Circular dichroism studies of papaya mosaic virus coat protein and its polymers

J. Mol.

Riol.

(1981)

147, 337-349

Circular Dichroism Studies of Papaya Mosaic Virus Coat Protein and Its Polymers J. W. ERICKSON~~,

J. B. BANCRO~

1Department

of Plant

AND

M. J. STILLMAN*$

Sciences

AND

‘Department of Chemistry The C~nioersity of Western Ontario lo&on, Ontario, Canada (Received

14 April

1980, and in revised form

26 August

1980)

The near-u.v. circular dichroism of papaya mosaic virus coat protein is dominated by intrasubunit interactions involving tryptophan and, to a much lesser extent, phenylelanine. The conformations of the 14 S protein discs at pH 4.0, pH 6-O and pH 8.0 are judged to be similar by circular dichroism. At pH 8.0, however, the conformation of the protein is influenced by the extent of aggregation, which is reflected in the intensity, but not the shape, of the circular dichroism spectrum. The increase in intensity level that accompanies aggregation at pH 8-O mainly appesrs to reflect modifications of tryptophanyl interactions. On the other hand, no spectral changes result when discs polymerize to form helical tubes at pH 4.0, indicating that a major conformational change is not requisite for helix formation. The above results are interpreted in view of the various protein and virus assembly reactions in which papaya mosaic virus coat protein participates. Denaturation of the coat protein results in quenching of optical activity in the near-u.v. region, and also in marked changes in the U.V. spectrum of the protein. These results indicate that the majority of the tryptophan and tyrosine residues are

buried

within

a hydrophobic

environment

in the undenatured

protein.

1. Introduction The coat protein of papaya mosaic virus self-assembles into a variety of polymers (Erickson et al., 1976; Erickson & Bencroft, 1978). A phase diagram describing the effects of pH and temperature on PMV$ protein polymorphism is presented in Figure 1. The 14 S polymer, which is most probably a two-layer ring or disc composed of 18 subunits, predominates over a wide range of pH, temperature, ionic strength and protein concentration. During virus assembly at pH 8.0, this disc may t Present address: Department of Biological Sciences, Purdue University, W. Lafayette, Ind. 47907, U.S.A. $ Member of the Centre for Interdisciplinary Studies in Chemical Physics at the University of Western Ontario. $ Abbreviat’ions used: PMV, papaya mosaic virus; TMV. tobacco mosaic virus: CD, circular dichroism. 337 0 1981 Academic Press Inc. (London) Ltd. 0022%2836/81/100337-13 $02.00/O

rl.

3

4

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ERIC’KSOS

/;7’

6

7

5

.-1/J.

8

9

IO

PH PIG. protein inverse pH 4.0 at, pH

1. States of aggregation of PM\’ protein: phase diagram. Effects of temperature and pH on polymerization are illustrated. Thick arrows indicate dependence (direct for upward arrow and for downward arrow) of the protein equilibrium at pH 8.0 on various factors. The diagram from to pH 6.0 represents the protein in a metastable equilibrium. Long term dialysis of PM\’ protein 4.0 to pH 6.0 at 5°C results in rod formation (Durham & Bancroft, 1979).

be responsible for specific encapsidation, which occurs near the 5’ end of PMVRNA. At pH 6.0, it participates in non-specific, multiple initiations with any singlestranded, natural or synthetic, nucleic acid (Erickson $ Bancroft, 1978.1981 ; Erickson et al., 1978; AbouHaidar & Bancroft, 1978). We wished to know whether the pH 6-O and pH 8.0 discs exhibited structural differences that. would account for their widely different eneapsidation properties, but which are too subtle for detection by hydrodynamic methods. Accordingly we employed circular dichroism in the near-u.v. region (250 to 340 nm) as a probe for inspecting the tertiary structure of these polymers in the environs of aromatic amino acid side-chains (Strickland, 1974). The CD of the helical protein tube, as well as of dissociated and denatured protein, were also investigated. Analytical ultracentrifugation was employed throughout these studies to monitor the polymerization level of the protein.

2. Materials (a) Preparation

and Methods of PM

V coat protein

Coat protein was prepared from purified PMV by the acetic acid m&hod (FraenkelConrat, 1957) as described (Erickson & Bancroft, 1978). In the experiments using high protein concentration, the protein was concentrated with 5Oo/o saturated ammonium sulfate, 001 M-Tris (pH 8.0). The precipitate was centrifuged, resuspended in a small volume of water, extensively dialyzed and stored at 5°C.

CD

STUDIES

OF

(b) Analytical

PM\’

PKOTEIX

339

uZtrucentr+g~tion

Analytical centrifugation was done in a Spinco model E ultracentrifuge under the described conditions. Protein samples at concentrations of 1.0 AZsO nm unit/ml and below were run in 30 mm double-sector cells; 12 mm double-sector cells were used for protein at higher concentrations. Dissociated protein at pH 3.0 was sedimented in a synthetic boundary cell. Values for the viscosity and density corrections in 5 M-urea were taken from the International Bureau of Standards tables. (c) Measurement

of protein concentrations

and turbidity

Protein concentrations were determined with a Gary 219 recording spectrophotometer with an automatic baseline subtraction. Az80 “,,, values were calculated after correcting for which were carried out in the same scattered light. For the turbidity measurements, instrument, the sample cell was thermostatically jacketed, and temperature control was maintained using a refrigerated, recirculating water bath. (d) CD spectroscopy CD spectra of protein samples were measured over the range 250 to 340 nm, at about recording spectropolarimeter equipped with a 1.0 =12*0 unit/ml, with a JASCO ORD/UV-5 Sproul SS-20 CD modification. Spectra in this region were often noisy, so that it was necessary to average 2 or more spectra from the same sample. For the variable temperature studies, temperature control was maintained with a water-jacketed cell holder and a refrigerated, recirculating water bath. Sample temperatures were measured at the beginning and end of each spectrum. The maximum fluctuation during a recording was W6deg.C. Positive air pressure was applied to the sample chamber to minimize temperature fluctuation and to prevent condensation on cell windows. All other spectra were recorded at room temperature between 24°C and 26°C. CD spectra at different protein concentrations were measured in cells of differing path lengths. The combination of path length and concentration was chosen so that their product was around l.Ocm.Azso unit. For @l A,,, unit/ml, however, a 5.0 cm path length was employed, and the resulting spectrum was scaled accordingly. All CD sample and baseline spectra were digitized manually. Baseline subtraction, spectra averaging, normalization, computation of difference spectra, and plotting of the final spectra were done by computer. In some cases, it was necessary to perform curve smoothing. CD is expressed as molar dichroic absorbance, dc( =c~--E~). baaed on ~74,,,,,~,=22,000 (Rees, unpublished observations) and ,l$rb,,,, = 075 (Erickson, unpublished results). Since the CD spectrometer measures ellipticity, 0, AC values were derived from the molar ellipticity [e] using the conversion A~=[0]/3300. The use of molar Ar, rather than the more commonly used mean residue Ar, conforms to the guidelines suggested by Strickland (1974) for analyzing protein CD data for the near-u.v. range. Units of Ar are in 1 mol-’ cm-‘.

3. Results (4 Effect of PH

The near-u.v. CD spectrum of the 14 S polymer of PMV protein at pH 60 exhibited a complex fine structure (Fig. 2(a)) to be discussed. Raising the pH to 8.0 had little effect on the overall shape, or envelope, of the CD spectrum. However, the intensity of the spectrum was shifted markedly to a more positive level. The difference spectrum (Fig. 2(c)) indicated that the differences observed were due to rotational strength changes in distinct transitions, and not to a rising baseline as might

be attributed

to a scattering

effect.

-2-5

Wavelength Wovelength

(nm)

(nm)

FIG. 2. (a) CD spectra of PMV protein at pH RO, 0.01 M-‘his buffer (-) and at. pH 6.0, 0.01 MIMES buffer (- - -). Protein concentration was 1 A 380 unit/ml, temperature was 25°C. (b) Schlieren profiles of PMV protein from (a) sedimented at 50.740 revs/min at %@5”C. Upper. 14s species at pH 6.0; lower, 14 and 25 S aggregates at pH 8.0. Photo was taken after 8 min. (c) CD difference spectrum computed by subtracting the pH 60 from the pH %O spectrum taken from (a).

Sedimentation analysis (Fig. 2(b)) demonstrated that the pH rise was accompanied by the expected polymerization of the 14 S protein to 25 S aggregates. The aggregate reported at pH 8.0 (Erickson & Bancroft, 1978) is probably composed of a number of species, some of which sediment slightly faster than 25 S. For simplicity these polymers are referred to collectively as 25 S aggregates. (b) ISffmt of protein concentration To determine whether the spectral changes accompanying the pH change from 6-O to 8.0 correlated with t,he state of polymerization of the protein, CD spectra were obtained at various protein concentrations at pH 8-Oand at room temperature (Fig. 3(a)). Over the range @l to 5 A,,, units/ml, the overall de for the CD spectrum increased with increasing protein concentration just as it did at a constant protein concentration when the pH was raised from 6-O to 8-O (cf. Fig. 2(a)). There was little change in the spectral envelope. Analytical sedimentation was employed to monitor the extent of polymerization of protein samplesafter their CD spectra had been recorded, The protein progressed from a state of dissociation, 2 to 3 S, at 01 Azso unit/ml, to an equilibrium between 14s and slower speciesat @25Azso unit/ml, and finally to the equilibrium, first

CD

STUDIES

OF

PMV

PROTEIS

5.0

2.5

0

2

-2.5

- 5-c

1

250

#

1

270

1

I

I

290 Wavelength

1

310

I

t

330

(nm)

FIG. 3. (a) CD spectra of PMV protein at various protein concentrations at pH 8.0,001 M-Tris buffer, 25°C. Spectra 1 to 6 represent protein concentrations of 10. 5, 1.0, 05, 0.25 and @lOAZsO unit/ml, measured in 0.1, 0.2, 1.0, 20, 4.0 and 5.0 cm cells, respectively. The CD spectrum of pH 6.0 protein is included for comparison (- - -). (b) Schlieren profiles of PMV protein from (a) sedimented at 50,740 revs/min, at 24 to 26°C. Patterns from top to bottom represent protein at 10,5, 1.0, @5, @25 and @lo A,,, unit/ml, respectively. Photos were taken after 6 to 10 min, except for the bottom photo, which was taken after 26 min. Bar angles varied.

seen at 1 =1280 unit/ml, between 25 S aggregates and the 14 S polymer and slower sedimenting forms. Further increases in protein concentration increased the proportion of 25 S aggregates until maximal levels were attained at about 5 A2s0 units/ml (Fig. 3(b)). It is clear that the intensity of the CD spectrum of PMV protein is related to the extent of polymerization at pH 8.0. The spectrum of the pH 6.0 14 S polymer at 1 A zsO unit/ml was intermediate between the 0.5 and 625 A2s0 unit/ml spectra of the pH 8.0 protein. Sedimentation analysis indicated that the pH 8.0 14 S polymer was in equilibrium with 25 S aggregates at 05 A2s0 unit/ml, and with species smaller than 14 S at 0.25 A 280 unit/ml. It follows that the intensity difference in the (‘D spectra of the pH 60 and pH 8.0 proteins at 1 .42s0 unit/ml (Fig. 2(a)) may be attributed entirely to the presence, at pH 8.0, of 25 S aggregates that do not exist at pH 6.0. We conclude, then, that the pH 6.0 and pH 8-O 14 S discs exhibit similar CD spectra. The correlation between CD intensity and extent of polymerization can be seen clearly when the percentage increase in AC at 272 nm and the percentage protein sedimenting as 25 S aggregates are plotted together as a function of protein concentration (Fig.4(a)).

.I.

IV.

5 N ki100 Gg 75 % P ; 50

ET

El 0.25

Protein

100

0

75 5 % z

l 1

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Temperoture

(0)

20

25

0

P 8

(“C)

(b)

FIG. 4. (a) Correlation of dichroic absorption of PMV protein with 25 S formation as a function of protein concentration. Percent maximum increase in dr of the 272 nm peak (a) was calculated by defining the maximum increase in AQ,~ nm e g ual to the absolute difference between the de272nm of the 10 A,,* units/ml (predominantly 25 S) and the 925 AzsO unit/ml (predominantly 14 S) spectra. Percent of 25 8 formation (0) was estimated from measurements of peak areas in the schlieren patterns. All material sedimenting between 145 and 2.5 S boundaries was included as 25 S aggregates, as depolymerization was assumed to occur at the boundary region (Erickson, 1978). (b) Correlation of dichroic absorption of PMV protein with solution turbidity as a function of temperature. Percent maximum increase in AQ~,~~,,, (0) was calculated by defining the maximum increase in Ae,,z nm equal to the absolute difference between the AE~,~ nm of the 25°C and 5°C spectra. Percent maximum increase in A 31,, nm (0) was calculated by defining the maximum increase in A 3 ,,, nm equal to the absolute difference between the A3LOnm of the 25°C and 5°C protein solutions.

(c) Effect of temperatwe CD spectra of PMV protein at pH 8.0 and at 1 A,,, unit/ml were recorded at various temperatures, since temperature also influences the protein equilibrium at pH 80 (Fig. 1). Increasing the temperature, from 5 to 25”C, resulted in an increased intensity of the CD spectrum without any change in the spectral envelope. This intensity increase also correlated with the temperature-dependent formation of 25 S aggregates (Fig. 4(b)), the latter being measured by turbidity (Erickson & Bancroft, 1978). (d) Effect of NaCl The addition of NaCl to 62 M at pH 8.0 induces dissociation of the 25 S aggregates to 14 S and smaller forms (see Fig. 1 ; and Erickson & Bancroft, 1978). Such treatment produced an overall negative shift in the intensity of the protein CD spectrum without changing the envelope (Fig. 5), further supporting the correlation drawn above.

(e) Effect of turbidity Tt was important to demonstrate that the polymerization of PMV protein at pH 8.0 were caused by increased light scattering by the larger difference CD spectrum (Fig. 2(c)) argued against experiment was conducted in which the protein,

intensity increases observed upon not due to a rising baseline effect polymers. The pH 80 minus pH 60 this latter possibility. A separate at pH 6.0, was warmed to 29°C to

(‘D

-5.01 250

STI:DIES

OF

270

PMV

290 Wavelength

343

PROTEIN

310

330

(nm)

FIG. 5. CD spectra of PMV protein in the presence (spectrum 1) and absence (spectrum 2) of @2 Mtemperature was 25°C. The NaCl at pH 89,091 M-Tris buffer. Protein concentration was 1 AZaO unit/ml, CD spectrum of the pH 60 protein is included for comparison (- - -).

increase the solution turbidity from @02 to 018 A,,, unit/ml. CD spectra were recorded both before and after the turbidity was increased (Fig. 6). A slight decrease in intensity of the CD spectrum occurred when the turbidity was increased. This effect was more pronounced at lower wavelengths as expected for a light scattering phenomenon. In contrast, the pH 8.0 spectra consistently increased in intensity as the extent of polymerization and, therefore, turbidity increased. Also, the turbidity increase that was observed when the pH was raised from 6-O (O-02A,,, unit/ml) to 8.0 (0*05A 31a unit/ml) at 1 A,,, unit/ml, 25”C, for example, was much lower than in the above experiment. These observations suggest that the spectral intensity changes were not caused by turbidity effects.

(f) Effect of helix formation Higher order polymers, consisting of RNA-free helical tubes, are formed by PMV protein at pH 4.0 (Erickson et al., 1976) and, in the presence of @2M-NaCl, the initial helix-forming reaction is temperature-dependent and reversible (Erickson, 1978). CD spectra of PMV protein at pH 4.0, 0.2 M-N&Cl, were recorded at 5 and 20°C (Fig. 7(a)). Helix formation was followed in the ultracentrifuge (Fig. 7(b)). At ci”C, the 14 S polymer predominated, and the CD spectrum was slightly more negative than the pH 6.0 spectrum. At 2o”C, where the protein polymerized into 80 S tubes, the CD spectrum was only slightly more negative again than the 5°C

.I \\’ 1.:I< 1(‘KS () s 67’ .4 /,. 5-o

- 2.5

-5.0

250

270

290 Wavelength

FIG. 6. Effect of turbidity pH 6.0,001 M-MES buffer, which caused its turbidity

310

330

(nm)

on the CD spectrum of PMV protein at pH 6.0. Protein at 1.0 A,,, 20°C. had an A3,0 = 002 (). This solution was t,hen warmed at 310 nm to increase to 0.18 (- - -).

unit/ml, to 29°C.

spectrum. This difference was attributed to the turbidity of the protein solution at 2O”C, which rose markedly with time, and not to the helix formation which did occur. (8) Effect of dissociation

with and witholrt

denaturation,

The 22,000 M, monomer of PMV protein is found only in the presence of denaturants or at very low concentrations. In order to observe the CD of dissociated protein, 5 M-urea and low pH (2.5 to 3.0) were employed. In either of these conditions the protein sedimented as a 1.8 S species (Fig. 8(b)) and no CD signal could be detected above background (Fig. 8(a)). (The minimum signal amplitude that can be rkliably discerned on our spectrometer is @3 millidegree.) Dissociation of the protein to the monomer does not, by itself, quench its CD signal, since a distinct CD spectrum ww obtained from protein at pH 8.0 which had been dissociated by lowering the protein concentration to 01 A2s0 unit/ml (Fig. 3(a)). These results suggest, therefore, that denaturation accompanies dissociation of the protein in urea and at low pH, but not at pH 8-O.In support of this contention, the acid-dissociated protein exhibited a U.V. absorption spectrum that differed markedly from that given by the pH 6-O(or pH 8.0) protein (Fig. 9, inset) whereas the U.V. spectra of protein at @I A,,,, unit/ml and 10 A,,, unit/ml were identical, after normalization, to that at 1 AzsO unit/ml (data not shown). Analysis of the U.V. difference spectrum (Fig. 9) revealed intense peaks at 291.5, 286 and 281 nm,

’

CD

STUDIES

OF

PMV

PROTEIS

345

5.c

2.5

4

c

-2.5

-5.c

I

250

,

1

1

270

,

1

290 Wavelength

,

310 (nm)

A(:. 7. (a) CD spectra of PMV protein discs and heiical polymers unit/ml, pH 49,0.01 M-citrate buffer, 92 M-NaCl and at 5°C (spectrum rod formation (spectrum 2). The CD spectrum of the pH 69 protein is (b) Schlieren profiles of PMV protein as in (a), Upper, 14 6 species at Sedimentation was at 35,600 (5°C) and 29.506 revs/min (20°C). Photos respectively.

indicating that major perturbations upon denaturation.

of

tyrosine

I

330

and

at pH 4.0. Protein at l.OAZsO 1) was warmed to 20°C to induce included for comparison (- - -). 5°C; lower, SOS species at 2OO”C. were taken after 19 and 13 min.

tryptophan

r&dues

resulted

4. Discussion The envelope of the near-u.v. CD spectrum of PMV protein is similar for all forms of the protein examined between pH 4.0 and pH 8.0, including the dissociated protein at pH X.0. Therefore, most of the optical activity of the various protein polymers can be ascribed to interactions of aromatic amino acid residues within, not between, subunits. Although the spectrum is complex, comparison with CD spectra of various proteins and model compounds (for a review see Strickland, 1974) allows some peak assignments to be made. The CD spectrum of PM\’ protein, which contains two tryptophan, four tyrosine and 12 phenylalanine residues per subunit (Rees, unpublished results), appears to be dominated by tryptophan. The ‘L, transitions of tryptophan (Strickland et al., 1969) could account for the positive background CD in the 260 to 280 nm region of the spectrum. The negative bands at 287 and 294 nm have the appropriate frequencies and spacing to be assignedto the ’ L, transitions of tryptophan (Strickland et al., 1969). The tyrosine ‘L, transitions, which overlap the tryptophan ‘L, and ‘L, transitions between 275 and 290 nm (Horwitz et al., 1970), eould be responsible for the shoulders appearing in this

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’

1 250

f

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I 270

lSKI(‘KSOS

67’

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1

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t 290 Wavelength

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(nm)

FIG. 8. (a) CD spectra of dissociated PMV protein. Protein at 1.0 A2s0 unit/ml, %“C, dissociated in buffer (-). or at pH 3.0, 001 M-glycine buffer (- - -). 5 M-urea, pH 8.0, 0.01 M-T& (b) Schlieren profiles of dissociated PMV protein. Left. 1.8 t; species of the urea protein from (a); in a synthetic boundary cell. right, 1.8 S species of the pH 3.0 protein from (a), sedimented Sedimentation was at 52,640 revs/min. Photo at left was taken after 153 min.

region, and may also contribute to the negative band at. 287 nm in the CD spectrum. The fine structure below 270 nm is attributable to phenylalanine (Horwitz et al., 1969). Negative (‘D bands from phenylalanine at’ 255, 261 and 268 nm superposed against the positive background CD could generate the observed peaks at 259 and 265 nm. The 272 nm peak is probably due to overlapping CD signals from phenylalanine and tryptophan, and possibly tyrosine, side-chains. Similarly, the apparent peak at 291 nm is probably a fine structure effect resulting from superposition of the negative ‘L, bands of tryptophan against some positive background CD at long wavelengths. This background CD could also originate from the ‘L, tryptophan transitions, which can extend into the 300 nm region (Strickland et al., 1969). Such long wavelength tryptophan CD may be responsible for the appearance, in earlier experiments in which temperature was not controlled, of a weak, broad band centred around 300 to 305 nm in the CD spectrum of the pH 8.0 protein (AbouHaidar et aZ., 1979). The CD spectra of the 14 S discs at pH 4-0, pH 6-O and pH 8+0 are quantitatively similar. Thus, although the disc participates in different assembly reactions at the different pH levels, the ground state conformation of the disc, at least insofar as the aromatic domains are concerned, is constant throughout this pH range. Judging from the anomalous titration behavior of PMV protein (Durham & Bancroft, 19’79),

CD

STlJDIES

OF

PMV

PBOTEIN

347

0.6

250 Wavelength

Wavelength

3oc (nm)

(nm)

FIG. 9. Ultraviolet difference absorption spectrum obtained by subtracting the pH 2.5 (- - -) from the pH 6.0 (-) uv. absorbance spectrum (inset) of PMV protein. Sample cell contained 062 A2s0 unit/ml protein at pH 60, 061 M-MES buffer; reference cell contained an equal concentration of protein at pH 25, 061 M-glycine.HCl buffer. Baseline difference spectrum, 901 M-MES, pH 6Q, txr.sus 601 Mglycine.HCI, pH 2.5, was subtracted automatically. Path length, 1 cm; slit width, 64 nm, automatic gain ; signal averaging period, 2.5 s ; scan rate, 0.1 rim/s.

the mechanism through which pH mediates control over the mode of polymerization of the disc, both in the presence and absence of nucleic acid, probably involves carboxyl groups. We conclude that changing the ionization levels of such groups confers different reactivity potentials to the disc, and that these different potentials do not necessarily reflect different conformations of the protein in the 14 S disc. Whereas the pH level does not apparently influence the conformation of at least one particular aggregate of PM\’ protein, the aggregation level of the protein at a given pH can. The latter point refers to the case at pH S-0, where the extent of protein aggregation is reflected by the intensity of the CD spectrum. As the protein polymerizes from monomers to discs to 25 S aggregates, the CD signal intensity increases to a more positive overall level. These increases, which are not due to increased scattering, could result from addition of positive CD or from subtraction of negative CD. The increase in background CD between 255 and 295 nm, and the peaks at 274, 281 and 290 nm in the CD difference spectrum (25 S minus 14 S polymers), indicate a more positive CD signal due to tryptophan in the larger aggregates. There are only two tryptophan residues per subunit, and therefore enhancement of the positive CD due to the tryptophan ‘I+, transitions probably occurs in the 25 S aggregates. Such enhancement could be brought about simply by decreased conformational motility of the tryptophan interactions (Strickland,

318

.I

\\’

ERl(‘KSOS

KY’.!/,

197-t). The absence of wavelength shifts in the (‘I) spect,ra make the generation of a new interaction in\-ol\ring tryptophan seem unlikely. In addition to tryptIophan involvement, the I)eaks at 261 and 268 nm. and the shoulder at 255 nm, in t.hcb (‘I) difference spectrum. indicate that t’he signal due t)o phenylalanine is decreased in the larger aggregates. However,. since there are 12 phenylalanine residues IN’ subunit, enhanced positi\,e (‘D due to some residues cannot be distinguished from weakened negative (‘D due to others and this also applies to new interactions involving phenylalanine. Only the resultant effect is observed. Thus it appears that the aggregation of 14 S discs into 25 S aggregates is accompanied by alterations in interactions involving t,ryptophan and. to a lesser extent, phenplalanine sidechains. In contrast to the changes in optical activity that’ accompanied aggregation of the protein discs at pH 8.0, polymerization of the I4 S discs into helical tubes at pH 1.0 did not significantly alter the protein (‘D. These results suggest that a different modeofaggregation (suchasaclose-packedcircleofdiscs. forexample) must obtain for the 25 S aggregates than that which occurs in the helical polymer, whose formation does not apparently perturb the optically active interactions that, are found in the disc. Therefore, conformational changes associated w&h helix formation are limited by the above considerations. On the other hand. when the TM\’ protein disc polymerizes into helices, the (‘D intensity changes markedly (Vogel & Jaenicke, 1974), presumably reflecting the observed interchain and postulated intrachain contacts that are modified during the disc to helix transition (Bloomer et al., 1978). While an active role for certain aromatic residues in the TM1 polymerization reaction cannot be ruled out (\Togel K: Jaenicke. 1976), the absence of accompanying (‘D changes during helix formation for PM\’ protein means that aromatic side-chains are probably not of the general significance that anornousl,v 1963: Durham CV Bancroft, 1979) are in the titrating carboxyl groups ((‘aspar, helix-forming reactions of viral coat proteins. Dissociation of the protein with urea or low pH quenched the (‘D signal intensity. The protein is apparently unfolded under these conditions, a conclusion supported by sedimentation analysis. The monomer should sediment at about 25 S (based on its molecular weight (22,000 M,) and partial specific volume (0.73 em3/g, Erickson. 1978)). However, in both of the denaturing conditions employed the protein sedimented at 1.8 S, indicating a high degree of asymmetry or hydration. The absorption spectrum of the acid-dissociated protein is also diagnostic of denaturation, the blue shift in A,,, arising from the transfer of buried aromatic sidechains from a hydrocarbon-like environment to an aqueous medium (Donovan. 1969). Further. from t,he freak magnitudes of the absorption difference spectrum, we calculate that one to two of the tryptophan residues and three to four of the four tyrosine residues per subunit are inaccessible to solvent in the disc polymer (assuming Ac,,~ nm= - 1600 and Aez8, “,,,= - 740 for the transfer of 1 mol of tryptophan and tyrosine. respectively, from the protein interior to the solvent (Donovan, 1969): and. estimating At overlaps from model compound data (Herskovitz 8: Sorensen. 196X)). Disruption of interactions in the protein interior. involving the majority of aromatic amino acid residues, apparently accompanies denaturation and provides a plausible mechanism for the observed quenching of

(‘D STCDIES

OF

PM\’

PROTEIS

349

the CU signal. A similar effect of denaturation on U.V. absorption (Brakke, 1979). and on (>D (Budzynski, 1971), was observed for the TMV coat protein, whose tyrosine and tryptophan residues are buried within a hydrophobic girdle in the disc polymer (Bloomer et al., 1978). The (‘D spectra of the double rings or discs of PMV and T&IV proteins are remarkably similar to each other, and also to the (‘D spectrum of the 14 S polymer of the coat protein of clover yellow mosaic virus, a PVX group virus whose protein also apparently makes discs (Bancroft et al., 1979). Thus, the disc polymers of coat proteins of helical viruses may share structural features that are reflected in their (‘D spectra. We thank Mr W. R. Browett for discussion. This work was supported by the Natural Sciences and Engineering Research Council, Canada and the University of Western Ontario Academic Development Fund grants (to J.B.B.) and by the Natural Sciences and Engineering Research Council, Canada and Research Corporation grants (to M.J.S.).

REFERENCES AbouHaidar, AbouHaidar, Bancroft, J. Bloomer. A.

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