over a moderate range of the population of the unpaired bases.
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Muroga et al. temperature dependence of the length and number of constituent rods in Model B. The local structure of CGMMV-RNA is more vulnerable and tends to be more easily modified or destroyed at an elevated temperature, compared with TMV-RNA. Accordingly, the nucleation reaction to trigger the construction of CGMMV is more likely to be retarded or inhibited, especially at an elevated temperature, and consequently, the infectivity of CGMMV would be more reduced than that of TMV. From these results, a possibility is suggested that the structural stability of CGMMV-RNA and TMV-RNA might be correlated with the infectivity of the corresponding virus, CGMMV and TMV, respectively. 5. Conclusions The thermal stabilities of the local structures of cucumber green mottle mosaic virus RNA, CGMMV-RNA, and tobacco mosaic virus RNA, TMV-RNA, were compared with each other in the temperature domain from 20 to 50 °C. The temperature dependence of the helix content and mean-square radius of the cross section shows that the local structure of CGMMV-RNA is more vulnerable than that of TMV-RNA. Such a different thermal stability of their local structures more clearly manifests itself in the temperature dependence of the length and number of the constituent rods when the structures of the two RNA chains are represented by a model which consists of rods joined with freely hinged joints. From these results, a possibility is suggested that the structural stability of CGMMV-RNA and TMV-RNA might be correlated with the infectivity of the corresponding virus, CGMMV and TMV, respectively. Acknowledgment. We are indebted to Dr. H. Inoue, Dr. Y. Hiragi, and Assistant Professor S. Ichikawa of the University of Tsukuba for valuable discussions and helping us with the SAXS experiments. References and Notes
Figure 4. Comparison between the Kratky plot of (A) CGMMV-RNA in a 0.1 mol L-1 phosphate buffer solution (S ) 0.2) and (B) TMVRNA in a 0.1 mol L-1 phosphate buffer solution (S ) 0.2), which were recorded at (1) 20 (O); (2) 30 (4); (3) 40 (+); and (4) 50 °C (×), and the theoretical scattering function for Model B, where the data of TMVRNA is reproduced from the extant data.6 The solid curves are theoretical ones computed for Model B using the molecular parameters listed in Table 1. Data set 0 in Figure 4B shows data sets 1-4 overlapped by multiplying the scattering intensity by a suitable constant factor.
Thus, the different thermal stability of the local structures of the two RNA chains more clearly manifests itself in the
(1) Butler, P. J. G. J. Gen. Virol. 1984, 65, 253. (2) Okada, Y. In The Plant Viruses; Van Regenmartel, M. H. V., Fraenkel-Conrat, H., Eds.; Plenum Publishing Co.:New York, 1988; Vol. 2, pp 267-281. (3) Meshi, T.; Kiyama, R.; Ohno, T.; Okada, Y. Virology 1983, 127, 54. (4) Tinoco, J. I.; Borer, P. N.; Dengler, B.; Levine, M. D.; Uhlenbeck, O. C.; Crothers, D. M.; Gralla, S. Nature (London), New Biol. 1973, 246, 40. (5) Kurisu, M.; Ohno, T.; Okada, Y.; Nozu, Y. Virology 1976, 70, 214. (6) Muroga, Y.; Sano, Y.; Inoue, H.; Suzuki, K.; Miyata, T.; Hiyoshi, T.; Yokota, K.; Watanabe, Y.; Liu, X.; Ichikawa, S.; Tagawa, H.; Hiragi, Y. Biophys. Chem. 1999, 83, 197. (7) Muroga, Y. Macromolecules 1988, 21, 2751. (8) Muroga, Y. Macromolecules 1992, 25, 3385. (9) Muroga, Y. Macromolecules 1992, 25, 6063. (10) Muroga, Y. Macromolecules 1994, 27, 2951. (11) Muroga, Y.; Muraki, T.; Noda, I.; Tagawa, H.; Holtzer, A.; Holtzer, M. E. J. Am. Chem. Soc. 1995, 117, 5622. (12) Muroga, Y. Biopolymers 2001, 59, 320. (13) Sano, Y. Carbohydr. Polym. 1997, 33, 125. (14) Sano, Y. Carbohydr. Polym. 1999, 38, 183. (15) Sano, Y. J. Colloid Interface Sci. 1990, 139, 14. (16) Sano, Y. Biopolymers 1993, 33, 69. (17) Hiragi, Y.; Inoue, H.; Sano, Y.; Kajiwara, K.; Ueki, T.; Kataoka, M.; Tagawa, H.; Izumi, Y.; Muroga, Y.; Amemiya, Y. J. Mol. Biol. 1988, 204, 129. (18) Hiragi, Y.; Inoue, H.; Sano, Y.; Kajiwara, K.; Ueki, T.; Nakatani, H. J. Mol. Biol. 1990, 213, 495. (19) Sano, Y.; Inoue, H.; Kajiwara, K.; Urakawa, H.; Hiragi, Y. J. Biochem. 1994, 115, 1058. (20) Sano, Y.; Inoue, H.; Kajiwara, K.; Hiragi, Y.; Isoda, S. J. Protein Chem. 1997, 16, 151. (21) Beachy, R. N.; Zaitlin, M. Virology 1977, 81, 160. (22) Goelet, P.; Karn, J. Gene 1984, 29, 331.
Local Conformation od CGMMV-RNA Compared with TMV-RNA (23) Goelet, P.; Iomonossoff, G. P.; Butler, P. J. G.; Akam, M. E.; Gait, M. J.; Karn, J. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 5818. (24) Meshi, T.; Ohno, T.; Okada, Y. Nucleic Acids Res. 1982, 10, 6111. (25) Zimmern, D. Nucleic Acids Res. 1975, 2, 1189. (26) Wilson, T. M. A.; Plaskitt, K. A.; Watts, J. W.; Osbourn, J. K.; Watkins, P. A. C. NATO ASI Ser., Ser. H 1990, 41, 123. (27) Osterburg, R.; Sjoberg, B.; Garrett, R. Eur. J. Biochem. 1976, 68, 481. (28) Min, J. W.; Haegeman, G.; Ysebaert, M.; Fiers, W. Nature 1972, 237, 82. (29) Noller, H. F.; Woese, C. R. Science 1981, 212, 403.
J. Phys. Chem. B, Vol. 111, No. 29, 2007 8625
(30) Holley, R. W.; Apgar, J.; Everett, G. A.; Madison, J. T.; Marquisee, M.; Merrill, S. H.; Penswick, J. R.; Zamir, A. Science 1965, 147, 1462. (31) Jacques, R.; Fresco, R.; Alberts, B. M.; Doty, P. Nature 1960, 188, 98. (32) Porod, G. In Small Angle X-ray Scattering; Glatter, O., Kratky, O., Eds.; Academic Press: New York, 1982; pp 17-51. (33) Timasheff, S. N.; Luzzatti, V. Biochem. J. 1960, 76, 57P. (34) Timasheff, S. N.; Witz, J.; Luzzatti, V. Biophys. J. 1961, 1, 525. (35) Muroga, Y.; Tagawa, H.; Hiragi, Y.; Ueki, T.; Kataoka, M.; Izumi, Y.; Amemiya, Y. Macromolecules 1988, 21, 2756.
