Physicochemical properties of N4 coliphage protein ghosts

45 °

BIOCHIMICA ET BIOPHYSICA ACTA

13BA 35847 PHYSICOCHEMICAL P R O P E R T I E S OI; N 4 C O L I P H A G E P R O T E I N GHOSTS

A. P E S C E AND G. C. S C H I T O

[stituto di Microbiologia dell" Universith di Genova (Italy) ( R e c e i v e d D e c e m b e r 7th, 197 o)

SUMMARY

I. The protein portion of the DNA coliphage N 4 has an intrinsic sedimentation coefficient of 192 S, an intrinsic viscosity of 7.2.1o -2 dl/g, a diffusion coefficient of 4.9" I°-S cm2/sec, a buoyant density in CsC1 of 1.274 g/ml and a molecular weight of 38.1 .io a. 2. It has a typical protein absorbance spectrum and consists of at least five distinct polypeptide subunits. 3. These data, together with amino acid analysis, indicate that the phage ghosts consist of a hollow protein shell similar in structure to the protein moiety of the whole virus.

INTRODUCTION

Viable particles of N 4 bacteriophage contain a single molecule of linear doublestranded DNA of about 4 o - l o 6 daltons wrapped by a protein envelope of complex morphology and composition 1. Separation and chemical characterization of the main structural component constituting the head membrane of the phage has now been undertaken. Since difficulties were encountered in the preparation of nucleic acid-flee material from the intact particle, the possibility of employing the artificial ghosts of the virus as the source of protein subunits has been considered. It is the purpose of this communication to describe experiments carried out in an effort to compare the mass and hydrodynamic properties of the bacteriophage to that of the e m p t y capsid and to present evidence concerning the chemical composition of the ghosts. In the following paper, the terms " e m p t y capsids", "shells" and "protein moiety" have been used as synonyms of phage ghosts. MATERIALS AND METHODS

Wild-type N4r+ bacteriophage was used to infect Escherichia coli strain KI2S. Procedures for the growth and purification of intact phage particles have been described by SCHITO2. Biochim. Biophys. Acta, 236 (1971) 45o-457

PROTEOLYSIS OF RABBIT SECRETORY i g A

451

Artificial N 4 ghosts were produced from concentrated suspensions (7-1o mg/ml) of purified virus following disruption of the phage structure through several cycles of freezing and thawing, digestion of the extruded DNA with deoxyribonuclease, concentration of the e m p t y protein shells by ultracentrifugation and final pycnographic purification by band sedimentation in a preformed CsC1 density gradient 3. A Beckman DB recording speetrophotometer was used for absorbance determinations. Corrections for light scattering were made by extrapolation from the nonabsorbing regions of the spectrum. Analytical ultracentrifugation for measurement of sedimentation coefficients, buoyant density and diffusion constant were performed in a Spinco model E ultracentrifuge operated at a temperature of 20 ° (ref. 4). An Ubbelohde suspended level capillary viseometer was used to measure the flow times of N 4 ghost solutions and buffer (0.05 M borate, p H 7.8, containing o.I M KC1) at 20 °. The specific viscosity was expressed in terms of flow times as ~]sp = (t'--t)/t, where t' is the flow time of the sample, and t, that of the solvent ~ and was corrected for the difference between the density of the sample solutions and that of tile buffer. The graphic plots o f ~ s p / C a s a function of concentration were linear and at infinite dilution yielded the intrinsic viscosity. Analytical disc electrophoresis G,7 in 7 . 5 / oo / acrvlamide . gel containing 8 M urea at pH 8.6 was performed as previously reported 8. Amounts of I 5 mg of ghost protein were hydrolyzed in 6 M HC1 at IiO ° for 24, 48 and 72 tl. The amino acid composition of the mixtures was determined in a Beckman Unichrom analyzer as described by SPACKMAN •t a.l. 9.

W~velength (nm) Fig. I. Ultraviolet a b s o r p t i o n s p e c t r u m of N 4 ghosts in o.o 5 M borate buffer (pH 7.8) containing o. I M KCI, uncorrected (A) and corrected (B) for light scattering. Fig. 2. Schlieren sedimentation velocity p a t t e r n of purified N 4 bacteriophage ghosts at a concent r a t i o n of 1.1 m g / m l in o.o 5 M b o r a t e buffer (pH 7.8) containing o.I M KC1. R o t o r speed was 2o ooo rev./nlin ;bar angle, 7o°; t e m p e r a t u r e , 2o °.

Biochim. Biophys. Acta, 236 (1971) 450-457

452

A. PESCE, G. C. SCHITO

RESULTS AND DISCUSSION

Physical characteristics of N 4 coliphage ghosts The ultraviolet absorption spectrum for a dilute solution of N 4 ghosts is typical of that of a protein. An absorption maximum occurs at 278 nm and a minimum at 256 nm (Fig. IA). Absorbance measurements from 330 to 500 nm were also made on the capsid suspensions; a nearly linear relationship between absorbance values and 1/).4 was found. Extrapolation to 278 nm indicated that about 65% of the ghost absorbance at that wavelength could be ascribed to light scattering while the remainder was due to the protein contribution. Assuming that at 320 nm the total absorbanee was due to light scattering, a corrected absorption spectrum was constructed (Fig. IB) by multiplying Aa20 by (320/2) 4 according to Rayleigh's law. Absorption measurements were made at several concentrations of N 4 ghosts in order to obtain the absorbancy index. The average of 7 determinations gave a value of 1.12 cm2/mg at 278 nm. N 4 empty protein shells examined in the electron microscope (see SCHITOa) after negative staining appeared filled by phosphotungstic acid under conditions where the virion was not penetrated and were less uniform in dimensions than N 4 itself, probably because they are more fragile. Nevertheless, they consist mainly of hexagonal hollow particles with a radius of about 35o A. N 4 ghosts sedimented as a single almost symmetric boundary in the analytical ultracentrifuge (Fig. 2). Ten velocity sedimentation runs as a function of concentration were made on the capsid suspended at pH 7.8 in KCl-borate buffer. The measured sedimentation coefficients were corrected to s20,w and yielded a value of 192 ± 1°,o S upon extrapolation to infinite dilution (Fig. 3). Sedimentation at low salt concentration (o.o5 M borate buffer, pH 7.8) revealed the presence of small amounts of a heavier component (Fig. 4) migrating with an S°2o,w of 272 4- 1% S. Complete conversion of this material, probably representing a dimer of the basic ghost monomer, to particles sedimenting with an S value similar to that shown by

6.Or

I 5b I

I

~o

................. i ............

2

. . . . . . . . . . ; .......................2 ....

A

c(mg/ml) Fig. 3. Reciprocal of the s e d i m e n t a t i o n coefficient of N 4 coliphage ghosts as a function of concentration. Fig. 4- Sedimentation velocity p a t t e r n of N 4 coliphage ghosts at a concentration of 1.3 m g / m l in 0.05 M borate buffer, p H 7.8. R o t o r speed was 20 ooo rev./min; b a r angle, 70°; t e m p e r a t u r e , 20 °.

Biochim. Biophys. Acta, 236 (1971) 45o-457

PROPERTIES OF N 4 COLIPHAGE PROTEIN GHOSTS

453

1.817

L

t

1.0--

0

~b

....

4b

6'0

8'0

'

(l_lo2st)t~2(sec)l

Fig. 5- D e t e r m i n a t i o n of the diffusion coefficient of N 4 ghosts according to the m e t h o d of VAN HOLDE II.

Fig. 6. Schlieren pattern of N 4 bacteriophage protein shells banded at equilibrium in a CsC1 density gradient. The ghost suspension was centrifuged for 18 h (to constant band width) at 44 ooo rev./min at 25°. The mean density was 1.286 g/ml.

the e m p t y capsids was observed whenever the ionic strength of the solvent was conveniently raised. Since the determination of the diffusion coefficient of high-molecular-weight compounds in a Tiselius apparatus has been shown to be unreliable 1°, independent values of this parameter for N 4 protein shells were obtained from analysis of sedimentation diagrams b y the procedure of V a x HOLDEn. From five such experiments a mean D2o, w of 4.9 .IO s cm2/see was calculated (Fig. 5). A b u o y a n t density of 1.274 g/ml was c o m p u t e d for the phage ghosts from the position of the particles 12 banded at equilibrium in a CsC1 analytical density gradient (Fig. 6). Assuming the partial specific volume g of the e m p t y shells to be 0.733 ml/g, as c o m p u t e d from the amino acid composition 13, the molecular weight of N 4 ghosts, as estimated with the appropriate substitutions from the Svedberg equation, 31 = R T s / D ( I -- vo)

was found to be 3 8 . I - I o 6. This value is in the range of that to be expected from the chemical composition of the intact phage ( 4 7 % DNA) and from its particle mass (80. Io 6 daltons) 14 if the ghosts analyzed represented the bulk of N 4 protein portion. Viscosity measurements were made on N 4 capsid solutions from o.2 to 1.2% (w/v) in concentration. Extrapolation of the curve relating r]/c to c (Fig. 7) resulted in an ordinate intercept value of 7.2. lO .2 dl/g, the intrinsic viscosity of the ghosts. F r o m the reduced viscosity, the water of hydration was estimated on the basis of the Einstein-Simha equation 15 [rt] - v(P2 + ~P1)

where v is the shape factor, equivalent to 2.5 for spheres, g2 is the partial specific volume of N 4 ghosts, d is the n u m b e r of g of solvent bound per g of protein and 171 Biochim. Biophys. Acta, 239 (1971) 45o-457

454

A. PESCE, G. C. SCFtlTO

[ 12-

8

~

~

~

<.i

4i o ~

a ~-

~--i6

lO.c(g/dl)

~

Fig. 7. D e t e r m i n a t i o n of the l i m i t i n g v i s c o s i t y n u m b e r of N 4 coliphage ghosts dissolved in 0.05 M borate buffer (pH 7.8) containing o.i M KC1.

is the partial specific volume of water at 20 ° assumed to be 1.002 ml/g. From these data a value of 2.I 4 g of water per gram of protein was found to be associated with the empty protein shells. However, this figure might be somewhat in error, owing to the fact that the shape parameter used corresponded to that of a perfect sphere and was thus a minimal value. The hydrated diameters of spherical particles may be derived from d - - KT/3Jz~D°2o, w

where K, T and 77 are Boltzman constant, absolute temperature and solvent viscosity, respectively, and was found to be 745 A for N 4 ghosts. This value is comparable to that of about 700 A. which was calculated for the diameter of these particles negatively stained in the electron microscope 3. TABLE I AMINO ACID COMPOSITION ( M O L E S / I O O MOLES) OF

amino acid

N 4 ghosts

N 4 protein subunits*

Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe

5.47 1.32 3.37 lO.66 9.6o 8.3o 8.63 4.6o 8.67 8.oo 7.85 3.io 6.I7 7.o4 3.69 3.5 °

5.72 1.41 3-42 I 1.76 9.83** 6.76"* 9.15 3.o4 8.6o 8.32 -6.64 3.io .5.91 6.96 3.94 3.32

N 4 COLIPHAGE GHOSTS AND P R O T E I N S I J B U N I T S

* P r e v i o u s l y d e t e r m i n e d from p h e n o l extracts of purified N 4 bacteriophage le. ** Uncorrected for losses during acid hydrolysis. B i o c h i m . B i o p h y s . A c t a , "236 (I97 I) 450-457

PROPERTIES OF

N4

COLIPHAGE PROTEIN GHOSTS

455

Frictional ratios may be obtained from KT/D%o,w fifo --

~1( 162,'~2M 17/N) 1/a

where N is Avogadro's number. A frictional ratio of 1.799 was calculated for N4 protein shells. If the reasonable assumption is made that these particles are effectively spherical in solution, then the fraction greater than I might be due both to hydration, and/or to the asymmetry introduced in the molecules by the presence of the very small adsorption apparatus.

Chemical composition and stability of N 4 ghosts The amino acid composition of N 4 protein shells is given in Column I, Table I. The data are mean values, corrected for partial destruction of amino acids from three analyses of 72-h HC1 hydrolyzates. The values obtained agree reasonably well with the composition reported by SCmTO et al. TM for the purified phenolic protein subunits (Table I, Column 2) of the phage. Preparations of N4 capsids were dissociated in 8 M urea, pH 8.6, at 5 °o for I h (see further), and the resulting polypeptides were subjected to electrophoresis in 7.5 % acrylamide gel. When stained columns were scanned in the Gilford gel scanning attachment at 562 nm the absorbance profile shown in Fig. 8 was obtained. Five protein fractions with distinct electrophoretic mobility were resolved. The pattern of migration and the quantitative distribution of the polypeptide components were indistinguishable from those reported for the phenolic subunits of the phage s. A limited investigation was made on the conditions under which the empty shells remain homogeneous in sedimentation velocity. The ghosts did not show any gross structural change, as reflected in their hydrodynamic properties when subjected

Fig. 8. Absorbance profile of N 4 coliphage ghost polypeptides subjected to electrophoresis in 7.5 % polyacrylamide gel c o n t a i n i n g 8 M urea at p H 8.6. The gel was scanned in a Gilford spectrop h o t o m e t e r e q u i p p e d w i t h a linear t r a n s p o r t m e c h a n i s m at 562 n m in a glass cuvette. The direction of electrophoresis was f r o m right to left. Biochim. Biophys. Acta, 236 (I97 I) 450-457

456

a . PESCE, G. C. SCH1TO

for e x t e n d e d periods of time (up to 2 4 h) to concentrated (8 M) urea or I".~, sodium dodecyl sulfate at n e u t r a l p H at room temperature. These conditions are known to cause dissociation in m a n y protein systems b u t do not seem to be effective on other purified phage capsids 17 21. I n these solvents, however, some degradation occurred when the t e m p e r a t u r e of i n c u b a t i o n was raised (Fig. 9), and dissociation was essentially complete following exposition of the ghosts to I °/o sodium dodecyl sulphate or 8 M urea at 5 °o for I h. Tile n a t u r e of the breakdown products was i n d e p e n d e n t of the methods of d e n a t u r a t i o n , a n d the resulting material sedimented as a single s y m m e t r i c b o u n d a r y (Fig. IO) m i g r a t i n g with an s20,w of 2.1 which is the typical S value of N 4 coliphage protein s u b u n i t s 16. Splitting of the virus coat seems to be an all-or-none p h e n o m e n o n since no c o m p o n e n t s e d i m e n t i n g between i n t a c t ghosts a n d polypeptide monomers was ever observed nor was a n y sodium dodecyl sulphate or urea-resistant fraction detected.

Fig. 9. Schlieren sedimentation pattern of N4 protein shells dissolved at a concentration of 1.3 mg/ ml in o.o5 M borate buffer (pH 7.8) containing 8.o M urea and heated at 37° for i h. Synthetic boundary cell; rotor speed, 2o ooo rev./min; bar angle, 7o°; temperature, 20~. Fig. IO. Schlieren sedimentation pattern of N4 protein ghosts dissolved at a concentration of 1.4 mg/ml in o.o5 M borate buffer (pH 7.8) containing 8.o M urea and heated at 5°o for i h. Synthetic boundary cell;rotor speed, 56 ooo rev./min, bar angle, 7o°; temperature, 2o°. I t m a y be concluded from these studies t h a t the e m p t y envelopes analyzed represent the protein portion of N 4 a n d consist of a hollow shell similar in dimensions to the viable phage particle. The capsid seems to be composed of at least 5 protein s u b u n i t s differing in charge d i s t r i b u t i o n b u t similar in size a n d shape. These findings confirm the d a t a recently presented b y SCHITO et al. s showing, b y salt fractionation a n d N - t e r m i n a l amino acid d e t e r m i n a t i o n , t h a t the envelope of the phage is made u p of several distinct polypeptide chains. The physical a n d chemical evidence reported here clearly indicates t h a t the e m p t y capsids comprise the whole protein moiety of N 4 coliphage. These particles seem therefore suitable as a source of the monomers c o n s t i t u t i n g the architecture of the virion. E x p e r i m e n t s are in progress in an effort to separate the different a n a t o m i c a l regions of N 4 ghosts a n d to analyze the protein c o m p o n e n t s in terms of their p r i m a r y structure. The results o b t a i n e d will appear in due course.

Biochim. Biophys. Acta, 236 (1971) 450-457

P R O P E R T I E S OF

N4 C O L I P H A G E

P R O T E I N GHOSTS

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ACKNOWLEDGMENTS

This investigation was supported by grant 69.o2222.115.127I from the Consiglio Nazionale delle Ricerche, Rome, Italy. The competent technical assistance of Dr. L. Radin and Mr. E. Debbia is gratefully acknowledged. REFERENCES I z 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21

G. C. SCHITO, G. RIALDI AND A. PESCE, Biochim. Biophys. Acta, 129 (1966) 482. G. C. SCHITO, Virology, 3 ° (1966) 157. G. C. SCHITO, Giorn. Microbiol., 14 (1966) 75H. 14~. SCHACHMAN, Ultracentrifugation in Biochemistry, A c a d e m i c Press, N e w Y ork, 1959. I. BENDET AND M. LAUFFER, Biochim. Biophys. Aeta, 55 (1962) 211. L. ORNSTEIN, Ann. N . Y . Acad. Sci., 121 (1964) 321. B. J. DAVIS, Ann. N . Y . Acad. Sci., 121 (1964) 404 . G. C. SCHITO, A. PESCE, G. SATTA AND C. A. ROMANZI, X V Natl. Congr. Microbiol. Abstracts of papers, Torin o (1969). 1). H . SPACKMAN, W . H . STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. i'. F. DAVlSCN .ANn D. FREIEELOER, J. Mol. Biol., 5 (1962) 635. K. E. VAN HOLDE, J. Phys. Chem., 64 (1961) 1582. J. B. IFFT, D. H. VOET AND D. J. VINOGRAD, J. Phys. Chem., 65 (1961) 1138. E. J. COliN AND J. D. EDSALL, Proteins, Amino Acids and Peptides, R e i nhol d, N e w York, 1943. G. C. SCHITO, G. RIALDI AND A. PESCE, Biochim. Biophys. Acta, 129 (1966) 491. C. TANFORD, Physical Chemistry of Macromoleeules, W i l e y, N e w York, 1961. G. C. SCHITO, A. M. MOLINA AND A. PESCE, Biochem. Biophys. Res. Commun., 28 (1967) 611. D. J. CUMMINGS, Biochim. Biophys. Acta, 68 (1963) 472. R. D. DYsoN AND K. E. VAN HOLDE, Virology, 33 (1967) 559. M. VILLAREJO, S. I-{UA AND E. A. EVANS, J. Virol., I (1967) 928. E. KELLENBERGER, Virology, 34 (1968) 549. L. L. LARCOM, [. J. BENDET AND S. MUMMA, Virology, 41 (197o) i.

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