Studies of virus structure by Laser-Raman spectroscopy

J. Mol. Biol.

(1976) 102, 103-124

Studies of Virus Structure by Laser-Raman

Spectroscopy

II.? MS2 Phage, MS2 Capsids and MS2 RNA in Aqueous Solutions G. J. THOMAS JR. B. PRESCOTT Department of Chemistry Southeastern Massachusetts University North Dartmouth, Mass. 02747. U.S.A.

PATRICIA

E. MCDONALD-ORDZIE

ANU

K. A. HARTMAN

Department of Biochemistry and Biophysics Uniwrsity of Rhode Island Kingston, R.I. 02851, U.S.A. (Received 11 August 1975)

Laser-Raman spectra of the bacteriophage MS2, and of its isolated coat-protein and RNA components, have been obtained as a furl&ion of temperature in both H,O and D,O (deuterium oxide) solutions. The prominent Raman lines in the spectra are assigned to the amino acid residues and polypeptide backbone of the viral coat protein and to the nucleotide residues and ribosyl-phosphate backbone of the viral RNA. The Raman frequencies and intensities, and their temperature dependence, indicate the following features of MS2 structure and stability. Coat-protein molecules in the native phage maintain a conformation determined largely by regions of b-sheet (-60%) and random-chain (~40%) structures. There are no disulfide bridges in the virion and all sulfhydryl groups are accessible to solvent molecules. Protein-protein interactions in the virion are stable up to 50°C. Release of viral RNA from the virion does not affect either the conformation of the coat-protein molecules or the thermal stability of the capsid. MS2 RNA within the virion contains a highly ordered secondary structure in which most (-85%) of the bases are either paired or stacked or both paired and stacked and in which the RNA backbone assumes a geometry of the A-type. When RNA is partially or fully released from the virion its overall secondary structure at 32°C is unchanged. However, the exposed RNA is more susceptible to changes in secondary structure promoted by increasing the temperature. Thus the viral capsid exerts a significant stabilizing effect on the secondary structure of MS2 RNA. This stabilization is ionic-strength dependent, being more pronounced in solutions containing high concentrations of KCl. Raman intensity profiles as a furlction of temperature reveal that disordering of the MS2 RNA backbone and rupture of hydrogen-bonding between complementary bases are gradual processes, the major portions of which occur above 40°C. However, the unstacking of purine and pyrimidine bases is a more co-operative phenomenon occurring almost exclusively above 55%. t I’aprr

I in this series is Hartman

u6 nl. (1973). 103

104

G. J. ‘J’HO,1IAS JR ET AL.

1. Introduction The bacteriophage MS2 is a small, nearly spherical RNA virus (Knight, 1974). The viral particle (virion) has a diameter of approximately f 6 nm and a sedimentation value of about 80 S. It is composed of 180 identical “coat-protein” molecules of molecular weight 13,750 which make up the capsid, one protein molecule called the “A protein” of molecular weight 38,000, and one RNA strand of molecular weight 1.1 x lo6 (Steitz, 1968). The protein is important in the infection process and appears to enter the host, along with the RNA, leaving t,he capsid protein outside (Krahn et al., 1972). The RNA in the native phage is not sensitive to the action of RNAase (Argetsinger & Gussin, 1966). It has been postulated (Matthews & Cole: 1972) that the capsid structure is maintained by hydrophobic forces between the coat-protein molecules and by ionic interactions between positively-charged residues of these molecules (e.g. -NH:) and negatively-charged residues of the RNA (e.g. -PO,). However, little is known of specific interactions which may exist between RNA and protein or between adjacent protein molecules in the assembled virion. The extent and kind of secondary structures existing in the RNA and prot’ein molecules are also unknown. Laser-Raman spectroscopy has provided a useful means of determining structural information on RNA (Thomas & Hartman, 1973; Hartman et al., 1974) and proteins (Chen & Lord, 1974; Spiro, 1974). Initial Raman studies on the bacteriophages, R17 (Hartman et al., 1973) and Pfl and fd (Thomas & Murphy, 1975). also indicate the usefulness of the technique for structural studies of viruses. The Raman method depends upon the abilit’y to obtain, by laser-light scattering, a vibrational spectrum consisting of a series of lines or frequencies characteristic of the structure and environment of macromolecular subgroups. The Raman frequencies and intensities therefore indicate the kinds of functional groups present and the kinds of secondary interactions in which the molecular subgroups participate. For example, the Raman spectrum of RNA is sensitive to the amount of hydrogen-bonding interaction between bases, the amount of base-stacking interactions and the degree of order in the ribose-phosphate backbone (Thomas, 1970; Thomas & Hartman, 1973). Raman spectra of proteins distinguish the constituent amino acids from one another, as well as S-H from S-S groups and COOH from COO- groups. The relative amounts of a-helical, /?-sheet and random-chain conformation in a protein backbone may als3 be inferred from its Raman spectrum (Chen & Lord, 1974). In this report we compare the Raman spectra of native MS2 phage, heat-degraded MS2 phage (the so-called HDT particle: Steitz, 1968; Oriel, 1969), MS2 capsids free of RNA, and MS2 RNA free of protein. Spectra were obtained as a function of temperature and at different conditions of ionic strength in both H,O and D,O solutions. The results provide new information on t,he structures and interactions of MS2 and its component molecules.

2. Materials and Methods (a) Preparation of phuge The MS2 phage were grown in Escherichia coli K12 Hfr Hayes which were infected at a multiplicity of infection of ten when the exponentially growing cells reached a density of 3 x lo* cells/ml (Klett reading of 60). E. coli wore grown in shake flasks containing t Abbreviation

used: HD particle, heat-degraded particle.

LASER-RAMAN

SPECTROSCOPY

OF PH;1GE

MS%

lOr,

H medium (50 mM-Tris base, 0.338 mM-potassium phosphate, 18.7 niM-ammonium chloride, 85 m-sodium chloride, 2 mnn-magnesium chloride, titrated to pH 7 with cont. HCl and supplemented with 0.48% (w/v) glucose, 0.24% (v/v) glycerol, and 0.002~~ (w/v) thiamine) incubated at 37°C. CaCl,, at 2 mm, was added before phage were introduced. After infection, the Klett reading continued to increase and then began to decrease and became constant about 3 h after infection. 0.1 vol. of chloroform was then added t,o help break the cells and after 10 min of shaking the NaCl concentration was increased t.o 0.5 M by the addition of solid NaCl. The lysate was cooled to 4°C and centrifuged to remove cell debris (16,300 g, 15 min). Next, polyethylene glycol (PEG-6000, Bake1 (!ompany) was added to 6% (w/v) and the solution was allowed to precipitate by remaining was collected by contrifugatioIl overnight at 4°C (Yamamoto et aZ., 1970). The precipitate (8000 g, 10 min) and was dissolved in O-2 M-NaCl. The resulting solution was given a lowspeed centrifugation (23,000 g, 15 min) to remove debris and the phage were pelleted b!ultracentrifugation (150,000 g, 90 min). The pellet of phage was resuspended, and after a low-speed spin (12,100 g, 10 min), the supernatant wa,s centrifuged (150,000 g, QO min)

1

I 0

I

I IO

t Froctim

FIG. 1. Sucrose gradient (--- EN--- n --).

I 20

I

I 30

no

sedimentation profiles of MS2 phage ( -12 - ---tip) The sedimentation inoreaqes from right to left.

and HD particles

to pellet the phage. This pellet was suspended at a concentration of about 40 mg/ml in an H,O or D,O solution containing the required salt (see below). The concentration of phage was determined by dilution and ultraviolet (u.v.) absorption spectrophotometry assuming an extinction coefficient at 260 nm of 8.0 ml-’ mg-l cm-l (Strauss & Sinsheimer, 1963). The final product gave sedimentation profiles (5% to 20% sucrose gradients made in 0.1 M-potassium acetate, pH 7, centrifuged at 230,000 g for 55 min in a Beckman SW50.1 rotor) with a narrow band at about 80 S (Fig. 1) and an electrophorzsis profile (30,; polgacrylamide gel as per Hartman et al., 1970) with one sharp peak which suggests a homogeneous sample. RNA extracted from these phage gave a sharp band in gel electrophoresis both before and after heating to 80°C which suggests that the phage contained homogeneous high molecular weight RNA with no “hidden breaks”. Measurement of plaque-forming units per ml of solution for phage solutions of known U.V. absorbance (260 nm) allows the calculation of the percentage of phage particles that are infect,ivt, A typical value for these preparations is 30%.

106

G. J. THOMAS (b) Preparation

,JH. X7’

df,.

of heat degraded particles

Heat-degraded particles were prepared by heat’ing phage in 0.1 M-potassium ac(ltat(b. pH 7, in t’he Raman cell to 55°C for 10 min in N wat.cr hat)h, followed t)p cnooling in room ;lir,. Sucrose-gradient centrifugation of HD part ides hrforcs irradiation shoM-cad a band at 60 S with no loss of RNA or protein (Fig. I, ahove). .4ftrr irmtliat,ioll hy tllcl latier. SOIIW HNA had been released from the HD particles. RNA cxtractcd front Hl) particles u-as homogeneous with no “hidden hrea,ks” as drmonst~ratc~d by I”)ISacr?la,Inic~~, gctl c,lcct,ropllorrsis. (c)

Preparation

qf capsids

RNA was removed from t,he phage t,o produce capsids by treatment, with KOH (Samuolson & Kaesberg, 1970). The phage solutiou (A,,, - 30) was made busic by addit,ion of KOH to 8 mm (23°C) and was then neutralized by t,he addition of 0.01 &I-Tris, pH 7.1. The liberated RNA was destroyed by the addition of 1.24 pg RNAasc/ml. The solution u’as then centrifuged to pellet the capsids (150,000 g for 3 h). The resulting pellet \vas dissolved in a small volume of H,O or D,O solution from which a Ramarl sample tribe was filled. Sucrose gradient sedirnentatioll profiles of capsid preparations observed t)l II.\‘. absorbance at’ 230 nm showed one band at about, 45 S. Absorbance at, 260 nm W&S very low suggesting t,hat, virtually all RNA had hc~c~ntxnmovr>fl. This was confirmed by Raman spectra of the capsids as described below. (d) Preparation

of KSA

Protein was removed from the RNA by treat’ing the phage (in 0.2 M-potjassium acetat,c, pH 5) with water-saturated phenol (redistilled) follow-ed by precipit’at,ion of the aqueous acrt,ate, pH 5, and phase with ethanol. The RNA was redissolved in 0. 2 til-potassium again precipitated with ethanol. The RNA a-as dried by passage of air over t,he precipitate. The dried RNA was t,hen dissolved in H,O or D,O solut’ions as required. The final product was homogeneous and of high molrculwr weight ~1s measured by polyacrylamide gel rlcctrophoresis. (e) Instrumentation

and sample-handling

for

Raman

spectroscopy

Raman spectra were excited with either t,he 488.0 or 514.5 nm line of an argon-ion laser (Coherent Radiation, model CR2) employing 300 mW of power at the sample, and were Samples werP contained in glass capillary recorded on a Spex Hamalog spectrometer. tubes (Kimax no. 34507) of 1-O mm inner diameter, to a volume of approximately 10 ~1. The sample-filled cells were usually centrifuged at 4000 revs/min prior to laser illumination, to sediment particulate matter which otherwise detracts from the quality of the Raman scattering spectra. The temperature of the sample was maintained to *0.5”C, over t,he range 0 to lOO”C, using a device described previously (Thomas & Barylski, 1970). Further details of instrumentation are given elsewhere (Thomas, 1971).

3. Results and Discussion (a) Raman spectra of MS2 cupsids Raman

spectra

were

obtained

over

the

temperature

range

0 to 50°C

on purified

capsids dissolved in H,O solutions containing 0.75 M-KU at pH 7. both with and without 0.08 M-potassium acetate. The spectrum recorded for acetate-free solutions at 32°C is presented in Figure 2(a). The Raman frequencies and intensities of the capsid are independent of the temperature up to 50°C. However, upon heating to 60°C or higher, the capsids (or their protein subunits) were found to precipitate from solution, presumably as a result of protein denaturation. Therefore, satisfactory spectra of the denatured protein could not be obtained. Raman spectra were also recorded as a function of t’emperature on purified aapsids dissolved in D,O solutions containing 0.75 M-KU at pD 7. The spectrum recorded at

LASER-RAMAS

SPECTROSCOPY

Frequency

OF PHAGE MS2

107

(cm-0

Frc;. 2. (a) Ramen spectra of H,O solutions of MS2 cepsids at 32°C. Conditions: 0.75 M-KU. pH 7. 38 pg capsids/$; excit,ation wavelength A : 488.0 nm; spectral slit width do = 10 cm-’ ; wan speed s = 25 cm-‘/min; rise t,ime t ~: 10 s; amplificetion 4 =- 1 (300 to 1800 cm-l), A =- 3 (2500 t,o 2600 cm-l), A = l/3 (2800-3100 cm-‘). The spectrum is unchanged over the range 0 to 50°C (_ (t)) Reman spectra of II,0 solutions of MS2 capsids at, 32°C. Conditions: 0.75 wKCI. pI> i, 1 = 10 s; amplification 40 pg capsidn/~l; A 2 488.0 nm; do : 10 cm-‘; s = 25 cm-‘/min; I (300 to 1900 cm-‘). A =:- l/3 (2800 to 3100 cm-l). The spectrum is unchanged over the .4 rang? 0 to 50°C’.

32°C is presented in Figure 2(b). For D,O solutions, the Raman spectrum of the MS2 capsid is also independent of changes of temperature up to 50°C. At or above 6O”C, however, the capsids also precipitate from D,O solutions which again prevents spect’ra of t’he heat-denatured protein from being recorded. TV establish an internal intensity standard in Raman spectra of capsids, a study was made of the capsids dissolved in H,O solution containing 0.75 M-KC1 + 0.0% Mpotassium acetate at pH 7.1. The intensity of the Raman line of the phenylalanine residue at 1004 cm-l normalized to the int’ensity of the Raman line of the acetate ion at 929 cm Ml (11001/1929) was found to be invariant over the range 0 to 59°C. Thus the intensity of the 1004 cm-l line in the spectrum of Figure 2(a) or (b) may be used as a basis for comparing Raman intensities of other lines originating from vibrations of protein residues. In this way we have verified that the Raman spectrum of t,ht: capsid does not change significantlp from 0 to 50°C in either the presence or the absence of acetate ion. Ra.man frequencies of the MS2 capsids in H,O and D,O solutions at 32°C are listed in Table 1 (columns 1 and 4, respectively) together wit’h assignments (column 7) based upon previously published Raman data of proteins, polypeptides and related model compounds (Lord & Yu. 1970a,b; Simons et al., 1972; Yu et al., 1972a,b; Yu & .Jo. 1973; Chen et al., 1974; Chen t Lord, 1974; Yu, 1974). A more detailed dis(+ussion of the assigned frequencies is given below.

0 a”

0, x

(0.20) (0.04) (O-OS) (B)

2897 (0*05S) 2960 (0.76) 2980 (0.32)

1575 (1.19)

MS2 RNA

phage

2576 “880 2943 2974

(0.11) (1.76) (4.40) (2.26)

1560 (0.16) 1576 (0.66) 1622 (0.18) 1663(B)

MS2

2876 2938 2974 3062

1872 (1.77) (4.06) (2.325) (0.34)

(0.12)

1549 (0.26) 1575 (0.19) 1616 (0.37) 1665 (1.47)

MS2 capsids .-_~~ -

(0.86) (2.50) (1.60) (0.21)

(0.86)

1684

“899 2942 2976 3097

(1.68) (0.22) (0.55)

RNA

1574 1621 1657

MS2

DzO

l--continued

2879 2941 2973 3061

1874

1664 1574 1618 1660

MS2

(1.63) (4.01) (2.69) (0.31)

(0.13)

(0.395) (1.09) (0.52) (1.99)

phage

S-D str Cys S-H str Cys .iliphetic C-H Aliphatic C-H Aliphatio C-H .Iromatic C-H

Trp Phe, Trp, Tyr Tyr, Phe, Trp Amide I + I’

Protein

str str str str

AssignmentsJ

c: = 0 str u,

G. (’

U,G,i'

G, A c = 0 str u c = 0 str L-,0,

RNA

t Frequencies in cm-’ are accurate to = 2 cm - 1 for sharp or intensc lines and to 5 4 cm- 1 for weak or broad lines (Figs L’, 3, 5, 6). Numbers in parent,heae* are relative intensities with a value of 1.00 assigned to the 1004 cm - 1 line of phenylalaninr in the capsids and the phage or to the 1100 cm-’ line of thr phosphate groups in the RNA; B denotes broad lines and S denotes shoulders. $ Abbreviations: standard 3.letter symbols are used for the amino acids and l-letter symbols for the RNA bases. Also, r ~ ribose, P L.- phosphate, anal c--c, C--N, C-~ -S, S-H, S-D. C-H, C=O, CO, and S-~-H denote their respective functional groups. Where specific types of vibrational modes itrr known, these are indicat.ed by sym (symmetric), str (stretching), def (deformation), deg (degenerate).

2574 (0.10) 2881 (1.46) 2941 (3.52) 2978 (1.46)

1552 1682 1626 1661

MS2 capsids

Hz0

TABLE

LASER-RAMAN

SPECTROSCOPY

OF

PHAGE

MS2

111

(i) Amide group frequencies Intense Raman lines due to vibrations of the peptide group are called amide T. (usual interval, 1625 to 1675 cm-l) and amide III (1225 to 1300 cm-l) and each may have several components. These are useful in determining the presence or absence of Q. ,3 or random-chain conformations in proteins and polypeptides (Chen & Lord, 1974). For MS2 capsids in H,O solution (Fig. 2(a)) amide I appears as an intense line centered at 1661 cm-l, while amide III appears as a strong line at 1236 cm-‘. H moderate line at 1254 cm-l and a weak line at 1287 cm- l. In the amide III region t,he 1236 and 1287 cm -I frequencies and their corresponding intensities indicate a predomina,nce of /3 structure in the protein capsomers. whereas the 1254 cm -I romponent indicates also a substantial amount of randomly oriented chain segments (random-chain). That the protein conformation is determined mainly by regions of /3 and r;tndom-chain st,ructures is also consistent wit,h the appearance of an amide I line at 1661 cm-’ (Chen & Lord. 1974). Although @ and random conformation predominate, the presence of small am0unt.s of x-helical chain segments cannot be definitively excluded by the present data. When capsids are dissolved in D,O solutions, some of the amide N-H groups of the prot,ein may exchange to become N-D groups. Amide frequencies of t,hese deuterated capsids (amide I’ at 1655 cm-’ and amide III’ components at 968 and 987 cm -I, Fig. 2(b)) are consistent with the above interpretation. All amide frequencies and their corresponding spectral intensities are unaffected by increasing the solut’ion temperature from 0 to 50°C which shows that the amount of x. p and random-chain structure does not change appreciably up to 50°C. (ii) Kypqupncies of S-H

and C-S

groups

Of the 129 amino acid residues in each MS2 coat-protein molecule, two are methionine and two are cysteine (Min Jou et al., 1972). We confirm that the cysteine residues of the coat protein are indeed present in the reduced form in intact capsids by observing both t,he absence of Raman scattering in the vicinity of 500 cm-l that’ could be attrihuted to disulfide S-S stretching vibrations and the presence of Raman scattering at 2574 cm-l that can be assigned to sulfhydryl S-H stretching vibrations. For capsids in D,O solution, Figure 2(b) shows a shift of the sulfhydryl group frequency to 1872 cm-l as expected if the hydrogen atom of the S-H is replaced by deuterium (S-D). The deuterium exchange of S-H groups appears to be complete in the capsid at room temperature after two hours in D,O. This result is of interest since at, least, one other protein. glyceraldehyde-3’-phosphate dehydrogenase, has been shown to have both rapidly and slowly exchanging S-H groups in the native structurcb (Peticolas (1975) Biophys. Sot. Abs., Biopolymers Subgroup, 19th A7l.n. ;Meeting. Philadelphia). From bhe exchange rate we conclude that, the S-H groups of the coat prot’ein are exposed to the solution and are not, buried in hy&ophohic regions of the protein at 23°C. The weak and broad Raman line at approximately 710 cm-1 in both deuterated and non-deuterated capsids is assigned to C-S stretching vibrations and serves to identify both methionine and cysteine residues. (iii) Frequencies Previous

non-ionized

of carboxyl

groups

studies (Lord & Yu. 1970a,b) indicate that the ionized (---COO-) (-COOH) states nf the carboxyl group are distinguished from

and one

112

G.

J.

THOMAS

JR

E’I’

3L.

another by their characteristic Raman frequencies at approximately 141.5 and 1720 cm-l, respectively, in spectra of aspartic acid and glutamic acid derivatives. Similarly the ionization states of terminal carboxyl groups in polypeptides may 1)~: determined by this criterion. The four aspartic acid and five glutamic acid residues as well as the carboxyl terminus of each coat-protein molecule may exist in either the ionized or non-ionized form under the conditions employed here (pH or pD 7). The absence of a prominent Raman line near 1700 to 1725 cm-l that could be assigned to C=O stretching of COOH groups, and the presence of a weak Raman line at 1400 cm-l (Figs 2 and 5) which can be assigned to symmetrical stretching of COO- groups, would appear t,o support the existence of ionized carboxyl residues (Lord $ Yu, 197Oa). However, studies by us on acetic acid and sodium acetate show that the Raman lines in question (i.e. near 1700 and 1400 cm-l) may be too weak in comparison to Raman scattering by other functional groups to be clearly detected in the spectrum of the capsid. Therefore, a firm conclusion on the state of ionization of the carboxgl groups of MS2 should await further study of additional model compounds. (iv) Prequencies

of phenylalani)Le,

tryptophan

and tyrosine residues

Many of the prominent Raman lines of the capsid can be assigned to ring vibrations of phenylalanine, tryptophan and tyrosine residues (Table 1). These Raman frequencies are, with one exception (Yu et al., 1975), insensitive to changes in both the environment of the residue and the state of aggregation of the protein molecule (Lord & Yu, 1970aJ). Such considerations also provide a basis for use of the 1004 cm-l line of phenylalanine as an internal standard. The lines at 486, 620, 1004 and 1030 cm-l are assigned with confidence to the four phenylalanine residues of MS2 coat protein. The two tryptophan residues of each coat-protein molecule give lines at 757, 876, 1010, 1356, 1425 and 1552 cm-l. The tryptophan line at 1010 cm-l cannot be distinguished from the 1004 cm-l line of phenylalanine in the Raman spectrum shown in Figure 2 since a spectral However, with a slit-width of slit-width (resolution) of 10 cm-l was employed. 4 cm-l, we have clearly resolved the 1004 and 1010 cm-l components from one another. The p-hydroxyphenyl ring of tyrosine, of which there are four in each coat-protein molecule, gives lines at 641 and 827 cm-l. The lines at 1175, 1210, 1582 and 1625 cm-l are due to coincident frequencies of more than one of the above aromatic amino acids (Table 1). Other frequencies expected of the aromatic residues are obscured by more intense scattering from C-H deformations or C-C and C-N stret’ching vibrations of non-aromatic side chains, as discussed below (see also Table 1). We were unable to detect Raman lines from the five histidine residues that are present in the one molecule of A protein per capsid. (v) Other group frequencies Aliphatic C-H stretching frequencies of amino acid side chains are observed as intense bands centered near 2880, 2940 and 2976 cm-l for both H,O and D,O solutions. These are assigned, respectively, to CH, symmetrical stretching, CH, in-phase stretching and CH, degenerate stretching vibrations. Aromatic G-H stretching vibrations of phenylalanine, tryptophan and tyrosine are detectable only in the spectra of Da0 solutions by a weak line at 3062 cm-l.

LASER-RAMAN

SPECTROSCOPY

OF PHAGE

MS2

113

The intense Raman scattering observed near 1450 cm-’ (Fig. 2) is due predominantly to C-H deformation vibrations of CH, and CH, groups, degenerate and symmetrical modes, respectively. In addition, the prominent’ line near 1335 cm-l may contain a substantial contribution from methine C-H bending. The large number of Raman lines observed in the intervals 1050 to 1200 and 800 to 950 cm-l are difficult to assign to specific amino acids. However: C-N and C-C skeletal stretching vibrations of most of the wliphat(ic amino acids are expected t)o occur in these intervals. Other assignments to specific amino acids are givcln in Tahlc 1. (b) Secondary structure of MS.2 coat profeitt A crude estimate of the amounts of cc, /3 and random-chain conformations in t’he MS2 coat-protein molecule can be made from the amide .I11 data discussed in the preceding section. Assuming that. roughly equal a,mide III intensities at 1236 and 1254 cm-’ should be obtained from equal amounts of p and random-chain structures. the observed intensity rat’io would suggest a slight predominanc~e of j3 over randomchain structures in the capsid. Also, we estimat,cb t’hat HS much as 20% a-helical structure could go undetected even if present. since t’he amide 111 intensity associated with a-helices is generaliy weak and occurs in a region (approx. 1275 to 1300 cm-‘) partly obscured by the amide III component of p structures at. 1287 cm-l. A greater percentage of tc structure is considered unlikely in view of hhe position of the amide I frequency of 1661 cm-l. Accordingly, we estimate the secondary structure of the coat-protein molecule as roughly 60&2Oqd ,6. 40-&20?:;, random-chain and O&201/, a. It1 must be emphasized that these estimabed values are based upon assumptions about the amide III intensities which are not unambiguously established by present,ly available data. on model compounds (Chen & Lord. 1974). ant1 constitute at best, a rough approximation. Although the MS2 coat protein has not, been sequenced directly, the sequence has been inferred from the succession of codons in the gene for coat protein in MS2 RNA (Min Jou et al., 1972). The coat-protein sequence so obtained leads to the amino acid composition summarized in Table 2. With the availability of the sequence (Min Jou et al.: 1972) statistical methods may be employed to predict the protein secondary structure. One such method frequently employed is that of Chou t Fasman (197&h). Application of this method to the MS2 coat protein leads to a prediction for the secondary structure given in Table 3 (nomenclature of Chou & Fasman. 1974b). This corresponds to an overall secondary structure containing 65% /3 (sheets -1 turns). 19”;) random and 16*;, a. in agreement wit’h the estimate based on amide III Raman intensities. (c) Raman spectra

qf MS2 RNA

Raman spectra of purified MS2 RNA in solutions nont,aining 0.75 M-KC1 +- O-08 Mpotassium acetate were obtained at 10°C intervals from 0 to 80°C. Spectra recorded at, 0, 32, 50 and 80°C are shown in Figure 3> for b&h H,O (pH 7) and D,O (pD 7) solut,ions. The frequencies measured at 32°C are summarized in Table 1 (columns B and 5). Raman frequencies and assignments for other RNA molecules have been discussed in detail elsewhere (Thomas, 1970; Thomas et al., 1971; Hartman et al., 1973; Chen & Thomas, 1974). Assignments perbaining to MS2 RNA arc similar to the previously 8

Secondury

sfructure (after

predicted Chw

.for ;II~‘:% tout-protein

& E’asmwt~.

7& x‘l 90 9x 102 JO3 I 13 114 I18 119~ 1% 1%)

~molecule

1.97A)

x3 X!l !)i 101 11” 117 124 1’8

published results on Rl7 RKA (Hartman ut al.. 1973) and arc’ listed in Table 1 (column 8). For H,C solutions (Fig. 3(n)) when the temperat’ure is increased we observe the following changes in Raman lines of the purine and pgrimidine bases: the lines at 672 (assigned to G residues) and 1485 cn~l (A.(=) decrease in int,ensity: the lines at 554 (U,C), 598 (C). 727 (A), 786 (CC), 1511 (A) and 1531 cm’ (C) increase in intensity; and the line at 1238 cm-l (U,C) shifts to higher frequency. In addition,

J,;\SER-RAMAS

SI’ECTROS(‘Ol’Y --

_-

OF -___--

I’HA(:E

1 I5

MS2

,. .

50°C

32”~

0°C

\-,.

80°c

b)

:’ 32’~

0°C

I

! 2900

I 1500 Frequency

~----_-

--

-I

l 500 (cm-l)

PIG. 3. (a) Raman spectra of H,O wlut ions of JIS:! RX.4 at 0. 32, 50 and 80°C. C’onditions: 0.75 ~-Kc1 + 0.08 x-potassium acetate, pH 7, 34 pg RNA/$. Other rontiitions as in Fig. 2(b). (b) Raman spectra of D,O solutions of MS2 RNA at (I, 32, 50 and 80°C. Conditions: 0.75 31. KC1 -I 0.0X nf-potassium avetatr. pI) 7, 43 pg RNA,!,]. Other r.ortditir>nn as in Fig. Z(b).

the line at 815 cm-l and to a much lesser extent, the line at 1100 cm-l (Fig. 3(b)). due, respect’ively, to the diester (O-P-O) and dioxy (PO, ) groups of the phosphatjcl residue. decrease in intensity with increasing temperature. All of these spectral changes were estahlishrd 1)~ rise of thr 929 cm-” linp of pot,assium acet.atc as an intensity standard. Similar changes also occur in the Raman spectrum of RKd \vhen D,O is t,he solvent,, as shown in Figure 3(b). In addition, we observe here more clearlp the trmperature-dependence of Raman scatt,ering hetn,een 1550 and 1750 cm-l which is pa,rtly obscured when H,O is the solvent. All of the above spectral changes occur. for the most. part. bc%u.een 40 and W(‘.

38

-26

3.0

0.6

26

I 0

I

1 20

I

I 40

I I !Oc)

I 60

I

I 80

I.8

0

20

40

60

80

f(Y)

FIG. 4. Plots of the Raman intensities of selected lines of MS2 RNA uer.Qus temperature (t). The intensity (axis on left; axis on right) of each line is normalized to that of the internal standard (929 cm -’ line of acetato- ) in the same spectrum. Intensity units are arbitrary. (a) Raman lines showing sharp melting transitions. (0) 786 (H,O) C, U; (m) 727 (H,O) A: (A) 665 (D,O) G; ( ,I) 672 (H,O) G; (0) 1511 (H,O) A: (0) 1531 (H,O) C. (b) Raman lines showing gradual melting transitions. (A) 1476 (D,O) G, A; (u) 1100 (D,O) P; ( n ) 816 (H,O) P; (0) 1667 (DZO) U, G, C; (0) 1238 (H,O) U, C.

This can be seen for several of the Raman lines by reference to Figure 4, where the normalized Raman intensities are plotted as a function of temperature. These results indicate that the conformation of the MS2 RNA molecule, when in a protein-free environment, is sensitive to changes in temperature, particularly above 40°C. The nature of the conformational changes occurring in MS2 RNA may be identified by consideration of the following. In polynucleotide model compounds, base stacking of A, U and C residues causes certain Raman lines (e.g. 720, 780, 1230 and 1575 em-l) due to in-plane vibrations of the atoms in these bases to undergo losses of Raman intensity (hypochromism), whereas base-stacking of G residues causes certain of its Raman lines (e.g. 670 and 1480 cm-l) to undergo gains of intensity (hyperchromism) (Thomas, 1970; Small & Peticolas, 197&b; Lafleur et aZ., 1972; Morikawa et al.. 1973; Rice et al., 1973; Prescott et al., 1974). When base-stacked secondary structure is removed by heating, the unperturbed Raman intensities are restored. When both base-stacked and base-paired secondary structures are under consideration, the same correlations generally apply as has been shown for ribosomal RNA (Thomas et al., 1971) and transfer RNA species (Thomas et nl., 1973; Chen C Thomas, 1974). In addition, base-pairing causes a pronounced change in the pattern of Raman scattering in the “double-bond region” (1600 to 1700 cm-l) (Thomas, 1970).

LASER-RAMAN

SPECTROSCOPY

OF PHAGE

3152

117

Secondary structures in aqueous RNA and in crystalline RNA, as well as in polynucleotide model compounds, also generate intense Raman scattering at 815 cm-’ due to a specific geometry of the phosphodiester group in the ordered backbone (Thomas, 1970; Thomas $ Hartman, 1973; Chen & Thomas, 1974). When this geometry is changed by thermal denaturation, the 815 cm-l line disappears and is replaced by a line near 795 cm-l characteristic of the disordered backbone. We have shown that there is a quantitative correlat’ion bet%-een the intensity of the 815 cm-l line and the fraction of nucleotide residues in the ordered conformation (Thomas & Hartman, 1973), provided the ionic strength of solution is low, i.e. no excess counterions are present. In the present, case with 0.75 M-KC1 present in excess ~2 do not, expect the quantitative relationship to hold strictly. However, the relative intensity of the 815 cm-l line remains an indicator of the relat’ive amount’ of ordered secondary structure in MS2 RNA and a lower limit of 851: of 6he nucleotide residues may be inferred as participating in base-pairing and or base-stacking interact’ions (Thomas & Hart,man. 1973). Accordingly, the present data indicate that heating svlutions of MS2 RNA leads to the unstacking of A, U, G and C bases, to the rupture of int,ramolecular hydrogen bonds het,ween complementary bases and to the general breakdown of ordered structure in the RNA backbone. More specifically, Figure 4 reveals the following. (1) Loss of hypochromism (lines at 1531, 1511, 786. 727: 598 and 554 cm-’ for H,C) solut,ions and 1522, 776, 714 and 549 cm-l for D,O solutions) or hyperchromism (672 cm-l in H,O and 665 cm-l in D,O) indicates that base-stacking interactions of -4, U, G and C are eliminated for the most part above 60°C in a more or less co-operative manner. (2) Int,ensity decay in the phosphodiester frequency (approx. 815 cm-l) indicates t,hat disordering of the backbone is a gradual process occurring over the entire range 0 t,o 80°C and is not dependent only on the eliminat,ion of base-stacking intvract(ions. (3) Int’ensity changes in the carbonyl region (1657 cm-l) are gradual, reflecting the rupture of hydrogen bonds between base-pairs over a wide temperature range. (4) Intensity changes at 1238, 1485 and 1475 cm-l. which are difficult to interpret,. probably reflect the combined effects of elimination of base-stacking and basepairing, which altogether occur over a wide temperature range. with the major part occurring above 60°C. Finally, we observe that the intensity of the 1100 cm- l line of MS2 RNA decreases in a small but significant fashion with increasing temperature when 0.75 M-KC] is present. In the absence of high salt concentrations t.his line intensity is independent of temperature (Thomas & Hartman, 1973). Consequently, the data obtained here indicate that electrostatic interactions between K+ and the negatively-charged backbone (PO, groups) are sufficiently strong to perturb the Raman scattering intensity of the symmetrical PO; stretching vibrat,ion and that such perturbations are temperature dependent. (d) Raman spectra of MS’% phage In this section we present the Raman data obtained on aqueous MS2 phage at conditions of both high and low ionic strength, and discuss these results in relation to those obtained on capsids and RNA separately (sections (a), (b) and (c), above).

C:. J. THOMAS

.JH fi,“l’

.1 f,.

Ii00 Frequency (cm-l) FIG. 5. (a) Raman spectra of MS2 phage in Hz0 solutions of high ionic strength at 32°C. Conditions: pH 7, 0.75 M-KU, 60 pg phage/pl. Other conditions as in Fig. 2(a). The spectrum is unchanged over t,he range 0 to 50°C. (b) Raman spectra of MS2 phage in D,O solutions of high ionic strength at 32’C. Conditions: pD 7, 0.76 M-KCl, 79 pg phage/pl. Other conditions as in Fig. 2(b). The spectrum is unchanged over the range 0 to 50°C.

(i) Xolutions of high ionic strength Raman spectra were obtained on MS2 in solutions containing 0.75 M-KC1 (pH 7) at 10°C intervals from 0 to 55°C. The spectrum recorded at 32°C is shown in Figure 5(a). The spectrum of the virus is independent of the temperature up to 55°C. However, upon heating to between 60 and 65”C, the protein of the phage precipitates completely from solution, leaving only the protein-free RNA in the supernatant. The Raman spectrum of the supernatant is similar to that of pure MS2 RNA. with no detectable trace of protein. To provide a basis for standardizing the Raman intensities of the phage, spectra were also recorded as a function of temperature on solutions containing 0.08 Mpotassium acetate as well as 0.75 M-KCl. From these it was verified that the 1004 cm-’ line of phenylalanine residues in the phage protein is independent of temperature and suffices as a reliable internal standard for comparing other Raman intensities of the phage as a function of temperature. On this basis, the Raman scattering from the phage does not change as long as the phage remains in solution (i.e. up to 55°C). This is true of Raman lines assigned to both the protein subunits of the capsid and associated MS2 RNA. The resistance of the associated RNA to changes in secondary structure upon heating to 55°C is quite different from the susceptibility of proteinfree MS2 RNA to large changes in secondary structure as revealed by Raman spectra presented above (Figs 3 and 4) and shows that protein-RNA interactions stabilize the secondary structure of the RNA in the native phage.

LASER-RAMAS

SPECTROSCOPY

OF PHAUE

MS”

119

We observe further that the structurally informative Raman frequencies and intensities of the phage protein. riz. amide I, amide 111, S-H and S-D group frrquencies, and so forth (section (a) above), to the extent’ that they are not obscured by Raman scattering from phage RNA, are unchangtbd from their values in the capsid. Thus the presence of RNA in the phage has no detectable effect on the Raman scat,tering and therefore on the structure of t,he phage protein. Detailed assignments are included in Table 1 (columns 3. 6. 7 and 8). The results obtained with D,O solutions of phage at high ionic strengt,h (0.75 Min KCl) are analogous to those obtained with H,O solutions and a-e summarized Table 1. A representative spectrum is shown in Figure 5(h). Therefore. t,he previous evidence concerning the existence of COO- rather than COOH groups in the protein and the availability of S-H groups to the solution in the RXA-free capsid is a,lso indicat,ed for the nativr phage.

(ii) 8okutiort.s of 1ow ion%< strength Raman spectra of MS2 phage in H,O solutions containing 0.1 M-potassium acetate. pH 7 (and in D,O solutions containing 0.1 M-KU, pD 7) were obtained at intervals of 10°C in the range 0 to 50°C. At 60°C the phage protein again precipitates complete13 from solution. Since the Raman frequencies of t,hcb phage at low ionic strength conditions (and 32°C) do not differ from those observed at high ionic strength conditions. t,he data and assignments of Table 1 (columns 3, 6, 7 and 8) are applicable here as well. The spectral contributions from the phage protein are. as before, unaffected b> increasing t’he temperature up to 50°C. However. small but significant changes do occur between 40 and 50°C in the Raman scattering from RNA subgroups within the phage. These spectral changes are qualitatively similar to those observed for prot.einfree RNA (Fig. 41, though smaller in magnitude. The three m 1st obvious changes in the spectra are a 10 to 15Oj, reduction in the intensities of the lines at 814, 1324 and 1335 cm -I. These changes do not occur in the spectra of either high-ionic-strength phage or of capsids and therefore they indicate that the secondary struct’ure of th(a viral RNA molecule. or of some part(s) of it, is de-st’abilized between 40 and 50°C by lowering t,he ionic strength of solution. A plausible explanation for this rclsult is given in section (e). t&w.

(iii) C’omparison

of spectm of phage and of its comporLerLts

In order to provide a clear comparison of the differences, if any, between Raman scattering of the phage and its constituent, parts, we show in Figure 6 tracings of the spectra of phage and the sum of phage constituents (capsid plus RNA) at conditions of high ionic strength. In Figure 6, only t’he spectral ranges with little or no interference from Raman scatterin: of t.he solvent, (HZ0 or D,O) or buffer (acetate-) are shown. These tracings were made from the best available spectra as follows. Solid-line curves (phage) were reproduced with a flat baseline from the recorded spect.ra of phage at the conditions indicated. Broken-line curves (constit,uents) were synthesized by adding together the Raman spectra recorded from RNA-free capsid and proteinfree RNA in 0.75 ~-Kc1 with the same baseline. The synthesized spectra reflect thr actual weight ratio of protein to RN,4 present in the native phage.

1200 Frequency (cm-9

FIG. 6. Comparison of the Raman spectrum spectra of capsid and RNA components (----). and D,O (upper) solution of high ionic strength

of MS2 phage (-) From spectra recorded (0,75 *T-KU).

600

with the sum of Raman at 32°C in H,O (lower)

It is seen from Figure 6 that at 32°C the spectrum of the phage is not significantly different from the sum of spectra of the coat protein and RNA constituents. The apparent discrepancy in the amide I region of Figure 6 (upper), as well as apparent discrepancies in the amide I and 1355 to 1425 cm-l regions of Figure 6 (lower) are attributed to uncertainties in the spectral baselines. Refined spectral measurements will be required to prove the complete identity of these spectra. (e) Heat-degraded

MS2 phage

A study was conducted on MS2 phage in H,O solutions containing 0.1 M-potassium acetate, pH 7, in order to look for evidence from Raman spectra of the so-called “heatdegraded” or HD particles of the virus. Formation of HD particles from native phage in low ionic strength buffer by controlled heating at 50°C for several minutes has been reported on the basis of the differences in sedimentation properties of the R17 phage (Steitz, 1968) and of the MS2 phage (Verbraeken & Fiers, 1972) and in optical rotatory dispersion curves of the MS2 phage (Oriel, 1969) before and after such heat treatment. Presumably, following a heat treatment, the RNA is partially released from the capsid which remains intact (Verbraeken t Fiers, 1972). If the HD particle differs substantially from the native particle in terms of its overall composition of RNA and coat protein or in terms of the secondary structures of RNA and protein constituents, then we would expect such differences to be revealed in the Raman spectra. To investigate these possibilities we obtained Raman spectra of the phage at 32°C both before and after heating to 55°C for 10 minutes. In a second experiment

LASER-RAMAN

SPECTROSCOPY

OF

PHAGE

MS2

I 2I

the phage was heated to approximately 60°C for 10 minutes and cooled again to 32°C before its spectrum was recorded and compared with unheated phage. In each case, we could find no significant difference between the Raman spectrum of the unheated and heated-and-cooled sample. No change in the extinction coefficient at, 260 nm was observed for phage and HD part,icles. However, examination of t,he sedimentation coefficients of the heat-treated samples by sucrose gradient centrifugation revealed that formation of HD particles had in fact occurred (HD particks sediment at 60 S and profiles show little or no loss of RNA or protein before irradiation uyhereas native phage sediment at 80 S (see Fig. 1)). These results therefore indicat’ta that the secondary structure of the partially released RNA is not. sufficiently different from t’hat of completely bound RNA at 32°C to be detected by Raman spectroscopy. Apparently the overall secondary structure (amount, of base-stacking and basepairing) of MS2 RNA a,t 32°C is little changed by conversion from native form to HD particles. At’ the same time these results are consistent M?th the changes observed in thti Raman scattering of RNA when the MS2 phage at low ionic strength is heated to 50°C (section (ii), above). ,4b 50°C we would expect, some HD particles to be present in solution and these would have some RNA released from its normal association \r,ith the capsid. This released portion of the MS2 RNA is free t’o assume a less ordered secondary structure than occurs at 32°C. in the same way that protein-free RNA assumes a less ordered secondary structure at elevated temperatures. We note. however, that more prolonged heat treatment of the phage at 60°C. eventually leads t’o a complete breakdown of the virion with the prot#rin components precipitating from solution as reported earlier.

4. Conclusions The sensitivity of the Raman spectrum to conformational properties of nucleic acids (Hartman et al., 1974), proteins (Lord, 1972) and viruses (Hartman et al., 1973 ; Thomas & Murphy, 1975) allows us to reach a number of conclusions on MS2 st’ruct’ure from the present study. First, the conformation of coat-protein molecules in the MS2 capsids and the protein-protein interactions in the assembled capsid are stable up to 50°C. We find no evidence whatsoever of any alteration of the capsid structure between 0 and 50°C. The conformation of the coat protein in the capsid is determined mainly by regions of p-sheet and random-chain structures in the protein backbone. MS2 differs great11 in this respect from the filamentous DNA phages, fd and Pfl, the coat-protein molecules of which are a-helical and are reversibly altered in secondary struct,ure by changes in this temperature range (Thomas & Murphy, 1975). Second, there are no disulfide bridges either within coat-protein molecules of MS2 or bet’ween them, and all sulfhydryl groups are accessible for deuterium exchange. This suggests that S-H groups are not buried within bhe protein in hydrophobic regions which are inaccessible to D,O solvent. We are as get’ unable to determint, whether MS2 contains -COOH groups for hydrogen-bonding with protona.ted cytosine residues, as has been proposed for turnip yellow mosaic virus (Kaper, 1972). Third, the conformation of protein-free MS2 RNA is similar to that of proteinfree RI7 RNA (Hartman et al., 1973) and 16 S and 23 S rRNA (Thomas et aZ., 1971: Thomas & Hartman. 1973). containing at, least 85”{, of t,he nucleotide rcsidutas in

I?!

(i.

.I. ‘I‘HoJl.\s

.JH /<‘I’ .-i I,.

regions of base-pai red and base-stacked swondary structure at J2 ( ‘. This xc~c~ot&r,, structure is gradually eliminated tj>- increasing the temp~mt,uw. Ai sut)st;intial disordering of the RNA backhone and elimination of tww-pairing wcurs up to .~i(j”(‘. hub t’he unstacking of purintb and pyrimidinc I)astas ocwrs for tllcl Ill(Jst part ;l\)()vc. 50°C.

lqourth. the stability of the protein struct’ure in the phage is the samt as for that of the capsid. i.e. the protein-protein interactions \vhich st,abilize thcb taapsid helow~ ss-‘(! are a,pparently not affected by t’he presence of RXA \z,ithin it. On the other hand, the association of RNA to the capsid in the native phagc stabilizes thcx secondary structuw of the RNA. which prevents both the disordering of the RNA t,at;khone :LII~ tht cllimination of base-pairing that normally occurs \~%en RlTA alone is hcat,ed t,o 50°C. This stabilizing effect is more pronounced in phage particles at high ionic strength t’han at low ionic strength. In the former case. no changes whatsoevw can bc dctct:ted in the RNA upon heating to 50°C. In the lather cmaw. small structural changes art’ detected in the RNA upon heating t’o 50°C. but, these changes are grwbly diminished in comparison to t’he struct,ural changes observrd for protein-fire: RNA \vhtw hcatcd to 50°C. This difference is due to the fact) that the phagr are morr stable in 0.75 MKC1 solution than in 0.1 &I-pot’assiurn acetate solution and do not, form HD particles at 55°C in H,C) sotuhions cont,aining 0.75 IV-KU (Hartman & ~~(~I)onald-Ordzit,. unpublished data). Fifth. the HD part’icles w%ich have been distinguished from natiw phage hy theit 1968: Verbraeken & Piers. 1972) and different sediment’ation propert,ies (St&z. optical rotaboy dispersion curves (Wet. 1969). are distinguishable from native phage by the temperature dependence of their Raman scattering. Sixth. we note that t,hc MS2 capsid. either \zith or wit’hout its complement of RNA. can be precipitated completely from the solutions examined here by heating to 60°C or higher for one or more hours. Since the prot~ein and RNA constituent’s of the phage are so easily distinguished from one another by their Raman spectra, these results have suggested to us tjhe plausible use of laser-Raman spectroscopy for assaying the RNA : protein ratio in virusrs or in thrir derived particles. Finally. 1z.c havtb drmonstrated that law-Raman spectroscopy in wmbination with i)iochemical met,hods can be used t,o obtain specific information ahout t8htb st,ructure and stabilizing interactions of an RNA virus in aqurous solution. This approach could also be utilized to obtain structural information on other ayucous nucleoproteins, such as nucleohistorws and ribosomen. which art’ difficult to investigate by alternative physicochemical methods. The assignments of Rama. lines of MS2 phagf to protein and nucleic acid sul)groups should be of considerable value in further studies of RNA4 hactrriophages utilizing vibrational Raman spectra. *Ireas of particular concrrn to US include the use of specific site-binding reagents. such as Hg2+ . and the measurement of rates of isotopic hydrogen exchange (Livrament,o & Thomas. 1974: Thomas & Livramento. 1975) at the C-8 positions of adenine and guanine residues of MS2 RNA and of 8-H groups in capsid probein to elucidat#e further the structure and~assembly of MS2 and R17 virions. TIP snppolt of the Kational National Institute of Allrrpy fully acknowledged.

Science Formdatioll (GB 41382 and (:H 29289A2). and tllc nrld Infectiorls Disrases (AI- 11855 and .%I- 11856) is grate

l,.\s~&RAhf.%S

SPECTR08COPY

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l’Hr\(:E

MS-?

REFERENCES Argrtsingrr. J. E. & Gussin, G. N. (1966). J. Mol. Biol. 21, 421-431. (‘lhen. M. C‘. $ Lord, R. C. (1974). J. Amer. Chem. Sot. 96, 4750-4752. C’hc-n, M. (1. & Thomas, G. a., .Jr (1974). Biopolymers. 13. 615-626. (‘h(>rb. M. (:.. Lold, K. C. dz Mondrlsohn, K. (1974). .1. .-lmer. C’hem. Sot. 96. 3038-3041. (‘lloll. F’. Y. 8r. li’asrnan. C:. D. (1974~). Biochemi.dry. 13. 2 11 222. (“tlorl. P. Y. & Fasman, (:. 11. (1974b). Biochemistry, 13. 222 245. Hartrklall. K. A.. Amaya. J. 6r Scllachter, E. M. (1970). Sciewx. 170. lil- 173. Riodwrn. h’iophys. Res. Cornmcc:~. Hartman. K. A.. (‘la>~ton. N. & Tl~omas. G. .I . . .Jr ( I!G:S). 50, !)42 -949. Hartmall, K. A.. Lord, K. C. Cy Thomas, G. J., Jr (1!174). 111 f’hysico-Chenlica( Z’ropwties .I., cd). x-01. 2. pp. 1 XI), Academic F’rcss, N.Y. of ,Ync/eic A rids (Duchesw. K+I~PI,. ,I. M. ( 1978). In RSA l’irtrses: Replicatiorc auf/ ,Strfrctccre (Blvcmcndal. H., .Jaspars. E. M. .J ., \‘anKammcn, .I. & Planta, K. .J . . et&). pp. 1!)- 41, Elwvic>r. N.Y. Knigllt. (‘. ,A. (1!)74). Molecular ITiro/ogy, McGraLv-HIl1, New York. Ii. .J. B Paranclr?cll. \V. (1972). I.iro/o.ogokrt. \*.. Mitslli. \‘.. Iitaka, Y. & Thomas. (:. .I.. #Jr (1973). Riopolymers. 12, 799-816. Biophys. 132, 8 1.5. Orit.1, 1’. .J. (196’)). Brch. B&hem. l’r.fwYltt. I<., GalnacJ1c~. li.. 111, rillll~~tlt~ll. .I. CY-rllcnrlils. (:. .I.. *Jr (1!)74). 13iopolymerx. 13. IX”1 1x45. Spectrosr. 1. Kiw. .I ,, Laflcln,. L.. Mrdeiros. (:. (‘. & Thorlias. ($. .I ,. ,JY ( 1973). .J. Raman 207 :!I;,. Harntlc~lsorl. (+. & Kaesherg, 1’. (1970). ,/. :2201. Hiol. 47. 87 $1. Simorw. I,., BergstrGm, G.. Blomfelt. (i., Forss. S.. Strnhdck, H. k \VarrsCn. Ci. (1972). f’r,,rrnrentatiorres I’hysico-Mathenlaticae. 42. 125 207. Small. 15. \\.. 8 Prticolas, LV. 1,. (1971~). RiopoZ~yrrte~.s. 10, 69 XX. Small. E. \V. & l’f%icolas, u’. I,. (1!)7lh). Riopo/ymers. 10, 1377 1418. rind Biochemical App/ir*atiotrs of I,n,ser.r (Mow-t>, (‘. B.. &). s, piro. ‘I’. (:. ( 11)i-f). In Chemical Acack~nlir Pwss. Ne\v York. Stt,itz. .I. .\. (1968). ,7. ?UoZ. Hiol. 33. !*47 951. Strauss. .J. H.. *Jr & Hinsl~eimw. l<. L. (1963). J. 11lol. H%o/. 7. 43~ 54. Wlo~uls. (:. .I.. *Jr (l!JTO). Biochim. Biophys. Acta, 213. 417 423. ‘rho~nt~s. (i. .J ., .J I’ ( 197 1). In Pllysical l’echniymes in Bioloykd Research. Optical ‘Jechniq,re,q ((Mvr. c:.. WI). 2nd cdn. x-01. l-4, Academic Press, New York. Tholrlilr. c:. .J.,
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\7(vhriit*kc1rl.

E. 6 Fiers, \v. (1972). ITirology. 59. 690 j()(). K. It., Alberts, B. M.. Bcnzingw. R.. l~awhorI1C~. I,. & Treiber, l.irology, 40. 734-744. Yu, X-T. (1874). .J. Amer. Chem. Sot. 96, 4664-4666. YII. N-T. h .Jo, R. H. (1973). J. .4mer. Chem. Sot. 95. 5039. 5037. ~~a1llNlnoto,

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JR

Eil’

:1 /,.

Yu, N-T., Liu, C. S. & O’Shea, D. C. (1972a). J. Mol. Biol. 70, 117~132. Yu, N-T. & Jo, B. H. & Liu, C. S. (1972b). J. Amer. Chem. Sot. 94, ‘7572-7575. Yu, N-T., Lin, T-S. & Tu, A. T. (1975). J. Biol. Chem. 250, 1782-1785.

,Vote added in proof: After this paper was submitted for publication, the recent work of Siamwiza, Lord, Chen, Takamabsu, Harada, Matsuura & Shimanouchi (1976, Biochemistry, in the press) wa,s brought to our attention. These authors have established a correlation between the Raman scattering by tyrosine (820 to 860 cm-l region) and the hydrogenbonding interactions of its p-hydroxyl group. Accordingly, the appearance of a strong Raman line at 827 cm-l in spectra of MS2 capsids, without a counterpart of comparable intensity near 853 cm-l, may be taken as evidence of strong hydrogen bonding in the capsid between tyrosyl OH groups as donors and other protein groups as negat,ivc acceptors (possibly -COOH groups). These interactions presumably occur in “buried” or hydrophobic regions of MS2 that are inaccessible to solvent (H,O). Such interact,ions are frequently encountered for protein molecules that are rich in /l structure (op. cit.). It thus becomes important to identify the potential acceptor groups for such hydrogenbonding interaction. We also wish to mention that few if any of the cyt,osine residues of the RNA in MS2 exist in the protonated form. Protonated cytosine residues give an extremely strong line near 1255 cm-l, which is not seen in the spectrum of the RNA (Fig. 3) or of the change in the state of phage (Fig. 4). The composite spectra (Fi g. 6) show no significant protonation of the cytosine residues between RNA in the phage particle and in the protein-free state. It therefore seems unlikely that a significant fraction of the 860 cyt’osine residues in an MS2 virion are protonated and hydrogen-bonded to protonated carboxyl groups as proposed for turnip yellow mosaic virus by Kaper (1972). A more complet,e discussion of this determination as applied to turnip yellow mosaic virus is given in a paper by Turano, Hartman & Thomas, which will appear in the June 1976 issue of J. Phys. Chem.