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Orientation of the RNA in tobacco mosaic virus
J. Mol. Biol. (1960) 22, 166-172
Orientation of the RNA in Tobacco Mosaic Virus EUGENE M. SUHAOETER-~, IRWIN J. BENDET AND MAX A. LAUFRER
Department of Biophysics, University of Pittsburgh, Pithburgh, Pennsylvania 15213, U.&A. Received 6 December 1965, and in revised form
30 August 1966)
The configuration of the ribonucleio acid within tobacco mosaic virus was studied by comparing the ultraviolet diahroism of flow-oriented virus and repolymerized, nucleic acid free tobacco mosaic virus protein. The dichroism of the RNA within tobacco mosaio virus, obtained by subtracting the protein contribution from the dichroic spectra of the virus, has been interpreted as being due to a preferred orientation of the planes of the purine and pyrimidine bases of the RNA “approximately” parallel to the longitudinal axis of the virus. The dichroism of the protein suggests a preferred orientation of the aromatic ammo acids, especially tryptophan, “approximately” parallel to the longitudinal axis. The dichroio ratio of tobacco mosaic virus was found to be influenced by the asymmetry of the particles in the flowing solution and by the geometry of the flow cell. With linearly aggregated particles at pH 5.15 and an optical flow cell of square cross-section, the dichroic ratios for tobacco mosaic virus in this study were appreciably higher than those previously obtained by others.
1. Introduction The structure of tobacco mosaic virus has been studied by a variety of physical chemical methods. The present understanding of the structural properties of the virns, its size, chemical composition and the helioal symmetry of its protein subunits and RNA strand have been recently reviewed (Anderer, 1963; Caspar, 1963; FraenkelConrat & Ramechandran, 1969; Klug & Caspar, 1960). Information concerning the configuration of the RNA strand within the virus has come principally from optical properties of the virus. According to X-ray analysis of oriented gels of TMVS and repolymerized protein, free of nucleic acid, the RNA strand follows the same path as the protein subunits, there being three nucleotides per protein subunit, or 49 nucleotides per helical turn. The radial electron density of the RNA, particularly of the phosphate groups, is located at a radius of about 40 A from the long axis of the virus, and is buried between turns of the protein which extends between radii of 20 A and 90 A. It has been suggested that the results of polarized light studies offer information pertaining to the configuration of the RNA bases within the native virus. Franklin (1955) interpreted the higher positive birefringence of oriented gels of TMV compared to those of gels of protein as due to preferential orientation of the planes of the bases parallel to the long axis. Since, however, biretingence measures an optioal t Present address: Department of Biology, Massachusetts Institute Mesa., U.S.A. $ Abbreviation used: TMV, tobacco mosc& virus. 166
of Technology, Gembride,
166
E.
M.
SCHACHTER,
I.
J.
BENDET
AND
M.
A.
LAUFFER
property of the entire specimen and is not specific for any particular oriented chemical grouping, the difference in birefringence, while it may be suggestive of orientation of the bases, could conceivably be due to the sugar or phosphate components of the RNA. Ultraviolet dichroism (e.g. see Beaven, Holiday & Johnson, 1956) of oriented specimens of TM7 has been studied by three groups of workers (Seeds & Wilkins, 1950; Perutz, Jope & Barer, 1950; Butenandt, Friedrich-Freksa, Hartwig & Soheibe, 1942) with very similar results. It was found that the dichroism was positive between 240 and 296 mp. All three groups concurred that the dichroism at 290 rnp was due to preferred orientation of tryptophan residues, but only the earliest group of workers suggested that the diohroism in the neighbourhood of 260 rnp was due to a preferred orientation of the nucleic acid. The two later groups stated a reluctance to assign the dichroism at lower wttvelengths to RNA orientation. Although dichroism is the expression of the orientation of certain light-absorbing chemical groupings, the spectral complexity due to the overlap of the absorption spectra of the nucleic acid bases and aromatic amino acids, as well as anisotropic light scattering, preclude the assignment of the dichroism at a particular wavelength to a specific chemical group. It was therefore decided to attempt to resolve the dichroism of the RNA from that of the whole virus by performing parallel experiments on the intact virus and on repolymerized protein free of RNA.
2. Materials
and Methods
(a) Preparation of virus and protein TMV was purified from infected Nicotiana tabacum by 3 or 4 cycles of differential centrifugation and EDTA depigmentation (Ginoza, Atkinson & Wildman, 1964). TMV protein, free of RNA, was prepared by both the alkali degradation-electrophoresis method (Schramm & Zillig, 1966). and the acetic acid method (Fmenkel-Conrat, 1967). For dichroism experiments, TMV solutions in water were olartied by centrifugation at 3000 g for 20 mm. The top third of the solution was removed with a dust-free pipette, diluted and dialyzed for 12 hr against two changes of potassium phosphate buffer (0.1 ionic strength, pH 5.16). Solutions of low molecular weight protein in water were clarified by filtration through a Millipore HA filter, diluted and dialyzed as were the virus solutions. The solvents for all sedimentation velocity experiments were O-1 ionic strength potassium phosphate buffers of varying pH. A Spinco model E ane;lytical ultracentrifuge equipped with schlieren optics was used for sedimentation velocity experiments. All runs were performed in 2’ aluminium centerpieces at 20°C. All reported sedimentation coefficients are uncorreoted and refer to the experimental conditions. For boundaries which were highly skewed toward the fastermoving species, the sedimentation coefficient refers to the maximum refractive index gradient of the distribution. (c) Ekctroon W&iC?-08COfJy Virus and protein were deposited from the same solutions 8s those used in dichroism studies, employing a method of adsorption and rapid removal of the high salt-concentration solvent. A drop of solution was placed on a Parlodion-covered nickel grid which wes then placed, solution side down, on a piece of filter paper and quiakly withdmwn. The hydrophobic Parlodion f?hn appeared to be dry after removal from the paper. The speoimen was then shadowed with uranium and examined in an RCA EMU-3F electron microscope. No salt crystals were evident in the electron micrographs. The magnification was determined by using a calibrated carbon replica of a diffraction grating irmnedietely after the virus and protein specimens were examined.
ORIENTATION
OF THE
RNA
IN TMV
167
(d) Comntra&on lneasuremen& Concentrations were determined either refiaotometrically by using a specific refractive increment of 1.86 x 10s3 (100 ml./g) for both the virus and the protein (Stevens, 1962), or spectrophotometrically from the absorption contribution to optical density by using absorptivities of 27 (g-cm/lOOml.)-l for TMV at 260mp and 13 (g-cm/lOOml.)-l for protein at 280 rnp (Fraenkel-Conrat t Williams, 1966): (e) Dichroiem apparatw A Cary 14M recording spectrophotometer was adapted for the dichroism experiments. No permanent modifications of the spectrophotometer were made; the optical corn. ponents for dichroism experiments, the polarizing prism and the optical flow cell being constructed to be accommodated in a specially constructed alignment assembly in the sample compartment. The light was polarized by a 20.mm aperture type A Clan Iu-ism (Karl Lambrecht Crystal Optics, Chicago, Ill.) placed immediately in front of the flow cell. The optical flow cell, constructed of fused silica (Lux Scientific Instrument Co., New York, N.Y.), has an optical flow channel of O-100 cm to a side square cross-section and 4.2 cm length. A light stop in front of the flow cell restricted the polarized beam to a width of slightly less than 0.100 cm. Quartz tubing is fused to the ends of the flow channel for connection to the flow-regulating system. The reference compartment of the spectra. photometer contained a standard 0.100 cm path length cuvette and a neutral density filter, which partially compensated for the optioal density of the prism. The flow-regulating system maintained a circulating flow of solution through the flow cell by forcing the solution from a closed reservoir under a variable pressure through the flow cell to a reservoir open to the atmosphere. A variable-speed pump returned the solution from the open reservoir to the closed one at a rate equal to the outflow rate. Initial exploratory experiments were also carried out in a thin rectangular cell after the design of Cavalieri, Rosenberg & Rosoff (1966) , which had a flow channel with approxi. mately the dimensions: length 4 cm, width 1 cm and depth (optical path) 0.01 cm. (f) Dichroiem wwaaurenzents (i) Conventione The spectrophotometric conventions employed are those suggested by Wetlaufer (1962), with the additional need of defining E = log (1,/I) = optical density, and A = E - T, where T is the turbidity. The turbidity, in an absorbing region of the spectrum, was calculated by extrapolating from the absorption-free region of 400 to 320 rnp (Ansevin, 1961) according to an inverse fourth power of the wavelength (Doty &z Ceiduschek, 1963). Ep/En = dichroic ratio. The subscript refers to the direction of the electric vector of the light either parallel (p) or perpendicular (n = normal) to the long dimension of the flow cell, which is the direction of preferred orientation of the particles. E, - E,, = dichroism. In order to compare solar absorptivities of the virus and protein irrespective of their state of aggregation, concentrations are expressed in moles of 17,630 g/mole protein subunits per liter, and moles of 18,490 g/mole viral subunit,s per liter. (ii) Procedure Two experiments were performed to determine the dichroism of the RNA within TMV, each with a preparation of virus and one of protein at pH 6.16. The optical density spectra, with the transmission direction of the prism successively parallel and normal to the cell axis, were recorded for the static solution and under conditions of flow, from 400 to 240 rnp. The conditions of flow for a given experiment were determined by measuring the dichroism at the absorption maximum as a function of flow rate. The solutions exhibited dichroism at the lowest regulatable flow rates, and reached a maximum dichroism at flows of 1 to 1.5 ml./sec, which remained constant up to a flow rate of about 4 ml./sec. Experiments were performed at flow rates in the middle of the constant range. The dichroism of samples of TMV at pH 7.26 were examined in the cell of square cross-section, in the cell of thin rectangular oross-section and on gels from centrifugal pellets, sheared between plane silica slides.
168
E. M. SCHACHTER,
I. J. BENDET
AND
M. A. LAUFFER
3. Results (a) Particle asymmetry It was desired to have very long particles for dichroism experiments, since the rotary diffusion constant is inversely proportional to the cube of the length. A survey of the sedimentation of TlW protein as a function of pH showed boundaries sedimenting slower than 170 S, characteristic of TMV at neutral pH, at pH values between 7.25 and 5.67. At pH 6.15 a broad component, skewed toward the fast side, of 487 s was observed, along with a minor trailing component of 252 s. When a solution of T&IV was examined at this pH, a similarly skewed boundary of 500 s was observed. Electron microscope examination showed mainly a linear aggregation for both T&IV and protein deposited from pH 6.15 phosphate buffer. The particles were two, three and up to nine times the characteristic length, 300 rnp, of T&IV in the viral preparation and were distributed within the same limits in the protein preparation. (b) Dichroism of
tObUGG0
mosaic virus and re~oIy~~rized protein
The dichroism results obtained for TMY and repolymerized protein at pH 6.15 for one set of experiments are summarized in Figs 1 through 3. The dichroism of both the virus and protein is positive from 240 to 294 rnp. From 295 to 301 or 302 rnp the dichroism of both is negative. Figure 3 indicates that the dichroic ratios of the virus and protein are very similar between 301 and 280 mp, whereas at lower wavelengths the dichroic ratio of the virus is higher than that of the protein. In the two experiments, the molar absorptivities were reproducible within from 5 to 10% and the dichroic ratios within 4% over the entire spectral range. The diohroic ratios of virus solutions oriented in the flow cell of square crosssection at pH 5.15 were appreciably higher than those at pH 7.25 (Fig. 3). Dichroic ratios obtained from experiments on TMY solutions of neutral pH in the thin cell of rectangular cross-section were of the same magnitude as those obtained by previous workers but not as high as those obtained in the cell of square cross-section. The results of attempting to orient virus gels were not reproducible; the dichroic ratios varied from specimen to specimen and none was as high as those obtained with flowing pH 5.15 solutions. There was a very large and variable contribution to optical density due to light scattering and reflections from textural irregularities of the sheared gels. (c) &$ois?n of the RNA within the virus The dichroism of the RNA within TMY was determined by subtracting the molar absorptivity of the protein from the molar absorptivity of the whole virus for each prism orientation. The results derived from the same experiments as in Figs 1 and 2 are summarized in Fig. 4. The dichroism of the RNA is positive at all wavelengths. The dichroic ratios agreed within 7% over the spectral range in the two experiments. 4. Discussion The determination of the orientation of a specific chromophoric group in a complex macromolecule such as TMV from its dichroism requires both a knowledge of the angular distribution of the oriented particles and the isolation of the spectral contribution of that group (Fraser, 1963). Concerning the former, it would have been extremely useful to have been able to prepare almost perfectly oriented gels such as are employed
ORIENTATION
OF THE
RNA
IN TMV
169
0
FIG. 1
320
FIG. 2
40
260
280
300
Wavelength
FIG. 3 Fm.
1. Dichroic
spectra
of TMV.
The curves marked “Parallel” and “Normal” indicate the direction of the electric veotor of the light with respect to the axis of the flow cell for the flowing pH 6.15 solution. The curve marked 0 is the molar absorptivity of a static solution. Fro.
2. Dichroic
spectra
and
repolymerised
TiWV
The curves marked “Parallel” and “Normal” indicate the direction light with respect to the axis of the flow cell for the flowing pH 6.15 0 is the molar absorptivity of a static solution. FIG. 3. Diehroia ratios of TMY and repolymerized data presented in Figs 1 and 2, and the dichroic ratios
protein. of the eleotric solution. The
vector of the curve marked
protein at pH 5.16, corresponding of TMV at pH 7.26.
to the
in X-ray dif&otion studies. This required capillary cells about 100 times thinner than used in X-ray studies, or about 10 p thick. Attempts at overcoming the technological problems were unsuccessful and no acceptable specimens could be prepared. The use of flow orientation was chosen as a compromise, because it was possible to reproduce orientation conditions and to vary solvent properties. The cell developed for these flow orientation studies, with its channel of square cross-section, does not provide a simple flow pattern that easily allows one to estimate the degree of orientation of the particles from hydrodynamic theory. However, its plane parallel
170
E.
M.
SCHACHTER,
I.
J.
BENDET
AND
M.
A.
LAUFFER
faces, provide good optical properties for use in a commercial spectrophotometer, and with it larger dichroic rstios were obtained than with cells previously used such as those of thin rectangular cross-section. In view of the hydrodynamic problems of determining the angular distribution of the viral and protein rods, the orientation of the ultraviolet absorbing chromophores can only be qualitatively described as having their planes either “approximately” parallel or “approximately” perpendicular to the longitudinal axis of the virus. The second major problem in interpreting dichroism is that of spectral isolation of the absorbance of a particular molecular group as well as correcting for intensity losses other than absorbance. There are 18 aromatic chemical groups, each possessing a unique position and orientation, which contribute to the absorption and dichroism of a structured unit of TMV: three tryptophans, four tyrosines, eight phenyhd~nines and three RNA bases. Since the spectra of all the absorbing groups overlap, the dichroism at any given wavelength is an “average” of those of all the groups; the dichroism of a single chromophore cannot be detected and isolated from the polarized light spectra of the whole virus. Both TMV and repolymerized TMV protein exhibit positive anisotropic light scattering (Higeshi, Kasai, Oosawa & Wada, 1963; Wippler, 1953), T, > T,, necessitating a grester scattering correction to Ep than En. In the region of positive dichroism for TMV and TMV protein, the changes in the dicbroic ratio due to correcting for anisotropic light scattering are not of a sufficient magnitude to intluence the interpretation in terms of chromophore orientation in this study. The region of negative dichroism near 297 mp, however, being of low intensity, is not seen unless sc&ering corrections are applied to the data (Schachter, 1965). No attempt has been made to interpret the results on the basis of distinguishing intrinsic and form dicbroism in a manner analogous to birefringence studies in solvents of increesing index of refraction. While experiments of this nature are yet to be performed on TMV, preliminary results with the bacteriophage fd have indicated its dichroism to be invariant with increasing concentrations of salt, suggesting that for fd, at least, form dichroism is not an important consideration. In comparing the nucleic acid-free analogue to the whole virus, structural homology between the protein portion of the virus and repolymerized protein has been assumed. The dichroic ratio profiles of the protein and virus are essentially the same between 300 and 280 rnp (Fig. 3), where mainly the aromatic amino acids are absorbing. X-Ray diffraction end birefringence measurements on high-humidity gels of TMV and protein (Franklin, 1955), also suggest that the absence of nucleic acid does not alter the arrangement of the protein portion. Although “stacked disk’ arrangement of protein subunits has been observed for dry particles in the electron microscope (Markham, Hitchborn, Hills & Frey, 1964), and dry gels show a disordered X-ray pattern and negative birefringence (Franklin, 1955), it is felt that at present there is no evidence to suggest a lack of homology between the two kinds of particles in solution. The resolution that has been obtained for the two analogues used in this work allows only a separation of the two kinds of groups, the aromatic amino acids and the nucleic acid bases. The high positive dichroism of the nucleic acid from the difference between the virus and protein establishes that the RNA bases are aligned “approximately” parallel to the long axis of the virus. At present we sre unable to decide whether the decrease in diohroic ratio as a funotion of wavelength (Fig. 4)
ORIENTATION
OF THE
RNA
represents either multiple transition moments of different orientation handling of the light-scattering correction.
240
FIG. 4. Dichroic Figs 1 and 2.
260
200 Wavelength
171
IN TMV
300
or inadequate
320
(m/l)
spectra and dichroic ratios of the RNA within
TMV derived from the data of
The positive dichroism of the protein confirms the view of the earlier workers (Seeds & Wilkins, 1960; Perutz et al., 1950; Butenandt et al., 1942), that the three tryptophan residues are on the average aligned ‘Lapproxim&.ely” parallel to the virus axis. The maxima in the dichroio ratio profile of the protein coincide with the maxima and fine structure peak of the absorption spectra of tryptophan. The region of negative dicbroism at 297 rnp occurs at a wavelength where most of the absorbanoe is contributed by tryptophen and therefore most probably represents an n-n* transition in the indole ring. The dominance by tryptophan of the protein spectrum does not allow an orientation to be assigned to the tyrosine or phenylalanine residues. Hopefully, in the future, amino acid replacement mutants of aromatic by non-aromatic amino acids may provide sets of analogues for examining the orientation of specific aromatic amino acid residues. This paper is Publication no. 124 of the Department of Biophysics, University Pittsburgh. It is taken in part from the Ph.D. thesis of one of us (E.M.S.), University Pittsburgh, 1966. The work was supported by U.S. Public Health Service Grant GM 10403.
of of
no.
REFERENCES Anderer, F. A. (1963). Advanc. Protein Chem. 18, 1. Aneevin, A. T. (1961). Ph.D. Thesis, University of Pittsburgh. Beaven, G. H., Holiday, E. R. & Johnson, E. A. (1965). In Th Nucleic Acida, ed. by E. Chargaff & J. N. Davidson, vol. 1, p. 532. New York: Academia Press. Butenandt, A., Friedrich-Freksa, H., Ha&wig, SD. & Soheibe, G. (1942). Hope-Se& 2. 274, 276. Caspax, D. L. D. (1963). Advanc. Protein C&m. 18, 37. Cavalieri, L. F., Rosenberg, B. N. & Rosoff, M. (1966). J. Amer. C%em. Sot. 78, 6236. Doty, P. 8c Geiduschek, E. P. (1953). In The Proteins, ed. by H. Neurath & K. Bailey, vol. 1, part 2, p. 444. New York: Academic Press. Fmmkel-Conrat, H. (1967). virology, 4, 1. Fraenkel-Conrat, H. BE Ramechhandran, L. K. (1969). Adwnc. Protein C&na. 14, 176.
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AND
M. A. LAUFFER
Fraankel-Conrat, H. & Williams, R. C. (1966). PTOC. Nai. Acad. Sci., Wad. 41, 090. Franklin, R. E. (1966). Biochim. &iO$dh~S. Acta. 18, 313. Fraser, R. B. D. (1963). J. Chem. Phys. 21, 1611. Ginoza, W., Atkinson, P. & Wildman, S. (1964). science, 119, 269. Higaahi, S., Kascti, M., Ooeawa, F. & Wada, A. (1903). J. Mol. BioZ. 7, 421. Klug, A. & &spar, D. L. D. (1960). A&am. Vi’irw Res. 7, 226. Markham, R., Hitchboru, J. H., Hills, G. J. & Frey, S. (1964). Virology, 22, 342. Perutz, M. F., Jope, E. M. & Barer, R. (1960). Diet. Faraday Sot. 9, 423. Schachter, E. M. (1966). Ph.D. Thesis, University of Pittsburgh. Schramm, G. & Zillig, W. (1966). 2. Naturf. 196, 493. Seeds, W. E. & Wilkins, M. H. F. (1960). Disc. Fa?radaySoc. 9, 417. Stevens, C. L. (1962). Ph.D. Thesis, University of Pittsburgh. Wetlaufer, D. B. (1902). Advanc. Protein Own. 1’7, 303. Wippler, M. C. (1963). J. Phy8. Radium, 14, 66s.
Orientation of the RNA in Tobacco Mosaic Virus EUGENE M. SUHAOETER-~, IRWIN J. BENDET AND MAX A. LAUFRER
Department of Biophysics, University of Pittsburgh, Pithburgh, Pennsylvania 15213, U.&A. Received 6 December 1965, and in revised form
30 August 1966)
The configuration of the ribonucleio acid within tobacco mosaic virus was studied by comparing the ultraviolet diahroism of flow-oriented virus and repolymerized, nucleic acid free tobacco mosaic virus protein. The dichroism of the RNA within tobacco mosaio virus, obtained by subtracting the protein contribution from the dichroic spectra of the virus, has been interpreted as being due to a preferred orientation of the planes of the purine and pyrimidine bases of the RNA “approximately” parallel to the longitudinal axis of the virus. The dichroism of the protein suggests a preferred orientation of the aromatic ammo acids, especially tryptophan, “approximately” parallel to the longitudinal axis. The dichroio ratio of tobacco mosaic virus was found to be influenced by the asymmetry of the particles in the flowing solution and by the geometry of the flow cell. With linearly aggregated particles at pH 5.15 and an optical flow cell of square cross-section, the dichroic ratios for tobacco mosaic virus in this study were appreciably higher than those previously obtained by others.
1. Introduction The structure of tobacco mosaic virus has been studied by a variety of physical chemical methods. The present understanding of the structural properties of the virns, its size, chemical composition and the helioal symmetry of its protein subunits and RNA strand have been recently reviewed (Anderer, 1963; Caspar, 1963; FraenkelConrat & Ramechandran, 1969; Klug & Caspar, 1960). Information concerning the configuration of the RNA strand within the virus has come principally from optical properties of the virus. According to X-ray analysis of oriented gels of TMVS and repolymerized protein, free of nucleic acid, the RNA strand follows the same path as the protein subunits, there being three nucleotides per protein subunit, or 49 nucleotides per helical turn. The radial electron density of the RNA, particularly of the phosphate groups, is located at a radius of about 40 A from the long axis of the virus, and is buried between turns of the protein which extends between radii of 20 A and 90 A. It has been suggested that the results of polarized light studies offer information pertaining to the configuration of the RNA bases within the native virus. Franklin (1955) interpreted the higher positive birefringence of oriented gels of TMV compared to those of gels of protein as due to preferential orientation of the planes of the bases parallel to the long axis. Since, however, biretingence measures an optioal t Present address: Department of Biology, Massachusetts Institute Mesa., U.S.A. $ Abbreviation used: TMV, tobacco mosc& virus. 166
of Technology, Gembride,
166
E.
M.
SCHACHTER,
I.
J.
BENDET
AND
M.
A.
LAUFFER
property of the entire specimen and is not specific for any particular oriented chemical grouping, the difference in birefringence, while it may be suggestive of orientation of the bases, could conceivably be due to the sugar or phosphate components of the RNA. Ultraviolet dichroism (e.g. see Beaven, Holiday & Johnson, 1956) of oriented specimens of TM7 has been studied by three groups of workers (Seeds & Wilkins, 1950; Perutz, Jope & Barer, 1950; Butenandt, Friedrich-Freksa, Hartwig & Soheibe, 1942) with very similar results. It was found that the dichroism was positive between 240 and 296 mp. All three groups concurred that the dichroism at 290 rnp was due to preferred orientation of tryptophan residues, but only the earliest group of workers suggested that the diohroism in the neighbourhood of 260 rnp was due to a preferred orientation of the nucleic acid. The two later groups stated a reluctance to assign the dichroism at lower wttvelengths to RNA orientation. Although dichroism is the expression of the orientation of certain light-absorbing chemical groupings, the spectral complexity due to the overlap of the absorption spectra of the nucleic acid bases and aromatic amino acids, as well as anisotropic light scattering, preclude the assignment of the dichroism at a particular wavelength to a specific chemical group. It was therefore decided to attempt to resolve the dichroism of the RNA from that of the whole virus by performing parallel experiments on the intact virus and on repolymerized protein free of RNA.
2. Materials
and Methods
(a) Preparation of virus and protein TMV was purified from infected Nicotiana tabacum by 3 or 4 cycles of differential centrifugation and EDTA depigmentation (Ginoza, Atkinson & Wildman, 1964). TMV protein, free of RNA, was prepared by both the alkali degradation-electrophoresis method (Schramm & Zillig, 1966). and the acetic acid method (Fmenkel-Conrat, 1967). For dichroism experiments, TMV solutions in water were olartied by centrifugation at 3000 g for 20 mm. The top third of the solution was removed with a dust-free pipette, diluted and dialyzed for 12 hr against two changes of potassium phosphate buffer (0.1 ionic strength, pH 5.16). Solutions of low molecular weight protein in water were clarified by filtration through a Millipore HA filter, diluted and dialyzed as were the virus solutions. The solvents for all sedimentation velocity experiments were O-1 ionic strength potassium phosphate buffers of varying pH. A Spinco model E ane;lytical ultracentrifuge equipped with schlieren optics was used for sedimentation velocity experiments. All runs were performed in 2’ aluminium centerpieces at 20°C. All reported sedimentation coefficients are uncorreoted and refer to the experimental conditions. For boundaries which were highly skewed toward the fastermoving species, the sedimentation coefficient refers to the maximum refractive index gradient of the distribution. (c) Ekctroon W&iC?-08COfJy Virus and protein were deposited from the same solutions 8s those used in dichroism studies, employing a method of adsorption and rapid removal of the high salt-concentration solvent. A drop of solution was placed on a Parlodion-covered nickel grid which wes then placed, solution side down, on a piece of filter paper and quiakly withdmwn. The hydrophobic Parlodion f?hn appeared to be dry after removal from the paper. The speoimen was then shadowed with uranium and examined in an RCA EMU-3F electron microscope. No salt crystals were evident in the electron micrographs. The magnification was determined by using a calibrated carbon replica of a diffraction grating irmnedietely after the virus and protein specimens were examined.
ORIENTATION
OF THE
RNA
IN TMV
167
(d) Comntra&on lneasuremen& Concentrations were determined either refiaotometrically by using a specific refractive increment of 1.86 x 10s3 (100 ml./g) for both the virus and the protein (Stevens, 1962), or spectrophotometrically from the absorption contribution to optical density by using absorptivities of 27 (g-cm/lOOml.)-l for TMV at 260mp and 13 (g-cm/lOOml.)-l for protein at 280 rnp (Fraenkel-Conrat t Williams, 1966): (e) Dichroiem apparatw A Cary 14M recording spectrophotometer was adapted for the dichroism experiments. No permanent modifications of the spectrophotometer were made; the optical corn. ponents for dichroism experiments, the polarizing prism and the optical flow cell being constructed to be accommodated in a specially constructed alignment assembly in the sample compartment. The light was polarized by a 20.mm aperture type A Clan Iu-ism (Karl Lambrecht Crystal Optics, Chicago, Ill.) placed immediately in front of the flow cell. The optical flow cell, constructed of fused silica (Lux Scientific Instrument Co., New York, N.Y.), has an optical flow channel of O-100 cm to a side square cross-section and 4.2 cm length. A light stop in front of the flow cell restricted the polarized beam to a width of slightly less than 0.100 cm. Quartz tubing is fused to the ends of the flow channel for connection to the flow-regulating system. The reference compartment of the spectra. photometer contained a standard 0.100 cm path length cuvette and a neutral density filter, which partially compensated for the optioal density of the prism. The flow-regulating system maintained a circulating flow of solution through the flow cell by forcing the solution from a closed reservoir under a variable pressure through the flow cell to a reservoir open to the atmosphere. A variable-speed pump returned the solution from the open reservoir to the closed one at a rate equal to the outflow rate. Initial exploratory experiments were also carried out in a thin rectangular cell after the design of Cavalieri, Rosenberg & Rosoff (1966) , which had a flow channel with approxi. mately the dimensions: length 4 cm, width 1 cm and depth (optical path) 0.01 cm. (f) Dichroiem wwaaurenzents (i) Conventione The spectrophotometric conventions employed are those suggested by Wetlaufer (1962), with the additional need of defining E = log (1,/I) = optical density, and A = E - T, where T is the turbidity. The turbidity, in an absorbing region of the spectrum, was calculated by extrapolating from the absorption-free region of 400 to 320 rnp (Ansevin, 1961) according to an inverse fourth power of the wavelength (Doty &z Ceiduschek, 1963). Ep/En = dichroic ratio. The subscript refers to the direction of the electric vector of the light either parallel (p) or perpendicular (n = normal) to the long dimension of the flow cell, which is the direction of preferred orientation of the particles. E, - E,, = dichroism. In order to compare solar absorptivities of the virus and protein irrespective of their state of aggregation, concentrations are expressed in moles of 17,630 g/mole protein subunits per liter, and moles of 18,490 g/mole viral subunit,s per liter. (ii) Procedure Two experiments were performed to determine the dichroism of the RNA within TMV, each with a preparation of virus and one of protein at pH 6.16. The optical density spectra, with the transmission direction of the prism successively parallel and normal to the cell axis, were recorded for the static solution and under conditions of flow, from 400 to 240 rnp. The conditions of flow for a given experiment were determined by measuring the dichroism at the absorption maximum as a function of flow rate. The solutions exhibited dichroism at the lowest regulatable flow rates, and reached a maximum dichroism at flows of 1 to 1.5 ml./sec, which remained constant up to a flow rate of about 4 ml./sec. Experiments were performed at flow rates in the middle of the constant range. The dichroism of samples of TMV at pH 7.26 were examined in the cell of square cross-section, in the cell of thin rectangular oross-section and on gels from centrifugal pellets, sheared between plane silica slides.
168
E. M. SCHACHTER,
I. J. BENDET
AND
M. A. LAUFFER
3. Results (a) Particle asymmetry It was desired to have very long particles for dichroism experiments, since the rotary diffusion constant is inversely proportional to the cube of the length. A survey of the sedimentation of TlW protein as a function of pH showed boundaries sedimenting slower than 170 S, characteristic of TMV at neutral pH, at pH values between 7.25 and 5.67. At pH 6.15 a broad component, skewed toward the fast side, of 487 s was observed, along with a minor trailing component of 252 s. When a solution of T&IV was examined at this pH, a similarly skewed boundary of 500 s was observed. Electron microscope examination showed mainly a linear aggregation for both T&IV and protein deposited from pH 6.15 phosphate buffer. The particles were two, three and up to nine times the characteristic length, 300 rnp, of T&IV in the viral preparation and were distributed within the same limits in the protein preparation. (b) Dichroism of
tObUGG0
mosaic virus and re~oIy~~rized protein
The dichroism results obtained for TMY and repolymerized protein at pH 6.15 for one set of experiments are summarized in Figs 1 through 3. The dichroism of both the virus and protein is positive from 240 to 294 rnp. From 295 to 301 or 302 rnp the dichroism of both is negative. Figure 3 indicates that the dichroic ratios of the virus and protein are very similar between 301 and 280 mp, whereas at lower wavelengths the dichroic ratio of the virus is higher than that of the protein. In the two experiments, the molar absorptivities were reproducible within from 5 to 10% and the dichroic ratios within 4% over the entire spectral range. The diohroic ratios of virus solutions oriented in the flow cell of square crosssection at pH 5.15 were appreciably higher than those at pH 7.25 (Fig. 3). Dichroic ratios obtained from experiments on TMY solutions of neutral pH in the thin cell of rectangular cross-section were of the same magnitude as those obtained by previous workers but not as high as those obtained in the cell of square cross-section. The results of attempting to orient virus gels were not reproducible; the dichroic ratios varied from specimen to specimen and none was as high as those obtained with flowing pH 5.15 solutions. There was a very large and variable contribution to optical density due to light scattering and reflections from textural irregularities of the sheared gels. (c) &$ois?n of the RNA within the virus The dichroism of the RNA within TMY was determined by subtracting the molar absorptivity of the protein from the molar absorptivity of the whole virus for each prism orientation. The results derived from the same experiments as in Figs 1 and 2 are summarized in Fig. 4. The dichroism of the RNA is positive at all wavelengths. The dichroic ratios agreed within 7% over the spectral range in the two experiments. 4. Discussion The determination of the orientation of a specific chromophoric group in a complex macromolecule such as TMV from its dichroism requires both a knowledge of the angular distribution of the oriented particles and the isolation of the spectral contribution of that group (Fraser, 1963). Concerning the former, it would have been extremely useful to have been able to prepare almost perfectly oriented gels such as are employed
ORIENTATION
OF THE
RNA
IN TMV
169
0
FIG. 1
320
FIG. 2
40
260
280
300
Wavelength
FIG. 3 Fm.
1. Dichroic
spectra
of TMV.
The curves marked “Parallel” and “Normal” indicate the direction of the electric veotor of the light with respect to the axis of the flow cell for the flowing pH 6.15 solution. The curve marked 0 is the molar absorptivity of a static solution. Fro.
2. Dichroic
spectra
and
repolymerised
TiWV
The curves marked “Parallel” and “Normal” indicate the direction light with respect to the axis of the flow cell for the flowing pH 6.15 0 is the molar absorptivity of a static solution. FIG. 3. Diehroia ratios of TMY and repolymerized data presented in Figs 1 and 2, and the dichroic ratios
protein. of the eleotric solution. The
vector of the curve marked
protein at pH 5.16, corresponding of TMV at pH 7.26.
to the
in X-ray dif&otion studies. This required capillary cells about 100 times thinner than used in X-ray studies, or about 10 p thick. Attempts at overcoming the technological problems were unsuccessful and no acceptable specimens could be prepared. The use of flow orientation was chosen as a compromise, because it was possible to reproduce orientation conditions and to vary solvent properties. The cell developed for these flow orientation studies, with its channel of square cross-section, does not provide a simple flow pattern that easily allows one to estimate the degree of orientation of the particles from hydrodynamic theory. However, its plane parallel
170
E.
M.
SCHACHTER,
I.
J.
BENDET
AND
M.
A.
LAUFFER
faces, provide good optical properties for use in a commercial spectrophotometer, and with it larger dichroic rstios were obtained than with cells previously used such as those of thin rectangular cross-section. In view of the hydrodynamic problems of determining the angular distribution of the viral and protein rods, the orientation of the ultraviolet absorbing chromophores can only be qualitatively described as having their planes either “approximately” parallel or “approximately” perpendicular to the longitudinal axis of the virus. The second major problem in interpreting dichroism is that of spectral isolation of the absorbance of a particular molecular group as well as correcting for intensity losses other than absorbance. There are 18 aromatic chemical groups, each possessing a unique position and orientation, which contribute to the absorption and dichroism of a structured unit of TMV: three tryptophans, four tyrosines, eight phenyhd~nines and three RNA bases. Since the spectra of all the absorbing groups overlap, the dichroism at any given wavelength is an “average” of those of all the groups; the dichroism of a single chromophore cannot be detected and isolated from the polarized light spectra of the whole virus. Both TMV and repolymerized TMV protein exhibit positive anisotropic light scattering (Higeshi, Kasai, Oosawa & Wada, 1963; Wippler, 1953), T, > T,, necessitating a grester scattering correction to Ep than En. In the region of positive dichroism for TMV and TMV protein, the changes in the dicbroic ratio due to correcting for anisotropic light scattering are not of a sufficient magnitude to intluence the interpretation in terms of chromophore orientation in this study. The region of negative dichroism near 297 mp, however, being of low intensity, is not seen unless sc&ering corrections are applied to the data (Schachter, 1965). No attempt has been made to interpret the results on the basis of distinguishing intrinsic and form dicbroism in a manner analogous to birefringence studies in solvents of increesing index of refraction. While experiments of this nature are yet to be performed on TMV, preliminary results with the bacteriophage fd have indicated its dichroism to be invariant with increasing concentrations of salt, suggesting that for fd, at least, form dichroism is not an important consideration. In comparing the nucleic acid-free analogue to the whole virus, structural homology between the protein portion of the virus and repolymerized protein has been assumed. The dichroic ratio profiles of the protein and virus are essentially the same between 300 and 280 rnp (Fig. 3), where mainly the aromatic amino acids are absorbing. X-Ray diffraction end birefringence measurements on high-humidity gels of TMV and protein (Franklin, 1955), also suggest that the absence of nucleic acid does not alter the arrangement of the protein portion. Although “stacked disk’ arrangement of protein subunits has been observed for dry particles in the electron microscope (Markham, Hitchborn, Hills & Frey, 1964), and dry gels show a disordered X-ray pattern and negative birefringence (Franklin, 1955), it is felt that at present there is no evidence to suggest a lack of homology between the two kinds of particles in solution. The resolution that has been obtained for the two analogues used in this work allows only a separation of the two kinds of groups, the aromatic amino acids and the nucleic acid bases. The high positive dichroism of the nucleic acid from the difference between the virus and protein establishes that the RNA bases are aligned “approximately” parallel to the long axis of the virus. At present we sre unable to decide whether the decrease in diohroic ratio as a funotion of wavelength (Fig. 4)
ORIENTATION
OF THE
RNA
represents either multiple transition moments of different orientation handling of the light-scattering correction.
240
FIG. 4. Dichroic Figs 1 and 2.
260
200 Wavelength
171
IN TMV
300
or inadequate
320
(m/l)
spectra and dichroic ratios of the RNA within
TMV derived from the data of
The positive dichroism of the protein confirms the view of the earlier workers (Seeds & Wilkins, 1960; Perutz et al., 1950; Butenandt et al., 1942), that the three tryptophan residues are on the average aligned ‘Lapproxim&.ely” parallel to the virus axis. The maxima in the dichroio ratio profile of the protein coincide with the maxima and fine structure peak of the absorption spectra of tryptophan. The region of negative dicbroism at 297 rnp occurs at a wavelength where most of the absorbanoe is contributed by tryptophen and therefore most probably represents an n-n* transition in the indole ring. The dominance by tryptophan of the protein spectrum does not allow an orientation to be assigned to the tyrosine or phenylalanine residues. Hopefully, in the future, amino acid replacement mutants of aromatic by non-aromatic amino acids may provide sets of analogues for examining the orientation of specific aromatic amino acid residues. This paper is Publication no. 124 of the Department of Biophysics, University Pittsburgh. It is taken in part from the Ph.D. thesis of one of us (E.M.S.), University Pittsburgh, 1966. The work was supported by U.S. Public Health Service Grant GM 10403.
of of
no.
REFERENCES Anderer, F. A. (1963). Advanc. Protein Chem. 18, 1. Aneevin, A. T. (1961). Ph.D. Thesis, University of Pittsburgh. Beaven, G. H., Holiday, E. R. & Johnson, E. A. (1965). In Th Nucleic Acida, ed. by E. Chargaff & J. N. Davidson, vol. 1, p. 532. New York: Academia Press. Butenandt, A., Friedrich-Freksa, H., Ha&wig, SD. & Soheibe, G. (1942). Hope-Se& 2. 274, 276. Caspax, D. L. D. (1963). Advanc. Protein C&m. 18, 37. Cavalieri, L. F., Rosenberg, B. N. & Rosoff, M. (1966). J. Amer. C%em. Sot. 78, 6236. Doty, P. 8c Geiduschek, E. P. (1953). In The Proteins, ed. by H. Neurath & K. Bailey, vol. 1, part 2, p. 444. New York: Academic Press. Fmmkel-Conrat, H. (1967). virology, 4, 1. Fraenkel-Conrat, H. BE Ramechhandran, L. K. (1969). Adwnc. Protein C&na. 14, 176.
172
E. M. SCHACHTER,
I. J. BENDET
AND
M. A. LAUFFER
Fraankel-Conrat, H. & Williams, R. C. (1966). PTOC. Nai. Acad. Sci., Wad. 41, 090. Franklin, R. E. (1966). Biochim. &iO$dh~S. Acta. 18, 313. Fraser, R. B. D. (1963). J. Chem. Phys. 21, 1611. Ginoza, W., Atkinson, P. & Wildman, S. (1964). science, 119, 269. Higaahi, S., Kascti, M., Ooeawa, F. & Wada, A. (1903). J. Mol. BioZ. 7, 421. Klug, A. & &spar, D. L. D. (1960). A&am. Vi’irw Res. 7, 226. Markham, R., Hitchboru, J. H., Hills, G. J. & Frey, S. (1964). Virology, 22, 342. Perutz, M. F., Jope, E. M. & Barer, R. (1960). Diet. Faraday Sot. 9, 423. Schachter, E. M. (1966). Ph.D. Thesis, University of Pittsburgh. Schramm, G. & Zillig, W. (1966). 2. Naturf. 196, 493. Seeds, W. E. & Wilkins, M. H. F. (1960). Disc. Fa?radaySoc. 9, 417. Stevens, C. L. (1962). Ph.D. Thesis, University of Pittsburgh. Wetlaufer, D. B. (1902). Advanc. Protein Own. 1’7, 303. Wippler, M. C. (1963). J. Phy8. Radium, 14, 66s.