J. -MOE.Biol. (1977) 113, 123-140

Alfalfa Mosaic Virus Protein Polymerization R. A. DRIEDONKS,

P. C. J. KRIJGSMAN

AND

J. E. MEKLEMA

Department of Biochemistry State Uwiversity of Leiden Fassenaarseweg 64, Leiden, The Netherlands (Received 19 November 1976) The self-association of alfalfa mosaic virus coat protein was studied by sedimentation analysis and electron microscopy under a wide range of conditions. In the depolymerized state the protein exists as a molecular species with a sedimentation constant of roughly 3 S and with a molecular weight of (48.4 & 1.1) x 103. This value is, within experimental error, twice the value of the monomer (van Beynum, 1975). The dimer has a very stable configuration, as no evidence was found for a monomer-dimer equilibrium between pH values of 3 and 9 and values of ionic strength up to 1.0. One main type of association product (30 S) uas found with a molecular weight of (1.48 & 0.03) x 106. Therefore this particle accomodates 30 dimers which are arranged according to a point group symmetry of 532. The orientation of the 30 dimers within the icosahedral lattice must be such that lattice dyads coincide with the 2-fold axes of the dimers. Micrographs of the 30 S particles show a diameter of about 123 .&; analysis of Iinear arrays of these particles shows that at low resolution the particle is a hollow sphere with an average coat thickness of about 40 A. The influence of pH, ionic strength, protein concentration and the type of buffer on the polymerization was determined to some extent and is discussed. The assembly of dimers into the icosahedral particle is an entropy-driven process (Lauffer, 1975) ; this is concluded from studying the temperature-dependence of the free energy change. Under favourable conditions (phosphate buffer pH 5.5 and ionic strength 0.5) the average enthalpy and entropy changes for the insertion of one dimer into the lattice are about 6.4 kilocalories per mole and 50 entropy units, respectively, based on the unit mole fraction.

1. Introduction Studies on the assembly of relatively simple virus particles (Caspar & Klug, 1962) will ultimately yield a better knowledge of how proteins and nucleic acids associate to form well-defined nucleoprotein aggregates. Information about the pathway of assembly, together with structural knowledge, may eventually lead to the understandiug of t,he relation between structure and function of the virus. This aspect is of general biological interest, being an example of the morphogenesis of one of the smallest biological structures. Tobacco mosaic virus is one of the most thoroughly studied virus particles in these terms; it beIongs to the class of simpIe viruses. Its structure consists of a large number of helically arranged, identical protein molecules, which protect an RNA molecule. The assembly of this virus starts with a specific interaction of the RNA and the disk, 123

124

R. A. DRIEDONKS

E!P BL.

a well-defined protein aggregate consisting of two rings each of 17 subunits (Durham et al., 1971; Champness et al., 1976). In common with several other simple plant viruses

(brome mosaic virus, cowpea chlorotic mottle virus) the coat protein was found to be able to associate into empty capsids in the absence of the RNA (Pfeiffer & Hirth, 1974; Adolph & Butler, 1974). Previous work on virus assembly has been reviewed by, for example, Casjens & King (1975). We are studying the assembly of alfalfa mosaic virus, an RNA-containing plant virus. AMY? exhibits characteristics different from many other simple viruses. For example, the RNA is not protected by the protein coat against RNAase activity (Bol & Veldstra, 1969), and the virus particle can be dissociated by incubation with its own RNA (van Vloten-Doting L%Jaspars, 1972). One kind of polypeptide chain structure with a molecular weight of 24,250, as follows from studies on the primary (van Beynum, 1975), is incorporated in the virus structure. The isolated coat protein is able to polymerize into spherical particles (Hull, 1970; Lebeurier et al., 1971). The total genome of AMV consists of three different RNA molecules which, together with the protein, form cylindrically shaped particles of different length (Jaspars, 1974). The nucleoprotein particles examined by electron microscopy have a diameter of about 140 A and display rounded caps at their ends. Mellema (1975) postulated a model for the cylindrical part of the virion which was derived from digital image analysis of negatively stained virus particles. The model is based on a P6 net in which the protein is clustered in such a way that relatively large holes exist at the Z-fold and 6-fold lattice positions. Our intention is to study the assembly of this plant virus in order to obtain a detailed description of the interactions between the coat protein and the nucleic acid component, which will lead towards an understanding of the morphogenesis of the virus. In order to evaluate the behaviour of the coat protein in sohrtion we undertook experiments to characterize the association reaction under a wide range of conditions. A preliminary report of these results has been presented by Driedonks et al. (1976). In a subsequent study we will present data on the role of the nucleic acid component in the assembly reaction.

2. Materials and Methods (a) Isolation

procedure

and solvent conditions

The isolation procedure for the virus has been described by van Vloten-Doting & Jaspars (1972). The coat protein was isolated according to Kruseman (1969). The protein solutions were contaminated with O-3 to 0.6% of residual RNA (Driedonks et al., 1976). The protein was dialysed and stored as described earlier (Driedonks et al., 1976). No fundamentally different behaviour of the coat protein in the association reactions was found when they were studied with and without a reducing agent, e.g. mercaptoethanol or dithiothreitol ; therefore in most experiments no thiol reagent was present. The protein solutions used had not been stored for longer than 3 weeks after isolation. In all experiments protein samples were dialysed against a buffer with the required pH, and an ionic strength of 0.1, and subsequently against a similar buffer with the same pH and the required ionic strength. This was done because of the relatively high rate of the polymerization reaction at low ionic strength. Unless otherwise stated, the required ionic strength value was reached using only the ions concerned in the buffer type. The ionic

t Abbreviation

used: AMV,

alfalfa

mosaic virus.

9MV

COAT

PROTEIN

strength (I) of buffer solutions, containing ing equation :

ASSOCIATION

125

n-valent anions, was calculated using the follow-

where CA represents the molality of anions, Cn the molality of protons and the K, values are the respective dissociation constants of the acid at the temperature of the experiment. The following buffer types were used: sodium pyrophosphate/pyrophosphoric acid, sodium phosphate/phosphoric acid, sodium acetate/acetic acid, sodium oxalate/oxalic acid and sodium citrate/citric acid. The influence of the buffer type on the AC: value was studied. at pH 5.5 and an ionic strength of 0.5, being the optimal conditions for polymerization in pyrophosphate buffer. A solution of ATP in 5 m&r-sodium acetate (pH 55), with the same molarity as the pyrophosphate buffer, namely 70 mg/ml was used. The reaction enthalpy and entropy were determined in phosphate buffer (pH 55) with I = 0.5, and not in pyrophosphate buffer, as the latter buffer is thermally unstable. Protein concentrations were measured spectrophotometricelly; an extinction coefficient of Ei&& (1 mg/ml) = O-7 was used. Values for the free energy change (AC) of the polymerization reaction were derived using the relation AC = - RT In K, in which R represents the numerical value of the equilibrium constant, expressed on the basis of unit mole fractions of the participating species, R is the gas constant and T is the absolute temperature in deg. Kelvin. The equilibrium constant determines the relation C, = 6, + 30 (XX’,,)30, in which Co represents the total number concentration of protein subunits and Cn the number concentration of subunits present in the unpolymerized state. The exact meaning of the equilibrium constant K is outlined in the Discussion. (b) State diagram For the composition of the state diagram the extent of polymerization was measured in pyrophosphate buffer at 4°C at a total protein concentration of 1 mg/ml. Protein samples were examined from pH 5.0 to pH 8.0 at intervals of 0.5 and from ionic strength 0.2 to I.0 at intervals of O-1. AC values for pH 3.0 to 5.0 and for pH 8.0 to 9.0 were obtained by linear extrapolation. In the state diagram the phase boundary 3 S/30 S was drawn at 50% (w/w) polymerization. The position of the phase boundary along the c axis was obtained by substituting Cn = iCo in the above equations, leading to the relation Co = exp[AG~~08]~ The association behaviour of the protein at low ionic strength was studied turbidimetrically at a wavelength of 320 nm in IO-mm optical pathlength cells (Driedonks et aE., 1976). Gel filtration was performed in order to obtain a separation of different aggregation states of the protein. Protein samples were passed through a Sephadex G200 column (I.5 cm x 26 cm). Constant volume fractions were collected and a separation of the different types of aggregates was established by monitoring the absorbance of the protein at 280 nm. In this way purified solutions of 30 S particles were shown to remain homogeneous during 6 days at 4”C, thus allowing sedimentation equilibrium measurements. Protein solutions were concentrated by dialysis against a solution of 10% polyethylene glycol in the required buffer. Analytical ultracentrifugation experiments were carried out with a Spinco model E ultracentrifuge, equipped with Schlieren optics and an ultraviolet scanning system. In all experiments 12-mm optical pathlength cells were used. (c) Sedimentation

velocity

experiments

Measurements of s20,w values of the 30 S component were carried out with double-sector and single-sector centrepieces in the low (< 1 mg/ ml) and in the high concentration region, respectively, with rotor speeds of 44,000 and 48,000 revs/mm The s20,Wvalues of the 3 S component were measured with double-sector and single-sector, respectively, capillary

126

R. A. DRIEDONKS

,YT AL.

type synthetic boundary centrepieces, with rotor speeds of 56,000 and 60,000 revs/min. All experiments were carried out at 4°C. The sedimentation coefficients were determined by measuring the position of the maxima of the gradient curves in place of the second moment. The error thus introduced is less than 1% (Schachman, 1959). Sedimentation coefficients could routinely be determined using a computer program, based on a leastsquares fit of the logarithm of the distance of the boundary position to the axis of rotation wer8u.stime (Kruseman, 1969). The sedimentation coefficients were corrected for temperature and viscosity and extrapolated to zero concentration. Solvent viscosities were determined with an Ubbelohde viscometer. Relative amounts of the 3 S and 30 S components were determined either by measuring the relative absorbances at 280 nm, using a double-sector centrepiece, or by measuring the areas of the Schlieren peaks, using a double-sector capillary-type synthetic boundary centrepiece. In the former method data were corrected for radial dilution. Int he latter method photographs of the sample and of a reference sample (2 mg/ml) of pure 3 S component were taken 13 min after the rotor reached a speed of 52,000 revs/min. The Schlieren peaks of the 3 S component were traced on paper, cut, weighed and compared. The Johnston-Ogston effect has not been taken into account, as the sedimentation coefficients differ considerably and are found to be hardly dependent on the protein concentration (see Results). (d) Sedimentation epz&librium Molecular weights were determined using the conventional and meniscus-depletion sedimentation equilibrium technique (Yphantis, 1964) with the u.v.-scanning system. The experiments were carried out with g-channel centrepieces at a temperature of 4°C and rotor speeds of 17,000 revs/min for conventional, 26,000 revs/min for meniscus-depletion experiments with the 3 S component, and 4800 revs/min for meniscus-depletion experiments with the 30 S component. Conventional experiments lasted 50 h and meniscusdepletion experiments 40 h. Solvent densities were determined pycnometrically, using pycnometers of 5 and 10 ml. The partial specific volume of the protein (0) has been taken as 0.735 (Kruseman et al., 19’71), based on the amino acid composition. The U.V. detection system sampled the photomultiplier signal every 1.2 s and transferred these data to paper tape. In this way each scanning pattern could be converted into a set of 250 data points. This set was made compatible for further processing on an IBM 370 computer. With the aid of a computer program each number of sampled values required could be used to produce a plot of In c - r2, where c represents the protein concentration and r the distance from the centre of rotation. In the program the slope of these plots is calculated, using a least-squares subroutine and finally the molecular weight is calculated with the relation d In c 2RT xi!, = (1 - Bp)w2 dr2’ where a’w is the weight averaged molecular weight, R the gas constant, T the absolute temperature, p the density of the solution and w the angular velocity. The corresponding value of c was derived from the arithmetic mean of the values of In c used in the In c - r2 plot. The relative uncertainty in the determination of the concentration increases with decreasing protein concentration. Therefore more adjacent points of the sampled concentration curve were required to produce a reliable In c - r2 plot in the low concentration region near the top of the cell. Therefore sets of data (a,, c) were obtained by taking decreasing numbers of adjacent points for the calculation of il?lw, always starting from the bottom of the cell with a maximum of 27 and a minimum of 5 points, and at constant intervals. In this way molecular weights could be determined at protein concentrations as low as 0.06 mg/ml. For each set of conditions (pH and ionic strength) 5 initial protein concentrations were used, ranging from 0.2 to 0.6 mg/ml. Sets of data (z,, c) were evaluated from at least 10 scans per set of conditions. A computer program was used for the extrapolation of molecular weights and sedimentation coefficients and for the determination of the reaction enthalpy and entropy.

AMV

COAT

PROTEIN

ASSOCIATJON

127

In this program a linear regression line was calculated through a number of points, based on a least-squares method. The errors given for the intercepts (molecular weights and sedimentation coefficients) and slopes (enthalpy and entropy) of the regression lines were derived from twice the standard deviation of the slope, being the limits of the 950/, proba,bility region. The parameters of the regression lines are summarized in Table 1. (e) Electron microscopy and image analysis Negatively stained specimens were prepared using 1% uranyl acetate as the stain. The protein samples were prepared in stain over holes or mounted on carbon (see Me&ma $ van den Berg, 1974). A Philips EM300 electron microscope operating at 80 kV was used to obtain images of these specimens. Analysis of the images was performed by optical diffraction and by digital analysis. The procedures as well as the hardware involved in the analysis have been described by Mellema (1975). For the analysis of arrays of 30 S particles a number of images of well-preserved particles (as judged by optical diffraction analysis) were digitized and their Fourier transforms obtained (DeRosier & Moore, 1970). The length of the selected parts of the arrays did not exceed 5 to 6 spherical particles, because of the lack of straightness of the arrays. Neither the optical nor the digital transforms gave convincing evidence for the presence of a, constant difference in orientation of successive particles within the arrays. For example, in the case of linear polymers of haemocyanin particles, the transforms of the images exhibit clear off-meridional maxima, indicating a constant difference in azimuth between successive particles (Mellema & Klug, 1972). Therefore a number of cylindrically averaged (Finch & Klug, 1971) images were calculated with the assumption that, at, the working resolution, the only contributions to the equatorial and meridional data arose from Jo terms. In fact, meridionally symmetric maxima on these layer lines differed in phase by 0 or 2 v to a good approximation. The average layer line distance measured approx. I/l25 a-l. The data were made perfectly e-fold by assigning real values to the Fourier coefficients after searching for the best 2-fold origin. This is plausible because at low resolution the projection of a structure with 532 point group symmetry is 2-fold. Phase residuals which measured the departure of 2-foldness had values of 13, 14 and 30 deg. in 3 typical cases. The layer lines involved in the synthesis had spacings to an axial resolution of about E/25 11-l and a radial resolution of about l/30 8-l.

3. Results Preliminary data about the range of pH and ionic strength which determine the association behaviour of AMV coat protein have been reported by Driedonks et al. (1976). Here we present the final results based on more and extensive measurements. The aggregation features of the protein are presented in a perspective plot with variables of ionic strength, pH and protein concentration (Fig. 1). In this graph three main areas can be distinguished: the so-called 3 5, 30 S and precipitation area (I’). They are indicated by different markings in the Figure. The characteristics of the products are discussed below. The pH range studied covers the physiological p range. Once exposed to more extreme values of pH than those presented (higher t’han 9-O and lower than 3.0) the protein is no longer able to associate into the 30 S particle when brought back to the right conditions (e.g. pH 5.5 and I = 0.5). At increasing ionic strength the 30 S particle has a slight tendency to depolymerize. The nature of the buffer ions appears to have some influence on the rate and extent of polymerization, but not on the type of association product formed. The blank region in Figure 1 is characteristic for a single species with an &” Tv value of (2*78&0*05) S, based on a least-squares analysis (see Materials and Methods). Figure 2 represents a plot of sedimentation coefficients derived from pH values in the acid a,nd alk.aline region of the 3 S area. The ionic strength does not appreciably

X mean,

106

2.7765

24.9964

6.7288

1.4818~ 10”

48.4401 x 10”

31.8199

I’ mean. The 9504 probability

- 3.2096

1nK = f (l/T)

lines run through

-0.0511

AC = f (T)

The regression of the slope A.

-0,4179x

M,, 30 S = f (concn)

1

limits,

given

are derived

1.1102

0.0078

0.0652 x 106

1.9295 x 103

0.1054

0.0078

S.D. OfA

2, 3, 5, 6, IO(a)

in the text

15

15

102

159

6

17

points

No. of

regression lines, presented in Figures

- 12.0292 x 103

0.0372

M,, 3 S = f (concn)

-- 0.0004

szo,w, 3 8 = f (concn)

of the linear

szo,w, 30 s = f (concn)

Parameters

TABLE

from twice

13.6600

- 7.7461

deviation

1.3809 x lo6

44.9138 x lo3

32.0333

2.7753

Y mean

the standard

3.5320

283.2333

0.2416

0.2931

5.7333

2.9812

x mean

and (b)

(SD.)

AMV

COAT

PROTEIN

ASSOCIATION

12d

0

FIG. 1. Three-dimensional state diagram of AMV coat protein, representing the different oligomoric states of the protein as a function of pH, ionic strength and protein concentration. Three areas can be distinguished : the blank region, being the stability region for t,he 3 S component; the region indicated by 30 S and a region indicated by P, in which the protein precipitates. The 3 S and 30 S components are representive for the dimer and the 60.mer of the protein molecule, respectively. The 3 S--30 S phase boundary has been drawn at, 50% (w/w) Wansition of t,he 3 S into the 30 S component. The association behaviour of the protein has been studied in pyrophosphate buffer at 4°C a.s indicat.ed in Materials and Methods.

of this influence the sZO.Wvalues. The results of the molecular weight determination material are displayed in Figure 3. An extrapolated value at c = 0 of (48.4kl.I) x lo3 daltons was determined, based on a least-squares refinement. In this ca~lculat,ion measurements at different conditions of pH and ionic strength were used. The molecular weight derived is; to a good approximation, twice the value of 24:250: the molecular weight of the protein monomer (van Beynum, 1975). Therefore, we conclude t,hat the A&IV coat protein in the 3 S form exists predominantly as a dimer. The 30 S area in Figure 1 is typical for a roughly spherical particle. A micrograph of a negatively stained preparation is shown in Figure 4. An average diameter of 123 A with a standard deviation of 12 A can be measured from the micrographs. The weight spherical particle has an & w value of (31.8% 1.2) S (Fig. 5). Molecular determinations of the purified a,ggregation product (see Materials and Methods) are presented in Figure 6. A least-squares fit of the data gives a value of (1*4S&O.O3) x IO6 for the molecular weight of the 30 S component, extrapolated t,o zero concentration. This value strongly suggests that the particle consists of 60 copies of the single polypeptide chain (M, = 24,250). Therefore the 30 S particle must possess a 552 point group symmetry (Caspar & Klug, 1962). Apart from the products which are found in the precipitation region (Fig. I), which are derived from the spherical 30 S particle (see below) no other type of association product has been detected. In order to be certain that the 3 S-30 S system really represents a chemical equilibrium, a number of experiments were performed. As has been reported in our earlier work, the association of the 3 S int’o the 30 S particle follows the pattern of a linear polymerization reaction (see

R. A. DRIEDONKS

130

X:2’ AL.

I 5

I IO

c protein

(mg/ml)

FIG. 2. Sedimentation coefficients of the depolymerized coat protein corrected for viscosity and temperature, as a function of protein concentration. (0) Protein sampIes dialysed against sodium acetate buffer (pH 4.8), ionic strength 0.6; (0) protein samples dialysed against sodium pyrophosphate buffer (pH 8.5), ionic strength 0.6. Experiments were carried out at 4°C in a capillary type centrepiece at rotor speeds of 56,000 and 60,000 revs/min. Materials and Methods, section (d), lists the least-squares solution of the data set.

60

I-

I

I

I

0.4

O-6

0.0

c protein

brig/ml)

FIG. 3. Weight averaged molecular weight (a,,,) of the 3 S component as a function of protein concentration as determined with sedimentation equilibrium experiments. Protein samples were dialysed against : (f) Acetate buffer (pH 4.8), ionic strength 0.6; (0) 1 rnM-acetate buffer (pH 5.0); (0) pyrophosphate buffer (pH 8.4), ionic strength 0.7. Experiments were carried out at, 4°C at rotor speeds of 17,000 and 26,000 revs/n&. The extrapolated weight at C=O was determined by least-squares: the equation for this line is given in LVaterials and Methods.

e.g. Oosawa & Higashi, 1967). This means that the association reaction does not run to completion, but that there is aIways a’ certain amount of 3 S material present besides the end product. The extent of association has been shown to be dependent on the protein concentration. Therefore the equilibrium is completely characterized by the ratio of the amounts of 30 S to 3 S, together with the total concentration of protein. Also, upon separation of the two components (see Materials and Methods)

AMV

COAT

PROTEIN

ASSOCIATIOS

131

FIG. 4. Electron micrograph of 30 S particles, negatively stained with 1 y. uranyl acetake. The particles were hnng in a film of stain over a hole in the supporting fitm. Solvent conditions: pyrophosphate buffer (pH h-5), ionic strength 0.3. Both free 30 S pertides and arrays ofparticies are visible. Magnification, 260,000 x .

1

I 5

1

I

r

15 cprotein (mg/ml)

FIG. 5. Sedimentation coefficients of the spherical association product, corrected for viscosity and temperature, as a function of protein concentration. Protein samples were dialysed against, pgrophosphate buffer, pH 5.5 and ionic strength 0.50. Experiments were done at 4°C in doublesector centrepieces at rotor speeds of 44,000 and 48,000 revs/mix For the least-squares solution see Materials and Methods.

the 3 S material must again be able to undergo an association rea,ction; if the right condit,ions are met. Part of t.he isolated 30 S particles must shorn some extent of dissociation. Mea,sured over several days after the association reaction has started, t,he ratio of30 S to 3 S attained a constant value after a couple of hours and remained constant. The concentrated 3 S material separated from the 30 S rapidly undergoes a new

132

R. A. DRIEDONKS

ET AL.

c protein hg/mll

FIG. 6. VVeight averaged molecular weight (A?,) of the purified 30 S component as a function of protein concentration, as determined with meniscus-depletion sedimentation equilibrium experiments. The solvent consisted of pyrophosphate buffer, pH 5.5 and ionic strength 0.50. Experiments were done at 4°C and at a rotor speed of 4800 revs/min. For details of the slope and intercept of the least-squares fit see Materials and Nethods.

aggregation, whereas the 30 S material very slowly dissociates (several weeks) into 3 S. Also the 30 S to 3 S ratio varies in a predictable manner when the solvent conditions are changed. Therefore we conclude that the 3 S and 30 S particles form a reversible equilibrium and that no other products are formed after longer reaction times. In the case of linear or helical polymerization of identical protein subunits it can be shown that the association is a quasi-crystallization (see e.g. Oosawa & Higashi, 1967). Also in the case of the polymerization of protein subunits into a polyhedral aggregate this conception is valid and similar behaviour of the reaction partners can be derived (Oosawa & Higashi, 1967). In the case of 3 S to 30 S transition the beginning and end products can be easily detected and we can use these concentrations to estimate the equilibrium constant for the association process. For this equilibrium the following relation holds: C, = C, + 30K,,, CF, in which C, is the total number concentration of 3 S before the reaction, C, the number concentration of free dimers after the reaction and Ktot the equilibrium constant of the 3 S-30 S transition. Cz is the total number concentration of dimers associated within the icosahedron and consequently is measured in mol/mol. If the polymerization is represented by a, stepwise addition of one dimer to the icosahedral lattice, an equilibrium constant Ki can be introduced for each successive step. As shown in the Discussion, there is only a limited number of different K, values. However, all dimers occupy equivalent positions within the spherical lattice and therefore it is possible to define an average equilibrium constant (K) for the insertion of one dimer int,o the lattice. The above relation now becomes G, = C, + 30(KC,)30. From plots of C, versus C, or 30(KCD)30 the equilibrium constant K or the free energy may be evaluated as demonstrated in Figure 7. To a first approximation each set of data (C,,,C,) at different conditions of pH and ionic strength, with different types of buffer ions or as a function of temperature, directly yields a value of K7 and consequently for AQ, using the above relation.

AMV

COAT

PROTEIN

ASSOCIATION

C, x 106(mol/mol)

7. Quasi-crystallization behaviour of the coat protein. The number concentration of unpolymerized dimers and the number concentration of dimers incorporated in the 30 S state have been plotted against the total number concentration of dimers present,. In the expression C!, = CD + 30(KCD)30 the total number concentration of dimers present is given by C, : G, represents the number concentration of free dimers and 30(KCD)30 represents the number concentration of dimers polymerized into the 30 S component. Protein samples were dialysed against pyrophosphate buffer (pH 55), ionic strength 0.50, at a temperature of 4°C. Concentrations of the 30 S (0) and 3 S (@) components were measured with the analytical ultracentrifuge. The curves drawn represent theoretical curves based upon the theory of icosahedral polymerization. The horizontal line represents CD as a function of C,, whereas the other line in the Figure depicts the dependence of 30(KCD)30 on C,, assuming a K FIG.

of 3 x I OB.

The effects of the different types of buffer ions are presented in a gallery of Schlieren pictures in Figure 8. Expressed as free energy changes for the association of a dimer, it can be seen that the changes are larger for pyre- and triphosphate-containing solutions than for solutions containing carboxylic ions (Table 2A). Moreover, it is evident that the free energy change increases in the orthophosphate, pyrophosphate and ATP solutions, respectively. Also it appears in a number of cases that the free energy change was lower in solutions of monovalent ions (chloride) than in solutions of similar pH and I values and containing only buffering ions (results not shown). The effects of pH and ionic strength have been tabulated for two cases with varying pH (Table 2B). From these results it follows that there is an optimum at pH 5.5 and that polymerization takes place to a slightly higher extent at lower ionic strength. At these conditions also AG was determined as a function of temperature (examples al=e shown in Fig. 9). From the data presented in Figure 10 values were derived for AH and AS of (6*4&4,4) kilocalories per mole and (51& 16) entropy units, respectively, being the average enthalpy and entropy changes on insertion of one dimer into t,he lattice. The precipitation area has been shown to consist of linear and three-dimensional association products of the 30 S particles (Driedonks et aZ., 1976). The micrographs of the linear arrays of the 30 S particles were analysed in more detail (see Materials and LMethods). Figure 11 shows a micrograph of such a polymer ; the diffraction patDern is also presented. Apart from the clear meridional maxima in the transform, approximately equally spaced at l/125 A-l, so far no reliable information of the presence of

R. A. DRIEDONKS

134

(6)

(0)

ET

AL.

(11

8. Schlieren diagrams of protein samples in different buffers, showing t.he influence of anions on the extent of polymerization. Protein samples were dialysed against a buffer (pH 5.5 and ionic strength 0.50) at 4°C. The following protein concentrations and buffers were used: (a) 15 mg/ml in acetate; (b) 20 mg/ml in oxalate; (c) 20 mg/ml in citrate; (d) 20 mg/ml in phosphate; (e) 15 mg/ml in pyrophosphate; (f) 15 mg/ml in 5 mlvr-acetate cont,aining 70 mg/ml ATP. Photographs were taken about 13 min after the rotor reached a speed of 52,000 revs/n&. Schlieren angle 70”. For the determination of the free energy changes see Materials and Methods. FIG.

(0)

(b)

Ccl

9. Some examples of Schlieren diagrams from which the reaction enthalphy and entropy has been derived. The protein with initial concentration 7.6 mg/ml has been dialysed against phosphate buffer (pH 5.5 and ionic strength 0.50) at the following temperatures: (a) 0°C; (b) 9°C; (c) 24°C. Photographs taken 13 min after the rotor reached a speed of 52;OOO revs/min. Schlieren angle 70”. FIQ.

AX%’

COAT

PROTEIN TABLE

A.

Qumtitutive

effects of diflerent

Anion Acetate Oxalate Citrate Phosphate Pyrophosphate Triphosphate

135

ASSOCISTIOS 2

anions upon the association

Gb%/ml)

Wmdml)

15.0 20.0 20.0

11.0 3.24 1.74

15.0

0.32

K(

x

10m5)

1.4 5.0 9.3 6.2 31.3 50.2

of .4XV

coat protein,

AQ (lrcaljmol) -6.6*0.4? -7.3*0.41 -7.6&0.4t -7.4&0.4$ --8.3&0.4$ - f+s*o.4t

Temperature, 4OC; pH 5.5; ionic strength, 0.50. ,Uthough non-idea,lity may play a significant role at the concentrations used in the experiments no correction for this has been made in the calculation of LG. The reason for this is that the relative data illustrate the influence of the anions on the polymerization suffioiently. Correction for non-ideality will alter the magnitude of the data, but not the relationship between the data. t Data obtained from a single experiment; the error in the dG values is estimated and supposed to be of t,he same order of magnitude as those in phosphate and pyrophosphate. j Data derived from at least 15 experiments.

B. Quan,titative

effects of p,H mnd ionic strength upon the association of AMV for a typical batch of protein I = 0.50

pI3

co !mg/ml)

5

1 .oo

5.5 6-5 7~5

0.96 0.86 0.86

(m$ml) 0.48 0.24 0.45 0.69

I = 0.90

K( x 10-b) 29.8 60.7 31.7 19.9

coat proteirt

AG

jlical/mol) -8.3hO.4 --8.7+0,4 -8.3+0.4 -GO&O.4

co (w/ml) 1.07 0.95 1.10 0.89

CD (mgiml) 0.56 0.31 0.67 0.83

K( ’ 10-5)

(lic~~ol)

25.5 47.0 21.4 16.1

-8.2IO.4 -8.5zO.4 --8.1;0.4 -7.9io.4

Association studied in pyrophosphate buffer at 4°C. The polymerization takes place optimally at pH 5.5. The extent of polymerization increases with decreasing ionic strmength values. However, below I = 0.40 the 30 S particles tend to associate into larger aggregates. Consequently the relative amounts of 3 S and 30 S components cannot be reliably measured with the analytical ultracentrifuge under these conditions.

off-meridional imensities has been obtained. Therefore, it is not known how successive particles within such an array are oriented (see Materials and Methods). A number of these images were digitally processed and the dat’a of the principal layer lines (having meridional information) were used to calculate a cylindrically averaged densit,y map. One or‘ the results is displayed in Figure 11. It can be observed that the particles are hollow spheres, which have a shell of protein with an average thickness of about 46) A. Dominant contacts between particles within the array lie at a radius of about 25 A.

4. Discussion (a) Xtructural

information

In the association behaviour of AMV coat protein the 3 S + 30 S equilibrium occupies a, central position. The molecular weight of the 3 S component was determined earlier by Kruseman et al. (1971), who performed equilibrium measurements at

136

ET AL.

R. A. DRIEDONKS

I

I

270

280

3.4

I

290

3.5 I/T Lx IO')

I

300

3.6

FIG. 10. Reaction enthalpy and entropy of the 3 S + 30 S equilibrium. Data obtained from 3 different batches of protein (+, 0, 0). Protein samples with inital concentrations between 3.5 and 7.5 mg/ml were dialysed against phosphate buffer (pH 5.5 and ionic strength 0.50) at different temperatures. Concentrations of the 3 S and 30 S components were measured with the analytical ultraoentrifuge (see Fig. 9). (a) Free energy change as a function of the temperature, yielding AS = (51+16) entropy units; (b) van’t Hoff plot, from which a reaction enthalpy AH = (6.4+4.4) kcal/mol has been derived. For the least-squares solutions of both sets of data see Materials and Methods.

pH 5.6 in acetate with concentrations ranging from 0.26 to 0.32 mg/ml; they found evidence for a dimeric particle. Our measurements confirm that the dimer is a stable configuration over a wide range of conditions. Therefore it can be concluded that the dimers are most proba,bly the active units in the assembly process. For the determination of the molecular weight of the 30 S particle as well as the thermodynamic parameters, hydrodynamic measurements have been made. This type of analysis is known to affect the local concentration of the component’s and consequently the equilibrium state will be perturbed, ca,using a readjustment of the equilibrium conditions. As was stated in Materials and Methods, in solutions of purified 30 S, no 3 S particles derived from 30 S can be detected during at least six days, As the sedimentation equilibrium measurements took place over two days, the molecular weight of the 30 S particles will ha,rdly be influenced by this effect. The determination yields a value of roughly 30 times the molecular weight of the

AMV

COAT

PROTEIS

ASSOCIATION

-zzs (b) FIG. 11. Analysis of an array of 30 S particies. (a) LH~crograph of a negatively stained array of 30 S particles. Magnification 660,000 dlgitized image of this array has been Fourier transformed (DeRosier & Moore, 1970). contour pattern of the amplitude map of this transform. (c) The cylindrically averaged map of the array on the left obtained by using the information on the principal layer the transform. (See also Materials and Methods.)

x , The (b) The density Iines in

which is not surprising in view of the possible spherica,l lattices of such a structure. Caspar & Klug (1962) pointed out that on geometrical grounds only ieosahedml particles can possibly be characterized by T = 1,3,4,7, etc. A T = 3 shell will accomodate 90 dimers and is expected to have a dia’meter much larger than the value of 123 A determined in this study. For these reasons the conclusion that a 6C)-mer exists seems well justified. The orientation of the dimers in the icosahedron is such that the lattice dyads must coincide with the 2-fold axes of the dimers. The qua,ternary structure of the 30 S particle therefore resembles that) of the cylindrical part of the virus coat (Mellema, 1975). The conversion of the cylindrical lattice into as icosahedral one can be realized by converting the 6-fold lattice positions into &fold ones (Caspar & Klug, 1962). It is beyond doubt that the rounded caps of the AMY nucleoprotein particles have been built to this kind of design. Until now no reliable evidence has been obtained to indica,te that the electron microscope projections of the “30 S” particle possess icosahedral symmetry. In view of the relatively small diameter and the gross morphology of the structure (to a first approximation a hollow sphere) as compared to several isometric viruses (e.g. turnip yellow mosaic virus, tomato bushy stunt virus), the details in negatively stained prepamtions will be hard to assess. Figure 12 shows a schematic drawing of the proposed orientation of the protein dimers in t,he icosahedral net. The protein dimers a,re bound through four homologous interactions with their four neighbours.

dimer,

(To) Thermodynamic

considerations

In t,he polymerization process the number of new bonds crea,ted wit11 each addition of a, subunit into the spherical lattice ranges from one to four. Therefore the assoeiation can be described by four types of equilibrium constants, which reflect the

138

R. A. DRIEDO;?;KS

El’

AL.

FIG. 12. Proposed model for the orientation of the protein subunits in the 30 S particle. For the sake of clarity the Figure does not show the back view of the particle. 30 dimers have been arranged over the lattice of the 97 = 1 icosahedron in such a way that the dimeric dyads coincide with the Z-fold positions of the lattice. Each dimer is bound through 4 homologous bonds with its 4 nearest neighbours, represented by solid rods. Long-range interactions have been omitted in the model. The icosahedral net is indicated by broken lines.

formation of one, two, three or four homologous dimer-dimer interactions in the lattice. Averaged over-all steps in the polymerization process, the addition of a dimer to the spherical laGice leads to the formation of two dimer-dimer bonds. The average equilibrium constant, occurring in the relation 6, = C, + 30(KC,)30, is now K = exp(2 e,/kT), where e, represents the free energy change on forming one dimerFor a single dimer bond, k is the Boltzmann factor and T the absolute temperature. bond a reaction enthalpy of 1-O to 5.4 kilocalories per mole and an entropy of 18 to 33 entropy units can thus be derived for the association in phosphate buffer. It was remarked earlier in the Discussion, section (a), that the determination of the concentrations of 3 S and 30 S may not reflect the equilibrium values. As in the case of the molecular weight determinations of 30 S particles the question is, how fast is the new situation adjusting itself to equilibrium? If it takes place very slowly compared to the duration of the experiment, the error will be negligible. On the other hand, when the readjustment is a relatively fa#st process the observed G, value does not reflect the value at the sta,rt of the experiment. No substantial errors are made if C, is roughly independent of C,. In a number of polymerizing systems, e.g. actin, flagellin, tobacco mosaic virus coat protein and cowpea chlorotic mottle virus coat protein, this situation has been detected and it has been shown that the theory of polymerization equilibrium can be applied in these cases (see e.g., Oosawa & Asakura, 1975 ; Adolph $ Butler, 1974). Therefore it seems plausible that the theory is also valid in the 3 S-30 S system. The error introduced by a fast readjustment of the equilibrium can be estimated by the relation between C, and C,. Over the working range the error in the concentration of C, is 60/, at the maximum, which represents

AMV

COAT

PROTEIN

ASSOCIATION

139

t,lle difference between C, at a protein concentration of 20 mg/m! and C, at the conceutrat,ion at which polymerization starts (0.52 mg/ml). This leads t,o an insccurecy in AG of about 0.03 kilocalorie/mole, which is negligible compared with the other errors in the determination (about 0.4 kilocalorie per mole). Our observations on the influence of the types of anions on the association rea,ction in some wa,y resemble the results obtained by Lonchampt et al. (1972), who studied t,he polymerization of tobacco mosaic virus protein in pyrophosphate buffer. These authors also found that polymerization in this buffer proceeded very quickly compared w&h, for example, polymerization in phosphate. As the extent of polymerization is different in the various buffer types, the position of the phase boundaries in the date diagram depend on the buffer used. We have not found it practical, in view of the amount of work, to determine these boundaries in other buffer systems. However, some of our results with other buffers than pyrophosphate do suggest that the buffer Lype influences the size of the 30 S stability region but not its shape. The dimer is a stable product over a wide range of pH and ionic strength. Therefore it could be suggested that the binding of the monomers within the dimers is mainly stabilized by hydrophobic interactions. The features of polymerization resemble those of, for instance, disc formation of tobacco mosaic virus protein so far as both processes are entropy-driven (for a review, see Lauffer, 1975). The large entropy effect suggests that the icosahedral particle is stabilized either by hydrophobic or electrostatic interactions (Kauzmann, E9.59)_ At this moment it is still rather speculative to decide which kind of interaction is mainly responsible for stabilizing the quaternary structure. The polymerization reaction was found to be a quasi-crystallization process. Similar behaviour has been observed in cowpea chlorotic mott.le virus protein polymerization (Adolph $ Butler, 1974). At the moment it is not possible to separate the whole process into a nucleation and a growt,h step (Oosa,wa & Higashi, 1967). No intermediate products in the association reaction have been observed in solution ; consequently, it is hasd to visualize the type of mechanism involved in the polymerization of dimers into a polyhedral structure.

(c) Prosfpects In our previous paper (Driedonks et aZ., 1976) we reported the existence of a 12 S component. A number of experiments showed us that this product could not be found reproducibly. This confirms our earlier conclusion that the 12 S component is not a,n intermediate in the polymerization process. Until now no evidence has been obtained that AiMV coat protein is able to polymerize into particles of cylindrical shape. Lebeurier et al. (1971) has shown that incubaGon of AMY protein with Haemophilus i@uenzae DNA leads to cylindrical particles. This observation as well as our experiments on the role of nucleic acid in the assembly react’ion lead to the conclusion that cylindrical particles are only found in the presence of nucleic acids of sufficient length. The gross morphology of DEA-AMV coat protein complexes can be shown to be similar to that derived from the cylindrical part of the virions (unpublished data). The relatively high value of AG in the polymerizat,ion react.ion of the protein in pyro- and triphosphate-cont.aining solutions suggests that the phosphate groups in the RNA play an important role in the stabilization of the virion. At present, research is concentrated on the elucidation of the role

140

R. A. DRIEDONKS

E2’

AL.

of the nucleic acid component in the assembly process. Binding studies of protein with nucleotides are in progress in order to determine the thermodynamic parameters of the protein-nucleic acid interaction. This type of research, together with the results already obtained, should eventually lead to a better understanding of the mechanism of initiation and subsequent growth during AMV assembly. The authors gratefully acknowledge the expert assistance of Mrs T. Remmelzwaal and Dr H. J. Blanksma with the data processing. We thank Mr J. A. P. van der Voort for interfacing the ultracentrifuge with a digital output device and for his help with these experiments. REFERENCES Adolph, K. W. & Butler, P. J. G. (1974). J. Mol. Biol. 88, 3277341. Bol, J. F. & Veldstra, H. (1969). Virology, 37, 74-85. Casjens, S. & King, J. (1975). Amnu. Rev. Biochem. 44, 555-611. Caspsr, D. L. D. & Klug, A. (1962). Cold Sprirzg Harbor Xymp. Quant. Biol. 27, l-24. Champness, J. N., Bloomer, a. C., Bricogne, G., Butler, P. J. G. & Klug, A. (1976). Nature (London), 259, X-24. DeRosier, D. J. & Moore, P. B. (1970). J. Mol. Biol. 52, 355-369. Driedonks, R. A., Krijgsman, P. C. J. & Mellema, J. E. (1976). Phil. Trans. Roy. Xoc. ser. B, 276, 131-141. Durham, A. C. H., Finch, J. T. & Klug, A. (1971). Nature New Biol. 229, 37-42. Finch, J. T. & Klug, A. (1971). PhiZ. Trans. Roy.Xoc. ser. B, 261, 211-219. Hull, R. (1970). Virology, 40, 34-47. Jaspars, E. M. J. (1974). Adwan. Virus Res. 19, 37-140. Kauzmann, W. (1959). Advan. Prot. Chem. 14, l-63. Kruseman, J. (1969). Thesis, University of Leiden, The Netherlands. Kruseman, J., Kraal, B., Jaspars, E. M. J., Bol, J.. F., Brederode, T. Th. & Veldstra, H. (1971). Biochemistry, 10, 447-455. Springer Verlag, Berlin, Lauffer, M. A. (1975). Entropy-driven Processes in Biology, Heidelberg, New York. Lebeurier, G., Fraenkel-Conrat, H., Wurtz, M. 85 Hirth, L. (1971). Virology, 43, 51-61. Lonchampt, M., Lebeurier, G. & Hirth, L. (1972). PEBX Letters, 22, 297-300. Mellema, J. E. (1975). J. Mol. Biol. 94, 643-648. Mellema, J. E. & Klug, A. (1972). Nature (London), 239, 146-150. Mellema, J. E. & van den Berg, H. W. J. (1974). 3. Supramol. Struct. 2, 17-31. Oosawa, F. & Asakura, S. (1975). Thermodynamics of the Polymerization of Protein, Academic Press, London. Oosawa, F. & Higashi, S. (1967). Progr. Theoret. BioZ. 1, 79-164. 61, 160-167. Pfeiffer, P. & Hirth, L. (1974). Virology, Sohachman, H. K. (1959). UZtracentrijugation in Biochemistry, Academic Press, New York. London. van Beynum, G. M. A. (1975). Thesis, University of Leiden, The Netherlands. van Vloten-Doting, I,. & Jaspars, E. M. J. (1972). Virology, 48, 699-708. Yphantis, D. A. (1964). Biochemistry, 3, 297-317.