Stable intermediate aggregates formed by the polymerization of barley stripe mosaic virus protein

VIROLOGY

36, 620-638 (1968)

Stable

Intermediate

Aggregates

of Barley J. G. ATABEKOV,

Stripe

of Bioorganic

Mosaic

V. K. NOVIKOV, AND

Laboratory

Formed

by the

Virus

Polymerization

Protein

N. A. KIISELEV,

A. S. KAFTANOVA,

A. M. EGOROV

Chemistry of Moscow State University, and of Academy of Sciences, Moscow, USSR Accepted August

Institute

of Crystallography

22, 1968

Barley stripe mosaic virus was dissociated into protein and nucleic acid by treatment with 0.5-0.6 M calcium chloride at pH 6.5. After removal of the nucleic acid precipitate, the protein was purified by two isoelectric precipitations at pH 5.0-5.5. The protein exists as monomers in 0.01 M phosphate, pH 11.8. These have a molecular weight of 21.5 X lo3 and a sedimentation coefficient of 1.8 S; they contain 187-190 amino acid residues but do not contain cystine, cysteine, or methionine. Polymerization of monomers is favored by decrease of pH, adding monovalent salts (e.g., NaCl or KCI) to 0.25 M, adding divalent salts (e.g., CaClz) to 0.01 M, and storage at low temperature. Polymerization is not favored by CaCll at more than 0.1 M. Polymerization occurs into components with sedimentation coefficients of about 10 S, 20 S, 30 S, 35 S, and 40 S, and larger rod-shaped structures, some of which form paracrystals. Electron microscopy of preparations containing 30-40 S components shows disks made up usually of l-6 layers of monomers. These disks apparently crystallize in two distinct ways. INTRODUCTION

The mechanisms of degradation of virus particles and of viral protein polymerization have been studied during recent years, mainly with tobacco mosaic virus (TMV). It is accepted that TMV-protein polymerisation proceeds through stable intermediates of increasing size (Schramm and Zillig, 1955; Caspar, 1963; Harrington and Schachman, 1956; Lauffer, 1966). The conditions under which any particular aggregate is stable, however, and the real interrelations between various intermediate aggregates have not yet been clearly defined. In a series of papers the mechanism of polymerization-depolymerization of TMV protein was studied by Lauffer and coworkers (Lauffer et al., 1958; Ansevin et al., 1964; Banerjee and Lauffer, 1966; Stevens and Lauffer, 1965). In general molecular morphology TMV and barley stripe mosaic virus (BSMV) are similar, both belonging to the group of rodlike helical viruses. The

BSMV particle is d280 i long and has a diameter of 190 A. Gibbs et al. (1963), and Harrison et al. (1965) showed that BSMV particles have an axial canal and display cross-banding at intervals of about 2.5 rnp. The molecular weight of the BSMV particle is about 26 X 106; the intact particle contains RNA with a molecular weight of about 1 X lo6 (Atabekov and Novikov, 1966). In the present paper and the ones to come, some morphological and immunochemical aspects of the BSMV-protein (BSMVp) polymerization are described. MATERIALS

AND

METHODS

The culture of BSMV used in this study was obtained from a barley field crop. Leaves of Chenopodium amaranticolor Coste & Reyn were inoculated with the sap extracted from BSMV-infected barley plants at a dilution near the infection end point and thoroughly washed with water. Inocu620

AGGREGATES

FORMED

BY BSMV

lum from a single lesion was applied to plants of Hordeum vulgare var. Viner, in which serial subcultures were made. Plants were inoculated in the 2- to 4-leaf stage. 1’tiu.s puri&xhm. Systemically infected barley leaves were removed IO-14 days after the plants were inoculated. The virus was purified according to the procedure of At,abekov and Xovikov (1966). After 34 cycles of differential centrifugat’ion, BSRV preparations were crystallized by adding 0.1 M MgCIZ to BSXV suspension in 0.05 M Tris-HCl (pH 7.0-7.5). The needlelike paracryst#als formed in the presence of n2gC1, were separated by centrifugation at 10,000 rpm for IO min. The supernatant liquid was removed and the pellet was resuspended in 0.05 M Tris-HCl at pH ‘7.0-7.5 in the presence of 0.02 M EDTA. The cryst,allization was repeated twice and the crystallized virus was stored a6 5” until used. The Prepcwation of BSMV protein. method of salt-deproteinization (Atabekov and Novikov, 1966) in 0.5-0.6 M CaClz was used unless otherwise stated. The solution of 3 M CM& in 0.05 M Tris-HCl at pH 6.5 was added to concentrated (5-10 mg/ml) BSMV preparat’ions to obtain a final concentration of 0.5-0.6 M CaC&. Under these conditions BSMV was completely dissociated into prot’ein and nucleic acid within l-5 minutes. During this time the opalescence was lost and a fine granular precipitate of Ca nucleate appeared; this was removed by centrifuging for IO-15 minutes at low speed in the cold. The water-clear protein solution was then dialyzed at 5” for 2 days against the several changes of 0.05 M Tris-HCl, pH 5.0-5.5. During t,his time the prot’ein comes into its isoelectric range (pH 5.3) and precipitates. This precipitate jvas separated by centrifugation for 10 min at 10,000 rpm, and the supernatant liquid was discarded. The pellet was dissolved in 0.05 M Tris-HCl solution containing 0.02 AI EDTA at pH 8.0-9.0. The isoelectric precipitation was repeated and the final pellet n-as dissolved in 0.01-0.05 M Tris-HCl (pH 8.0-9.0). The solution obtained was centrifuged at 105,000 g for an hour. The supernatnnt, liquid was stored at’ 5”. A modification

PROTEIX

POLYMERIZATIQK

621

of this method was used to isolate infectious virus RNA (Novikov and Atabekov, 1968). A modification of the acetic acid method of Fraenkel-Conrat (1957) wax sometimes used to obtain BSMVp preparations. The virus was disintegrated in 33 % acetic acid and dialyzed as in the original method. After isoelectric precipitat,ion, the pellet of protein was dissolved in 0.01 31 Tris solution at pH 10.0. Then the solution was centrifuged at 105,000 g for an hour and the supernatant liquid \vas used. Xlllec~~ophotometry. Ultraviolet (UV) absorption spectru were obtained with SF-4A and Hitachi spectrophotometers using a cell of 1 cm path length. Concentrations were determined by measuring III absorption at the appropriate wavelengt’h and t)hen using the specific absorption coefficients: d (270 rnp, 1%) = 27.0 for intact BSRfV and &4 (280 rnp, 1%) = 17.5 for BSMVp. The absorption coefficient’s were determined from the dry weight of BSllZV and BSMVp xvater solutions of known optical density. Sedimentaticrn analyses were made in Spinco Model E and Hit’achi Model UCLi-l analytical ultracentrifuges. The sedimentation coefficients (& ,W) and sedimentat,ion constants (S,“0,N) 1% Iere determined in the usual way. 2’h.e cZi$usion coeficient (D,, ,,+.)n-as det,ermined by layering buffer over BSRlVp (2.5 mg/ml) in :L synthetic boundary doublesector cell in a Spinco E ultracentrifuge at 8.5”. The diffusion coefficient was corrected for viscosity of the solvent and t,cmperature. Diffusion was allowed to occur for about, 2 hours at, 4609 rpm. Calculations were done as described by Elias (1963) using the reduced area height ratio method. Electron microscopy. The preparations mere stained in either 2-5 % phosphotungstic acid (PTA) at, pH 7.0, or 2 % uranyl acetate (pH 4.S-5.0) or 2 % nmmoniunl molybdate. Electron micrographs were m:bde with a .JEI\I-6c inst,rument, at, 80 1~1’. Optical rotatory dispersion (OKI)). ORD measurements were obt,:rined with i1 J:LSCO ORD/UV5 recording spectropol:trimet~r in the spectral range of 500-300 mp. The data were treat,ed using >loffitt,‘s equation (C’rnes and Do@, 19611. The parameters of this

622

ATABEKOV

ET AL.

equation

were calculated using a X0 value of 212 rnp; the bo value was obtained from the slope and a0 from the intercept of plots of

4 a0 X0”

[m’lh

=

(X2 _

X02) +

(A2b:h;02)2

[m!lx x2 - x0 X02

240

260

280

FIG. 1. Ultraviolet BSMVp preparations 20 S protein.

: -,

3bo

320

of ---, TABLE

AMINO

Amino acids

iisparmc

acm

Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Cystine Cysteine Methionine

ACID

1 OF BSMV

COMPOSITION

Amino acid residues per 100 g of protein (g)

x2 ___ x2 - X0”

For the calculation of [m’], the reduced mean residual rotation, a value of 115 was used for mean residual molecular weight. The protein concentration was 34 mg/ml. Amino acid analyses of BSMVp. The protein was hydrolyzed with 6 N HCl at 105” for 60, 40, and 20 hours. Excess HCl was removed by repeated evaporation in vacua at 45-50” and the samples were dissolved in citrate buffer (0.2 M, pH 2.2). No cysteic acid was found in hydrolyzates of BSRlVp oxidized with performic acid with the method of Hirs (1956). Amino acid analyses were performed in this laboratory by Dr. V. P. Korzhenko with a Hitachi KLA-3B automatic amino acid analyzer. Tryptophan and tyrosine were determined by the method of Goodwin and Morton (1946). The nitroprusside reaction in 8 M guanidine chloride was also used as further evidence of the lack

3Ao mr

absorption spectra 10 S protein;

against

PROTEIN

Ammo acid residues per protein monomer (mol. wt. 22.10s) f

92 (%I

Average values

Rounded off values

13.L.3

A.”

bJ.3”

Lir‘G”

4.05 3.47 11.16 5.68 2.03 6.53 4.53 3.03 10.60 5.56 4.52 2.37 3.68 11.79 4.12 -

3.52 3.33 3.43 5.86 0.96 0.61 3.96 2.17 0.70 0.60 0.60 2.35 2.70 1.44 1.20 -

8.82 8.77 19.03 12.74 7.84 20.23 10.06 5.90 20.64 7.50 6.76 3.78 6.70 16.62 4.87 -

9 9 19-20 12-13 8 20 10 6 21 8 7 4 7 17 5 -

96.35

187-190

AGGREGATES

FORMED

BY BSMV PROTEIN

of cysteine in BSMVp. TMV protein was used as a reference protein (positive cont#rol) for the nitroprusside reactions.

POLYMERIZATION

ti23

10 S Protein

The viral protein existed as a monomer only in the presence of the disaggregating agents (see Table 2). RESULTS Different BSMVp preparations obtained Prot’ein preparations obtained by salt in 0.01-0.05 M Tris-HCl at pH 7.5-10.0 disintegration of BSMV display a typical after removal of disaggregating agent conprotein absorption spectrum between 230 tained a major component with a sedimenrnp and 290 rnp with the maximum at 282 tation coefficient in the range of 9.0-12.0 S rnp (Fig. 1). The maximum/minimum value (see Fig. 2, d,f,g; Fig. 3; and Table 2). The for BSMVp is about 2.8-3.2. In Table 1 S,“,,, value of 10.0 S was calculated for the results of amino acid composition anal- one BSMVp preparation in 0.01 M Trisyses of 4 separately isolated BSMVp prep- HCl, pH 8.5 (Fig. 3, inset). Below, t,his arations (18 hydrolyzate samples) are sum- aggregate of BSMVp will be referred to as marized. BSMVp contains comparatively 10 S protein. Small amounts of monomeric large amounts of polar amino acids and protein were sometimes detected in 10 S proline, but no cysteine, cystine, or methio- protein preparations (Fig. 2, d,f, arrows) as nine. Unlike TMV protein, BSMVp con- a small peak near the meniscus. The 10 S t’ains histidine (Table 1). protein is the smallest well-defined aggreThe partial specific volume (7) for the gate of BSMVp subunit.s. molecular weight determination was calculated to be 0.730 ml/g from amino acid 20 S Protein composition data of BSMV protein. The Further protein polymerization takes data of BSMVp sedimentation analyses are place after addition of NaCl or KC1 to 10 S summarized in Table 2. protein preparation. In 0.01 M Tris-HCl at pH 7.5-9.0 with 0.1 M NaCl (or KCl) the 1.8S Protein Subunit BSAIVp solutions always contained a proComplete dissociation of BSMV protein tein aggregate with a sedimentation coeffitakes place under the influence of different cient in the range of 20-23 S (Fig. 2, e,f; agent!s (see Table 2 and Fig. 2, a,b). The Fig. 3; and Table 2). An S& ,W value of component wit.h the sedimentation con- 22.8 S was calculated for one BSMVp stant of Sz00,~= 1.8 S (Fig. 3) is probably preparation in 0.01 M Tris-NC1 + 0.1 Jf a monomer of BSMV. The diffusion co- NaCl (pH 8.5) (Fig. 3, inset). This aggreefficient of the BSMVp monomer was gate will be referred to below as 20 S protein. Very small amounts of 10 S material calculated to be 7.57 X lo-’ cm2/sec. The molecular weight of monomeric pro- are also present in 20 S preparation (Fig, 2, tein at pH 11.8 was measured using Sved- e, f, arrows). In 0.1 Jr NaCl almost comberg’s equation (Svedberg and Pedersen, plete transformation of 10 S into 20 S 1940). A value of 21.5 X lo3 was obtained. protein occurs instantly. Partial 10 S --+ 20 S A similar value (22 X 103)was found earlier transformation takes place at lower concen(Kiselev et al., 1966) using the equation of trations of NaCl (0.01-0.025 Jf) (see Table 2). Scheraga and ZLIandeIkern (1953). Large amounts of 20 S protein. and Z-46 It should be noted that some preparat’ions of monomeric protein were not homo- S, and/or larger aggregates are formed geneous even at pH 11.8 (see Fig. 2~). For when the concentration of NaCl is increased determination of molecular weight, the most to 0.25-0.3 M. Small amounts of monomeric homogeneous protein preparation was cho- protein oft’en also appeared in the BS?tIVpsen. The close similarity between the mo- sedimentograms at 0.1 M and higher s:Llt, lecular weight values calculated from the concentration (Fig. 2, g, arrow). Prepamequations of Scheraga and Mandelkern and tions of 20 S protein also become highly of Svedberg proves that they were deter- het,erogeneous when stored at 5” and then mined correctly. contain addit,ional more rapidly sedimenting

SEDIMENTATION

BEHAVIOR

TABLE OF BSMVp

2 UNDER DIFFERENT

CONDITIONS~ S20.,” values

Type of buffer and treatment

Cont. of BSMVp 1. Monomeric BSMVp 0.01 M phosphate buffer

pH 11.8

(mg/ml)

1.8 ~-

0.05 M Tris-HCI

+ 0.6 M CaC12 pH 8.0

0.05 M Tris-HCl

+ 1 h4 MgClz

1% sodium dodecyl Tris pH 8.0)

sulfate

10.0 1.6 4.8 4.0*

pH 8.0

added to BSMV

3.0 4.0*

(0.01 M

10.0 II. 10 S BSMVp 0.01 1M Tris-HCl

pH 7.5

10.5 10.0 11.5

0.01

M Tris-HCl

pH 8.5

0.01

M Tris-HCl

pH 9.0-9.2

10.0 9.4

5.0 10.6 --

3.5 10.9

0.01

M Tris-HCl

pH 10.0

10.0 12.0

5.5 11.5

2.5 11.5

8.0

5.8

2.0

III. 20 S BSMVp 0.01 M Tris-HCI

+ 0.1

9.3

M NaCl pH 7.5

4.0 22.6

M NaCl pH 9.0-9.2

5.0 22.

2.5 21.5~

4.0 11.8

2.5

M NaCl pH 10.0

M Tris-HCI

+ 0.1 M NaCl

0.01

M Tris-HCI

+ 0.1

0.01

M Tris-HCI

f

0.1

22.4

22.0

0.01

pH 8.5

4.0 IV. Heterogeneous BSMVp Preparations 0.01 ICI Tris-WC1 pH 9.0 + 0.01 M NaCl

0.01

M Tris-HCl

0.01 IM Tris-HCl

0.01

M Tris-HCl

pH 9.0 + 0.025

pH 9.0 + 0.05

9.9

11.3,

22.7e

4.0 11.5, 22.9”

M NaCl

4.0 21.5’

M NaCl 22.0,

pH 9.0 + 0.25 II/I NaCl

4.0 31.0” 4.0

624

21.6 1.0

AG(;REC:ATES

FORMED

BY BSMV TABLE

PROTEIN

625

POLY?\IERIZATIOI”;

Z-Continued S2~.w values

Type of buffer and treatment Cont. of BSMVp (mp/ml) 0.01 M Tris-HCl

pll

9.0 + 1.0 M NaCl

BSi\l\-p stored in 0.01 Ai Tria-HCl month at, pH 8.6

+ 0.1 RI KaCl

12.0,

for 1

21.1,

22.0d*e

4.0 20.2, 34.4,

40.5d

3 5 * Every sedimentation coefficient in parts I, II, and III of the table corresponds to an individual BSMVp preparation separately isolated and analyzed in the course of this study. Freshly prepared protein preparations were used unless otherwise stated. b Abbreviation “-28” means that a brief (20 min) experiment was done for the rough examination of the prot,ein preparation; the peak with an S value of about 2 S was present at the meniscus in the sedimentogram at a speed of 56,100 rpm but the real SZO,,value was not calculated. c 0.1 M KC1 was added in this experiment instead of NaCl. d Most sedimentable material in 10 S aggregate. 0 Ditrerent samples of the same BSMVp preparation were used in these experimeuts. The preparation was stored for 48 hours at 5”.

cally counted. We assumed this number to components (see Table 2). The additional aggregates found in heterogeneous 20 S be 21 in our subsequent calculations. protein solutions fall into three groups Specific Protein-Polymerizing Action cfi Diaccording to their sedimentation coefivalent Cations Used in Low Concenirncients: (1) 27-31 S, (2) 34-37 S, (3) 4042 S tions (Fig. 3; Tables 2 and 3). Aggregates with 27-31 S and 40-42 S will be referred to It was found that addition of 0.005-0.05 below as 30 S protein and 40 S protein, M CaC12 or MgClz to BSMVp preparations respectively. immediat’ely induced extensive polymerizaElectron microscopic investigations of the tion. Different types of particles were remostly homogeneous 10 S and 20 S protein vealed when the preparations of polymerized preparat’ions revealed no visible particles. protein were examined (at pH 7.5-9.0) with Thus both the 10 S and 20 S protein aggre- the electron microscope. Often hollow progates are too small t,o be resolved or are tein cylinders were formed that’ had helically u&able and dissociate into subunits when packed subunits; the cylinders had the stained. diameter of BSMV-particles (Fig. 4) and On the other hand, t,he 30 S and 40 S were more than 1 P long (Fig. 5). aggregate molecules were visible in the elecAnother type of macromolecular aggretron microscope as hollow disks (and disk gate can occasionally be observed in reaggregates) with t’he diameter of the BSMV polymerized protein preparations (see Fig. particles (Fig. 8, a). The presence of 30 S 6). This t,ype of particle was original13 and/or 40 S aggregates in BSMVp sedimen- believed to be a long aggregate of double tograms was always correlated with the disks (Kiselev et al., 1966). When the st,rucpresence of disklike particles in BSMVp ture of these particles was studied by the electron micrographs. As a rule the disks optical diffraction method (Klug and De were connected in pairs. Rosier, 1966), it was found that they have Electron microscope images of negatively an extraordinary (two-start) helical strucstained single disks of polymerized BSMVp ture (Kiselev et al., manuscript in preparawere analyzed (Fig. 8, b) ; in the clearest tion) . images 20-22 individual protuberances could Rodlike particles of BSNVp can int,eract be counted around the periphery, but in side by side, giving rise thereby to formation others t,he number could not be unequivoof paracryst8alline aggregates (Fig. 7). The

626

ATABEKOV

ET AL.

FIG. 2. Sedimentation diagrams of BSMV protein preparations. (a-c) Monomeric BSMV protein. (a) BSMVp preparation containing 5 mg/ml protein in 0.01 M phosphate buffer (pH 11.8). The photograph was taken at a bar angle of 60’ at 20 min after reaching a speed of 42,040 rpm in the synthetic boundary cell. (b) BSMVp preparation (4 mg/ml) in 0.01 M phosphate buffer (pH 11.8). The photographs were taken at a bar angle of 70” at 5 min, 65 min, and 95 min after reaching a speed of 63,650 rpm. Two BSMVp preparations isolated separately by the same method were analyzed (top and bottom pattern). (c) BSMVp preparation containing 4.0 mg/ml protein in 0.01 M phosphabe buffer (pH 11.8). The photograph was taken at a bar angle of 40” at 25 min after reaching a speed of 42,040 rpm in the synthetic boundary cell. (d) 10 S BSMVp preparation containing 6 mg/ml protein in 0.01 M Tris-HCl (pH 8.6). The photographs were taken at bar angles of 70”, BO”, and 60”, respectively, at 14 min, 37 min, and 50 min after reaching a speed of 56,100 rpm.

AGGREGATES

FORMED

BY BWIV

PROTEIX

POLYMERIZATIOX

e FIG. 2-(Continued) (e) 20 6 HS%IVp preparation containing 4 mg/ml protein in 0.01 112Tris-HCl pH 8.6 + 0.1 M SaCl. The photograph was taken at a bar angle of 70” at I1 min after reaching a speed of 55,430 rpm. The arrow show-s the presence of some 10 S material in 20 S BSiVIVp preparation. (f) 10 S and 20 S BShKp preparations containing 4 mg/ml protein. Top pattern, in 0.01 AZ Tris-IICl + 0.1 M NaCl, pH 9.0 (20 S protein) ; bottom pattern, in 0.01 M Tris-HCl pH 9.0 (10 S protein). The photograph was taken at, an angle of 60” at 12 min after reaching a speed of 52,640 rpm. Note the arrows showilkg the presence of 10 S material in 20 S BSM\‘p and monomeric protein in 10 S BSRl1-p. (g) BSMVp preparation containing 10 mg/ml protein. Top pattern 10 S protein in 0.01 M Tris-IiCl (pB 9.0) ; bottom pattern, aggregated BSMVp in 0.01 M Tris-HCl (pH 9.0) + 0.3 M NaCl. The photograph was taken at au angle of 60” at 8 min after reaching a speed of 56,100 rpm.

paracrystals are needlelike and have dimensions large enough to be revealed by the light microscope. The process of paracrystal formation does not usually require more than l-10 min (in 0.014.05 M CaCI,). The polymerizing action of magnesium and barium chloride up011 BSMVp is similar to that of calcium chloride.

Protein paracrystal formation is marked by the appearance of a specific silky brilliancy of the BSMVp preparation. The paracrystals may be easily pelleted by low speed centrifugation. In the supernatant liquid thus obtained, some residual aggregated BSMVp material is present (Table 3). Paracryst,al formation was observed very

628

ATABEKOV

l

FIG. 3. Concentration dependence of BSMVp sedimentation at different states of aggregation. The sedimentation coefficients of different BSMVp preparations obtained in the course of study. The preparations were analyzed in 0.01 M Tris-HCl (pH 7.5-10.0) with 0.1 M NaCl and without NaCl for 20 S and 10 S BSMVp, respectively. The solvent for monomeric BSMVp was 0.01 M phosphate buffer (pH 11.8). (.) BSMVp contains a single peak in sedimentogram. (8) Heterogeneous BSMVp preparations. When different heterogeneous preparations of the same concentration were examined the symbol -8 was used to indicate the sedimenting components present together in one given preparation, and the symbols @I- (or -@-) indicated the components of other preparations. Inset: S,“,,, determination for the individual 10 S and 20 S BSMVp preparations.

often, but not invariably, after addition of 0.01-0.05 M CaCL to BSMVp solutions (at pH 7.5-9.5). Indeed, a remarkable amount of polymerization was invariably induced by low concentrations (0.005-0.05 M) of calcium chloride over a wide range pH even when the crystals were not formed (Table 3). Disklike aggregates morphologically similar to the double-disk particles (and stacks of disks) found when TMV-protein polymerizes (see for reference, Caspar, 1963) were present invariably in Ca-polymerized BSMVp preparations (Fig. 8 a). It must be

ET AL.

noted that mono-disk and triple-disk aggregates as well as disks consisting of 4-6 layers of subunits were present together Faith double-disk parbicles. Disklike aggregates were usually found mixed with rodlike particles of helical BSMVp-aggregates. However, in some experiments the Ca-polymerized BSMVp-preparations contained only disklike particles and did not contain the rodlike ones though the conditions for protein polymerization were always the same. Different types of crystalline aggregates can be formed by aggregation of disks in the presence of 0.01-0.05 M CaCL. An electron micrograph of one of these crystalline aggregates is presented in Fig. SC. The crystal, negatively stained with PTA, showed an approximately0 square network with a side of about 200 A. This aggregate seen from the angle of Fig. 8c, resembles the three-dimensional crystals with the body-centered tetragonal lattice reported earlier by Macleod et al. (1963) and Finch et al. (1966) for TMV protein. It is reasonable to suggest that the crystal shown in Fig. 8 c is built up by superposing several disk layers as is schematically shown in Fig. 8d. It must be noted, however, that we have no electron micrographs of side views of these crystals. Thus, we have no conclusive evidence proving that the crystal is built up of double-disk particles (and not of triple-disk particles, for example). The second type of aggregate composed of disklike particles is shown in Fig. 9. We believe that it is indeed a second type of aggregate of disks and not the other projection of a crystal of the type shown in Fig. 8 c. The disks are here seen in side projection (Fig. 9). It is noticeable that the disklike aggregates of the different aggregation states (double disks as well as triple disks and even disks consisting of 4, 5, and 6 layers of subunits) can serve as a packing unit forming the network in this aggregate (see Fig. 9 a, b). Note also the clearly seen disklike particles on the edges of the crystal shown in Fig. 8 c and the apparent absence of such formations on the edges of the crystal shown in Fig. 9, a-c. The presence of small regularly packed regions with a random orientation in relation to each other

AC:Gt:ECbATES

FORXEI)

BY BS?\ly

PROTEIN

TABLE SEDIMENTATION

2

I

) 10.0 / ( 10.0

3 ; 3.0 4 I --5r 4.0 (i ( 10.0 7

, ! I

3

PROPERTIES OF BSMV PROTEIN IN THE PRESEN(;E OF CALCIUM ION (SEDIYENTA~~~OS COEFFICIENTS IN SVEDREKGS)" Starting material before CaCl:! addition

(‘enc. Expt. of No. protein hdml)

1

639

POLY?*IERIZATION

I

CaCle concentration pH

(U) ____--~.-

0.05

0.005

0.3

0.15

~.~~-0.5

10s

’ BSM\’ in 0.01 .II Tris-ITCl , I ~ ’

6.5 /88 (245)bj ’

7.5

28.6, 70.0”~

8.5 1 8.7 9.0

--

I I I I

-

10,0,22.9,11.5 180.0”~ f

I

I ~ -2S, , -2s / 20.0 1

90.0** j 31.5, 41.6, 4G.0~ 19.7, 2G.1, 30.-l* (30.4, 40.9, 46.2h ( ~-

I ~I -

1 20 6 / BS?rlYp in ~ 0.01 ilil Tris-HCl + 0.1 M IiaCl

9.5 (26.@, * 8.5 , -

/ 21.5, 29.6, 40.2” (

--

I~--!_ ! I

j

i ._ I I

I I \ ---

9.0 20.0, 27.6, 32.3h 18.9, 29.0, 31.@ 119.5, 28.0, 35.7 j ~L a The initial 10 S and 20 8 preparations used in these experiments were homogeneous. Without parentheses: the results obtained in 1 hour after CaClz addition, in parenthesis: the resrdts obtained after 48-913 hours at 5”. b In these experiments the pellet of the BSM1*p crystals was separated by low speed cerrtrifugat.ion and the material remaining in supernatant was analyzed in the analytical ultracentrif~lge. c Before analytical centrifugation, 0.1 ii/l NaCl was added to dissolve the crystalline precipitate formed in the presence of 0.05 M CaCla . d These experiments were performed at the same time. e III this experiment 0.01 M CaC12 was used for BSMVp polymerization. J Long (0.2-2.0s) rodlike particles in the electron microscope. Q Short (0.2pj rodlike particles and a great number of disks in electron microscope. 6 Disks iu electron microscope. 8”

4.0

(Fig. 9, a, c) is characteristic of t’his aggregate. The conditions under which any particular type of crystal is built up of disklike units is not clear at. present. Dissociation of BSMVp Aggregates in Concentrated Salt Solutions It was noticed that BS?tlVp crystallization did not occur when the CaC12 concentration was increased to 0.1 M. The optical density at 320 rnp in BSMVp solutions was measured to test the protein aggregation at pH 7.5 under the influence of cations (Fig. 10). It can be seen that the turbidity of BS;\IVp increased greatly in the presence of

0.01-0.05 M CaClz and t(hen decreased sharply in 0.1 M CaC12. On the contrary, the ODa20 mP value remained constant and was relatively low over a wide range of B+ and iYa+ concentration (Fig. 10). Increase in Ca2+ concentrat.ion led to progressive dissociation of protein aggregates. In 0.5 M CaClz solution, the BSMVp was t,ransformed into monomers (Table 3). The influence of high salt concentration on macromolecular rodlike aggregates of BShlVp was also studied. The BSRIVp crystals obtained in 0.01 M Tris-WC1 (pH S.5) + 0.05 M CaC12 were dissolved in 0.1 II1 NaCl. This solut,ion contained :L great

630

ATABEKOV

FIG. 4. Electron

micrograph

number of helical protein particles detected with the electron microscope and showed a single broad peak of about 180 S in the sedimentogram. Aggregates of 180 S dissociated into three smaller aggregates (20.9 S, 27.8 S and 34.1 S) or into monomeric BSMVp in concentrated (1.5 ionic strength) NaCl and CaCh solutions, respectively. pH Dependence of the BSMVp tion

Polymeriza-

The polymerization of BSMVp is strongly pH dependent and reversible. The 20 S protein may be transformed into 10 S aggregate at pH 10.0 even in the presence of 0.1 M NaCl (Table 2). On the other hand, the components with sedimentation coefficients of 20 S and higher were readily formed even in the absence of metallic ions, when 10 S preparations were acidified to pH 6.4. Lowering the pH of 10 S or 20 S protein solution to 7.0 leads to the polymerization of BSMVp and to formation of double disks and stacked-disk structures. Rodlike helical particles were also observed at pH

ET AL.

of negatively

stained

BSMV.

6.0-6.5 in these preparations tron microscope.

with the elec-

Optical Rotatory Dispersion (ORD) The possibility that the conformation of a BSMV-protein subunit changes somewhat on aggregation was not excluded. Clear evidence for the conformational changes in proteins might be provided by ORD measurements and a-helix content determination. We made an attempt to estimate a-helix content in BSMVp at different stages of aggregation. The values of R/loffitt’s constants obtained for different aggregates of BSMVp are shown in Table 4. It can be seen that the bo value is the same in all BSMVp preparations and does not depend on the state of BSMVp aggregation: it is very low (Table 4). It seems reasonable to conclude that the a-helix content of BSMVp does not exceed 6-7%. This conclusion was made on the assumption that bo = -630 is the value corresponding to 100 % a-helix content in a protein (see Urnes and Doty, 1961).

FIG. 5. Helical

rodlike

phicles

of polymerized

The real value of a-helix content in BSA,IVp may be somewhat less as bo is calculat,ed wit,h an accuracy not exceeding 6 %. This renders doubtful the application of 0RD measurements for the comparison of conformlttional changes in BSMV proteins on the basis of a-helix content’. DISCUSSION

Our studies indicate that BSMV protein subunits can assemble themselves in vitro into structures resembling those of the intact virus. The process of BSMVp polymerization proceeds stepwise, and a number of &able intermediates of increasing

BSIIY

protein,

(0.0131 CaCls , pH7.5).

size may be formed. The following BShiVp aggregates were defined: (1) 9.0-12.0 d (designated as 10 S); (2) 20-23 S (designated as 20 S) ; (3) 27-31 S (designated as 30 S); (4) X-36 S aggregate; and (5) 40-42 S (designat,ed as 40 S). In 0.01 ;11 Tris-HCl solutions and in the presence of monovalent, cations with an ionic strength of about 0.01 at pH 7.5-10.0, BSMVp was polymerized into 10 S aggregate. In the presence of monovalent cations at’ pH 7.5-9.0 in a wide range of ionic strength (0.1-1.0) the BSMVp-polymerized predominantly into 30 S-aggregates (Table 2). The 10 8 protein intermediate is com-

632

FIG, 6. Rod-shaped pH 9.0).

ATABEKOV ET AL.

aggregates

of polymerized

BSMV

parable to the A protein of TMV and may be isolated from BSMV by any of the deproteinization methods used. From the data presented in Table 2 it can be seen that the S20,Wvalue varies considerably for different 10 S protein preparations. This also happens with TMV A protein (Caspar, 1963), where the sedimentation constant measured for dif-

protein

resembling

stacked

disks (0.01 M CaClz

ferent A protein preparations varied from 4 to 4.6 S. Caspar (1963) thought that this variability reflected differences in the state of aggregation between A protein preparations under different conditions. It was mentioned above that very small amounts of 10 S aggregate were sometimes present in 20 S-BSMVp sedimentograms and

ilGGREGATES

FIG. 7. Paracrystal pH 7.5).

FORMED

made up of individual

BY BShlV

rodlike

that traces of monomeric BSMVp (~2 S) can be revealed even in the relatively homogeneous 10 S and 20 S protein preparations. Different protein intermediates are probably always present, together in BSMVp solutions in equilibrium with each other. Unequal amounts of minor components could be present besides the major one (monomeric protein and 10 S protein in 20 S protein solutions; monomeric protein and 20 S protein in 10 S solutions) in different BSMVp preparations isolated by the same procedure. Thus, it seems reasonable to suggest that small uncontrolled variations between individual preparations occur and that BSMVp preparations isolated separately by the same method differ somewhat from each ot,her. It can also he seen from Table 2 that the sedimentation coefficients experimentally determined for different 10 S- and 20 S-preparations varied considerably and this variation was not directly related to the protein concent#ration and pH. It must be noted that t.he concentration dependence of SZO,n.for one given BSXVp preparation was not large (Fig. 3, inset). The possibility that the molecular composition of 10 S aggregate molecule varied to some extent’ in separate 10 S BSAIVp preparations was not excluded. Using some hydrodynamic principles, Caspar (1963) succeeded in det’ermining the molecular composition of some ThIV protein aggregates. Assuming that the bonding between the subunits in 10 S, 20 S, and other component’s of BSJIVp is similar t#o that of the Tfi2V helix and that t,he hydration of

PROTEIN

particles

POLYI\IERIZATION

of polymerized

BSXWp

(0.05 M C”:tCla ,

BSMV and TMV protein is the same (lo35%) we carried out the calculations with BSMVp aggregat’es according t)o Caspar’s procedure. From the electron microscopic studies on the intact virus (Harrison et al., 1965; Gibbs et al., 1963), the shape of the monomer can b$ approximated by, a prolate ellipsoid 75-80 A long and 25-26 A in diameter. The sedimentation constants calculated for the different aggregates are presented in Table 5. Approximate agreement between the calculated sedimentation constants of some of the suggested aggregates and the observed values for 10 S, 20 S, and 30 S, components provides some information on their real molecular organization. This information is restrictJive rather than otherwise. Thus, it, seems unlikely that t’he 10 S aggregate or 20 S aggregate has a single-disk or double-disk structure with 21 monomers in a turn (see Table 5). Sediment,ation coefficients have been calculated for different, hypothet,ic:tI aggregates composed of 7, 8, 9, and 10 monomers arranged either in compact concentric structures or in two layers. The values of axial ratio for t)hese aggregnt,es (7-10 mers) lvere in the range of l-2 and calculated S values in the range of 9)--12 S. Only one of those possible aggregates is shown (S-mer) in Table 5 by way of example. Thus, a number of hypothetical structures (‘i-10 mers) could correspond to 10 S BSMVp. It also seems likely that the X0 S aggregate consist~s of t,wo Icyers of subunits (11 subunit,s in one layer) being, thus, half of a double-disk (or helicnl-disk) r):wtiClC

634

ATABEKOV

ET AL.

FIG. 8. (a) Disklike structures; disks connected in pairs and “end-on” aggregation of such pairs of disks (0.05 M CaClz , pH 8.0). (b) Electron micrographs of selected disks of BSMV protein. (c) Threedimensional crystal built by superposing of several layers of disks (see the scheme in d) (0.05 M CaClz , pH 9.0).

AGGREGATES

FORMED

BY BSMV

PROTEITU’

POLY1IEKIZATION

E’rc;. 0. (a, c, d) Electron micrographs of BSXlV protein aggregates built of the disklike part irlw different thickness placed on t,heir sides (see the scheme in h, vorrespondirrg to the olltlillerl $wt it) (0.05 rll CaClz , pII 9.0). (d) Electron micrograph showing the first stages uf disk aggregatioll: WVOI disks placed on t,heir sides form the aggregate.

636

ATABEKOV

ET AL TABLE

4

OPTICAL ROTATORY DISPERSION CHARACTERISTICS OF BSMV PROTEIN

State of BSMVp aggregation

I

,

5

, I II

II I

I

I

0.03 a03al 1.0 2.0 FIG. 10. The behavior of BSMVp preparations in various concentrations of Ca2+ and Na+, K+ ions. The salts were added to 10 S protein preparation (0.01 M Tris-HCl, pH 7.5) and optical density at 320 mp was measured after 3 hours incubation at 20”. 0

0.01

(see Table 5). These considerations agree with our electron microscopic observations showing that disk particles are absent in 10 S and 20 S preparations. Moreover, the doubledisk structure of the 30 S component predicted by S calculations (see Table 5) agrees with the fact that a great number of disk particles always appears when 30 S component is present in the protein preparations. The real structure and functional role of each individual aggregate of BSMVp is not clear at present and we are unable to point out the real way subunits polymerize. At comparatively low ionic strength, divalent cations are extremely effective in polymerizing BSMVp (Table 3). By contrast, high ionic strength led to the dissociation of BSMVp aggregates. It is of interest that Ca2+ is much more effective than Na+ in both the polymerizing and depolymerizing capacities. These results have suggested that cations may play a specific role in the macromolecular structure organization of BSMVp aggregates. The polymerizing action of salts depends not only on their contribution to the ionic strength of the solvent, but also (and mainly) on the specific polymerizing ability of divalent cations. At pH 8.0-9.0 the calcium ion is capable of inducing formation of rodlike particles of helically polymerized

1. Monomeric unit (1.8 S at pH 11.8) 2. 10 S protein 3. 20 S protein 4. BSMVp polymerieed by 0.01 M CaCba addition at pH 8.5

Specific Moffitt’s coefficient rotation at 500m~ --b 0 -a0 - [oil 500 29.0

43.0

127.6

25.0 26.0 19.0

46.0 40.0 43.0

95.0 95.0 66.0

a A mixture of rodlike aggregates and disks seen in the electron microscope.

protein (HPP) at an ionic strength of about 0.01 or less. Under the same conditions (pH, ionic strength, time of incubation) monovalent cations induce only the first steps of the polymerization, 10 S and 20 S aggregate formation. It is not unlikely that in BSMVp polymerization calcium plays a role of ligand binding to the protein and changing its conformation. The changed conformation induced by the binding of Ca2+ might be favorable for the polymerization of BSMV protein. It seems likely that isolated monomer exists in solution in altered conformation in which it is not capable of forming ordered aggregates. The native conformation of BSMVp, which is altered in the presence of disaggregating agent (salt, urea, detergent, etc.) is probably reformed stepwise on polymerization. However, ORD-measurements were unable to reveal conformational differences between the different aggregates of BSMVp because of a very low content of a-helix in the BSMVp. The results of comparative immunochemical analyses of different stable intermediate aggregates are presented in two other papers (Atabekov et al., 1968a, b). It is shown there that all the BSMVp aggregates of 10 S BSMVp or more are serologically identical to each other and to BSMV. By contrast monomeric protein exhibits only partial sero-

AGGREGATES

FORMED

BY BSRIV TABLE

CALCULATED AND OBSERVED SEDIMENTATION

PROTEIN 5

COEFFICIENTS

OF AGGREGATES OF BSXG

Axial ratio (for oblate ellipsoid)

calculated

8

2, 0

9.6-10.4

21

8, 0

13.4-14.8

Proposed molecular morphology of the aggregate”

Xumber of monomers

63

POLYMERIZATION

PROTEIN

s

22

2, 0

18.3-19.8

42

4, 0

25.7-27.7

9.0-12.0 (10 S protein)

27.0-31.0 (30 S protein)

a The real arrangement based on the assumption that in TMV helix.

of monomers in any appropriate aggregate is not clear. The calculations are that the mode of packing of the subunits in BSMVp aggregates is similar to

logical identity aggregates.

BSRlV

with

and BSMVp

ACKNOWLEDG?VIENTS The authors thank Dr. 1’. Gurevitch for help in ORD measurements and in a-helix estimation in BSMVp and Dr. E. P. Senchenkov for making the electron micrograph shown in Fig. 9, A and C. We would also like to thank Miss T. I. Heifetz for assistance in translating this text. REFERENCES ANSEVIN, A. T., STEYENS, C. L., and LAUFBER, &I. A. (1964). Polymerization-depolymerization of tobacco mosaic virus protein. III. Change in ionization and in electrophoretic mobilit,y. Biochemislry 3, 1512-1518. ATABEKOV, J. G., and NOWKOV, Jr. K. (1966). The properties of barley stripe mosaic virus nucleoprotein and its structural components. Biokhimia, UISSR, 31,157-166. ATABEKOV, J. G., SCHASKOLSKAYA, N. D., DEMENTYEVA, S. P., SANCHENKOV, E. P., and SACHAROVSKAYA, (+. N. (1968a). Serological study of barley stripe mosaic virus protein polymerization. I. Immunodiffusion, immuno-

electrophoretic characteristics and absorption experiments. Virology 36, 587-600. ATAS~EKOV, J.G., DEMENTYEVA,~. P., SCHASKOLSKAYA, XL’.D. and SACHAROVSKAYA, G. s. (1968b). Serological study on barley stripe mosaic virus protein polymerization. II. Comparative antigenie analysis of intact virus and some stable protein intermediates. virology 36, GOI-612. BANERJEE, K., and LAUFFER, 31. A4. (19G6). Polymerization-depolymerization of tobacco mosaic virrls protein. VI. Osmotic pressure studies of early stages of polymerization. Riochemish-y 5, 1957-1964. C&PAR, D. L. D. (1963). Assembly and stability of the tobacco mosaic virus particle. .lclz~nn. Protein Chem. 18, 37-121. ELIAS, H. G. (1963). Ultracentrifugen~-;\Iethodeil. De&emu Monograph. B44, 1-i-k F~XCII, J. ‘I’. (1965). Preliminary S-rap diflraction studies on tobacco rattle and barley stripe mosaic virllses. J. Mol. Riol. 3, 612Ni19. FINCH, J. T., LEBERMAN, R., CHANC Ju-S~asti, and KLUG, A. (1966). The rotational symmetry of the two-turn disc-aggregat,e of TXL21. protein. Nature 212, 349-350. FRaExKEL-COi%RAT, 11. (195i). Degradation of

638

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tobacco mosaic virus with acetic acid. Virology 4, 14. GIBBS, A. T., KASSANIS, B., NIXON, H. L. and WOODS,R. D. (1963). The relationship between barley stripe mosaic and Lychnis ringspot viruses. virology 20, 194-197. GOODWIN, W., and MORTON, R. A. (1946). The spectrophot,ometric determination of tyrosine and tryptophan in proteins. Biochem. J. 40, 628-632. HARRINGTON, W. F., and SCHACH~XAN, N. K. (1956). Studies on the alkaline degradation of tobacco mosaic virus. I. Ultracentrifugal analysis. Arch. Biochem. Biophys. 65, 278-295. HARRISON, B. D., NIXON, H. L., and WOODS, R. D. (1965). Lengths and structure of particles of barley stripe mosaic virus. Virology 26, 284-289. HIRS, C. H. W. (1956). The oxidation of ribonuclease with performic acid. J. Biol. Chem. 219, 611-621. KISELEV, N. A., ATABEKOV, J. G., KAFTANOVA, A. S., and NOVIICOV, V. K. (1966). Studies on virus protein repolymerization and reconstruction of some rod-shaped viruses. Abstr. 6th Intern. Congr. Electron Microscopy, Kyoto, 147-148. &UG, A., and DE ROSIER, D. J. (1966). Optical filtering of electron micrographs: reconstruction of one-sided images. Nature 212, 29-32. M. A. (1966). Polymerization-deLAUFFER, polymerization of tobacco mosaic virus protein. VII. A model. Biochemistry 5, 2440-2446.

ET AL. LAUFFER, M. A., ANBEVIN, A. T., and CARTWRIGHT, T. (1958). Polymerization-depolymerization of tobacco mosaic virus protein. Nature

181,1338-1339. R., HILLS, G. J., and MARKHAM, R. (1963). Formation of true three-dimensional crystals of the tobacco mosaic virus protein. Nature 260, 932-934. NOVIICOV, V. K., and ATABEKOV, J. G. (1968). Salt-deproteinization of plant viruses. (Manuscript in preparation). SCHACHMAN, H. K. (1959). “Ultracentrifugation in Biochemistry.” Academic Press, New York. SCHERAGA, H., and MANDELKERN, L. (1953). Consideration of the hydrodynamic properties of proteins. J. Am. Chem. See. 75, 179-182. SCHRAMM, G., and ZILLIG, W. (1955). ifber die Struktur des Tabakmosaikvirus. IV. Die Reaggregation des nucleinsaurefreien Proteins. 2. Naturforsch. 100, 493-498. STEVENS, C. L., and LAUFFER, M. A. (1965). Polymerization-depolymerization of tobacco mosaic virus protein. IV. The role of water. MACLEOD,

Biochemistry 4, 31-37. SVEDBERG, T., and PEDERSEN,

K. 0. (1940). “The Ultracentrifuge.” Oxford Univ. Press, London and New York. Johnson reprint Corporation, New York. URNES, P., and DOTY, P. (1961). Optical rotation and the configuration of polypeptides and proteins. Advan. Protein Chem. 16, 401-544.