THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 1, Issue of January 10, gp: 663-671,1985 rrnted m U.S.A.

An Alkyl Imidate Labeling Study of the Organization of Phospholipids and Proteins in the Lipid-containing Bacteriophage PR4*

(Received for publication, May 29,1984)

Trisha Ne11 Davis$ and John E. Cronan, Jr. From the Department of Microbiology, university of Illinois, Urbana, Illinois 61801

The structure of the lipid-containing bacteriophage PR4 was studied using two alkyl imidates, ethyl ace- timidate (EAI), a reagent permeant to lipid bilayers and isethionyl acetimidate (IAI), which is impermeant to membranes. The virion is an icosahedral particle consisting of a protein coat surrounding a membrane of phospholipid and protein which in turn encloses the DNA genome. Upon exposure to the permeant reagent, EAI, 50% of the phosphatidylethanolamine (PE) mol- ecules reacted rapidly (half-life <10 min). A similar fraction of the PE also reacted with IAI, the imper- meant reagent. The remaining half of the PE molecules reacted slowly with EA1 (half-life of 80 min) and failed to react with IAI. All of the phage proteins reacted with both EA1 and IAI (except a DNA-associated pro- tein which reacted only with EAI). These labeling re- sults indicate that the phage membrane consists of a lipid bilayer and that at least a portion of each phage protein (except the DNA-associated protein) is exposed on the external face of the lipid bilayer. Several of the membrane proteins could be cross-linked either to the phage membrane PE after EA1 treatment or to phage phosphatidylglycerol after periodate treatment. The major structural protein of the phage was readily cross-linked to PG but failed to cross-link to PE sug- gesting that the protein specifically interacts with PG.

~~

The lipid-containing bacteriophage PR4 and closely related phages such as PRDl offer a simple and readily manipulated system to study membrane assembly and structure. These phages grow on the well-characterized hosts, Escherichia coli and Salmonella typhimurium, and are easily purified. Previous studies have concentrated on the composition of the virion and its assembly. The bulk of the structural information on the virion comes from electron microscopy (Bradley and Rutherford, 1975; Lundstrom et al., 1979) and studies on the structures produced by disruption of the virion with guanidine hydrochloride (Bamford and Mindich, 1982). These studies show that the virion is an icosahedral particle with a diameter of 65 nm (Bradley and Rutherford, 1975) in which the external icosahedral protein coat encloses a membrane composed of phospholipid and protein. This membrane in turn surrounds the genome, a double-stranded linear DNA of 14.5 kilobase pairs. (Lundstrom et al., 1979; Davis et al., 1982). The phos-

* This work was supported by Grant GM26156 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ National Science Foundation Graduate Fellow. Present address, Department of Microbiology and Immunology, University of Califor- nia, Berkeley, CA 94720.

pholipid compositions of the host and phage membranes are qualitatively similar. However, when compared to the host the phage membrane is enriched in PC’ at the expense of PE (Muller and Cronan, 1983). The phage lipids are derived from those present in the host membrane (Muller and Cronan, 1983). The virion contains at least 14 proteins, one of which accounts for 80% of the total virion protein and presumably forms the faces of the icosahedron (Davis et al., 1982). In this work, we have sought to answer two main questions important to the use of this phage to study membrane biogenesis. First, is the phospholipid component of the virion structured into a bilayer? Second, which of the virion proteins are in contact with (or enclosed by) the phospholipid layer?

The imidoesters, EA1 and IAI (Fig. 3), offer several advan- tages as probes of membrane structure because the probes have the same specificity and very similar reactivities but are complementary in their ability to penetrate membranes. Whereas both reagents react with primary amines, only EA1 is nonpolar and can cross hydrophobic membrane barriers. Due to the hydrophilic SO3 group, IAI is impermeant to both natural and synthetic membranes (Whiteley and Berg, 1974; Rothman and Kennedy, 1977; Roseman et al., 1975). Thus, proteins and amino phospholipids exposed on the exterior side of a limiting membrane can be identified by reaction with both reagents, whereas the phospholipids of the inner leaflet of the bilayer and those proteins protected by the hydrophobic barrier are only accessible to EAI. Another advantage to the use of these reagents specific to phage PR4 is that the probes are sufficiently small to penetrate the protein layer that encloses the lipid layer. There are, however, two difficulties in working with acetimidates (Peters and Richards, 1977). The reagents hydrolyze at significant rates and at pH values near neutrality, there are two possible products of the reaction of imidoesters with amines (Hand and Jencks, 1962). In this paper we demonstrate that these problems can be overcome (and even exploited) to provide valuable information on the structure of this phage.

EXPERIMENTAL PROCEDURES~

RESULTS

Rates of Hydrolysis of EAI and IAI-To accurately control amino group modification using the acetimidate reagents, the

The abbreviations used are: PG, phosphatidylglycerol; EAI, ethyl acetimidate; IAI, isethionyl acetimidate; PE, phosphatidylethanola- mine; SDS, sodium dodecyl sulfate; PB, phosphate buffer.

Portions of this paper (including “Experimental Procedures,” Table I, and Figs. 1-4, 9, and 10) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document NO. 84M-1606, cite the authors, and include a check or money order for $5.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

663

664 Imidoesters as Structural Probes of Phage PR4

rate of hydrolysis of the reagents must be known. However, the values given in the literature disagreed greatly. Hunter and Ludwig (1962) had reported a half-life of hydrolysis of 27 min at 25 “C (pH 8.0) for methyl acetimidate whereas Browne and Kent (1975) reported that EA1 hydrolyzed with a half- life of about 4 min at 22 “C (pH 8.5). We measured a half-life of 26 min for EA1 at 22 “C (pH 8.4) (Fig. l), a value in good agreement with that reported by Hunter and Ludwig (1962). We attribute the aberrantly short half-life reported by Browne and Kent (1975) to the accumulation of significant concen- trations of ammonia due to the high concentration (0.2 M) of EA1 used. The ammonia resulting from hydrolysis would rapidly react with the remaining EAI. In fact, one of the products observed by these workers was acetamidine, the product of the reaction between an acetimidoester and am- monia. Thus, Browne and Kent (1975) primarily measured the reaction of EA1 with ammonia rather than EA1 hydrolysis per se. We and Hunter and Ludwig (1962) used much lower acetimidate concentrations (25 and 12.5 mM, respectively). The half-life of IAI hydrolysis at pH 8.4 and 25 “C was 40 min (Fig. l), a value in reasonable agreement with the value of 26 min obtained by Whiteley and Berg (1974) at pH 8.4 and 23 “C.

Products of the Reaction between Acetimidoesters and PE- It has often been assumed that the reaction of an acetimi- doester with an amino group yields only a single product, the acetamidine. However, this is true only under quite alkaline conditions (pH > 10.5) inappropriate for most biological materials (Hand and Jencks, 1962). At lower pH values (pH 8-9) two products, the N-substituted acetamidine and the N- substituted acetimidate, are formed (Hand and Jencks, 1962; Browne and Kent, 1975). We have demonstrated the forma- tion of two products upon reaction of either EA1 or IAI with sonicated dispersions of 32P-labeled PE (Fig. 2). Since these unknown compounds also became labeled when [ 14C]imidoes- ters and nonradioactive phospholipids were used (data not shown), both compounds were derived from the reaction of PE with the imidoester. The products predicted from the reaction mechanism (Hand and Jencks, 1962) are N-PE ac- etamidine and N-PE ethyl acetimidate (IV and V, respec- tively, in Fig. 3 with R2 being phosphatidylethanol). The formation of unknown 1 was greater at pH 10 and also was formed at the expense of unknown 2 in the presence of ammonia (Table I), the behavior expected of N-PE acetami- dine. Unknown 2 was produced preferentially at pH 8.4 and was converted to N-PE acetamidine and PE by reaction with ammonia (Table I), properties consistent with N-PE ethyl acetimidate.

The reaction of PE with IAI at pH 8.4 also produced two compounds; both of which were labeled by either PE or IAI. One product cochrornatographed with N-PE acetamidine and thus was tentatively identified as N-PE acetamidine whereas the other product cochromatographed with N-PE ethyl ace- timidate and was tentatively identified as N-PE isethionyl acetimidate (data not shown).

Treatment of PR4 with Imidoesters-The experiments studying the reaction of phage amino groups with EA1 and IAI were designed to satisfy the three conditions necessary for the reaction to follow pseudo first-order kinetics (see “Experimental Procedures”). Briefly, purified phage were mixed with a large excess of imidoester. Every 30 min fresh imidoester was added, replacing the hydrolyzed imidoester and thereby returning the imidoester concentration to the initial concentration. The pH was maintained between 8.4 and 8.6 throughout the experiment by the frequent addition of HC1.

We first determined the rate of phage inactivation by EA1 or IAI (data not shown). A phage sample was removed for infectivity titer every 30 min before addition of additional imidoester. Treatment with 25 mM EA1 resulted in an expo- nential decrease in infectivity (half-life of 25 min) with ap- proximately 2% of the phage surviving the treatment for 150 min. Treatment with IAI gave much less phage inactivation; 60% of the infectivity remained after 150 min of treatment.

Although the acetimidate-treated phage samples had de- creased infectivity, the virions remained intact as determined by sedimentation velocity centrifugation. 32P-labeled phage treated with either EA1 or IAI (or left untreated) were sedi- mented through 5-20% sucrose gradients (Fig. 4). In all three cases, 50% of the radioactive phage loaded on the gradient was recovered in a single peak which coincided with the peak of infectivity (instability of phage PR4 on sucrose gradients has been observed previously (Davis et al., 1982)). The re- maining label, which was spread throughout the bottom third of the gradient, had the same lipid composition as the infec- tious peak of the gradient suggesting aggregation of the phage. No peaks consistent with the products of phage disruption or of empty (DNA-less) particles (Davis and Cronan, 1983) were found (Fig. 4).

Reaction of Phage PE with EAI-EA1 treatment of phage uniformly labeled with 32Pi gave a time-dependent conversion of the phage PE into two derivatives (Fig. 5). The amounts of PG and cardiolipin remained constant. The products of the reaction were quantitated by scraping and counting the areas of the chromatograms corresponding to PE and its two deriv- atives (located by autoradiography). In Fig. 6, the fraction of PE remaining is plotted on a logarithmic scale uersw the time of reaction. As discussed under “Experimental Procedures,” if all the PE had the same reactivity towards EAI, in the presence of excess EA1 pseudo first-order kinetics would result. That is, a plot such as that shown in Fig. 6 would yield a straight line (x-intercept = 1.0) with a slope dependent on the rate constant, the concentration of imidoester, and pH. However, the observed reaction was biphasic (Fig. 6). The slow component reacted with apparent first-order kinetics with a half-life of 80 min. The same half-life was calculated with or without the 30-min time point and, therefore, reaction of the fast component was essentially complete by 30 min and had a half-life of <10 min. The fraction of PE reacting with a half-life of 80 min (46% of the total) was obtained by extrapolation to the x-intercept. Therefore, the fast compo- nent comprised 54% of the total PE. Greater than 90% of the total PE in the sample reacted by 180 min. To ensure that the reaction observed was between EA1 and PE resident in intact particles, a sample of the treated particles was sedi- mented in a 5-20% sucrose gradient and the peak fractions were analyzed. Ninety-two per cent of the PE in these purified particles had reacted, in good agreement with the reaction measured in the total sample (90% reacted).

Reactiuity of Phage PE with IAI-IAX treatment of phage uniformly labeled with 32P, gave results complementary to those obtained with EAI. Experiments using two different concentrations of IAI are plotted in Fig. 6. Although seen more clearly at the higher reagent concentration, at both concentrations the slope of the curves decreased and leveled off after modification of approximately 50% of the total PE. Since the three conditions necessary for the reaction to follow pseudo first-order kinetics were met (see “Experimental Pro- cedures”) the observed change in slope strongly suggested that not all of the PE was available for reaction with IAI. Assuming that all the PE had the same intrinsic chemical reactivity, the proportion of PE accessible to IAI was calculated (see “Ex-

Imidoesters as Structural Probes of PhagQ PR4 66.5

SF CL

30 60 90 120

SF

CL

0

150180 un

I:I(;. rt. I’rotlucts o f the react ion of l’lt.1 l’b; w i t h 1-:121. 1’- hlwld ph;l:.cs (T1.7 X lO”/rnl: 55‘, inlwtiorls) u‘t’re treated wlth a high concentr;ltion (2.5 mM) o f EA1 for 1x0 min as descrihtd under “Ex- perimental Procedures.” ’The phospholipids were extrarted and sep- ;Irated Iyv t hin-laver chromatography and ar~toradiographed. The nurn0c.r.v refer to the time fin minJ when samples were taken and the reaction wm terminated; un. phospholipids of untreated phage; 0. origin: APE,’, N-I’F: acetamidine: I’E,’, phosphatidylethanolamine: /+,‘//’E,’, N-I’E ethvl acetimidate: /’(;. phosphatidvlglvcerol; (’/,, cardi- olipin: SF, solvent front.

perimental Procedures”) to be between 50 and 57% of the total PE present.

IAI Treatment Blocks a Portion of the PE from Reaction with EN-A further measure of the distribution of PE in PR4 was provided by treating phage first with a saturating concentration of nonradioactive IAI and then with a saturat- ing concentrat,ion of [I4C]EAI. The amount of radioactivity incorporated into phage PE was 52-549; of the amount incor- porated into a parallel phage sample not treated with IAI and thus 52-54% of the PE was inaccessible to IAI. As calculated from the concentrations of the reagents used and the data of Fig. 6 , 51 and 94% of the PE was expected to have been accessible to IAI and EAI, respectively. Thus, 46% of the PE t.hat react,ed with EA1 should not have reacted with IAI, a value in good agreement with the value of 52-54% we ob- t,ained.

Reactivities of the Virion Proteins with the Imidoesters- Most of the protein labeling experiments were not done under the same conditions as the lipid labeling experiments since,

01-

3 0 60 90 I20 150 180

unlike the lipids, the separation properties of the imidoester modified proteins were little altered. Thus, direct labeling with radioactive acetimidates was the onlv detection method available. Only limited use of the high concentrations of acetimidates used in the lipid experiments was possible due to the expense of the labeled reagent and the high specific activity necessary to detect labeling of minor phage proteins.

Exposure of phage samples to a single dose of a low concen- tration (1.7 mM) of either [1-I4C]EAI or [ I -14CJIAI gave maximal labeling in less than 10 min and resulted in no loss of infectivity. Although the amount of radioactive labeling of the virion proteins obtained with [“CIIAI was less than with [“C]EAI, both imidoesters labeled all of the phage proteins previously described (Davis ~t al., 1982). except perhaps some of t h e poorly resolved low-molecular-weight proteins (Fig. 7).

Very similar results were obtained in an experiment similar to the lipid labeling expeiments, in which the phage proteins were first reacted with a high concentration of nonradioactive IAI and then exposed to a high concentration of [I4C]EAI (Fig. 8). In this experiment onlv those amino groups unable to react with IAI would become IT-labeled. In the control experiment, phage not previously exposed to IAI were labeled with [“CIEAI. Analysis of the phage after purification showed tha t all of the major proteins in both samples were labeled. T h e very minor virion proteins P8 and PI1 were not detect- ably labeled with [“CJEAI and the label in proteins P4 and P6 could not be readily quantified due to the low specific activity of the reagent. However, it is clear from the high specific activity experiments (Fig. 7) that 1’4 and P6 reacted with both probes. Densitometry showed that the amount of label in each of the other virion proteins (per g of protein) was decreased (but not abolished) bv prior treatment with nonradioactive IAI (Fig. 9); therefore, only a subset of the amino groups of these proteins were blocked by IAI. The high- molecular-weight proteins (M, > 60,000) and several similar unidentified bands are believed to be virion proteins cross- linked to each other (see below).

During t.he latter stages of this work, it became clear that phage PRD1, a phage previously thought to have a much larger genome than phage PR4, was in fact a closely related, homologous phage (Hamford rt al.., 1981 ). Moreover. Ramford

‘1

A EAI IA

-

-Pl

L-- -P2

- =P3 - -- -p5

-P6

D -- =P7

/P9 -Pl(

A B C

cl al.. (1983) reported a I’KI)l virion protein that we had not previously ol’served in our analysis of I’H4. ‘I’his I’KDI pro- tein (called 1’8) is covalently hound to the ends of the I’III)l genomic 1)NA (13amf’ord (,I ~1.. lW3). The association with 1)NA prevents migration of the protein on SI)S-polyacryl- amide gels and accounts l’or its omission from previous anal- yses. Although I he lipid M)eling studies strongly suggested that IA1 was unable to penetrate the lipid hilayer. the discov- ery of the I)NA-associated protein allowed s more direct demonstration of the essential membrane impenet rahility 01 this rragent. All of the availa~)le evidence on phages I’II4 and 1’111)l indicate that the 1)NA is enclosed within the lipid

membrane of the phage ~Hamlorfl and \llnfllf.h, I!W!. I);IL IS

(‘1 al., 1982; I)avis >IIld (‘rOllilI1. IWll ,IIld. h~nf~f~. 1 hf, I JN.4

associated protein .shoulfl iII\f’ I)fa Iof atfd H 11 hln t hf. 111vm hrane. The I’H1 protein ~oIIIoII~~~‘II~ to I% 1’1 I’I{I)l I, i’rc’teln I’5A as ident Ilied by its mc’lc~lcular \selght .rrId IIIW of S\ II

thesis (I)avis, 19X:{). 1Ve shourci that I’.i:1 W;I~ IIc~~rrItl to 1)X:\ using the experimental approach clr~\r~lc’pcd lor I’rotcln I% (see “ICxperlmental I’rc)crdures”l anal Ihilt I’,;:! c ot~ltl I)? Ia beled with [ ‘Y’]I?AI hut f~~wIIt~all\ no I,Ihf~lIII:: u~t h [ “(‘JIAI was f’ound (E’lg. IO) (Iahellng of the I’rotf~ln ~11 h [ “(‘]I*::11 u;i\ >Wf’old greater than with [ “(‘]I:11 1 Kurt hf*rmr’rf*. t rfz,lt mc~nt of’phage with sat~lratln~f~onf~cntr;lt~~‘r,~ of ntrnr,ldlo,rf~tr~~ IA1 &cl not block lahellng with IOU f’onf ent ratllBn\ 1’1 [ ‘t’Ib:,.1I (The scarcity of this protfsln 111 I hf* \ lrlcln rfq~llrfd Iat’f~llng .II high specific act i\ 1t.b and hfaiif fa II’D f~bfif~ii1 rcitlcbfl 1 Sync f’ I’5A IS located with the 1)N.A is It hln t hca I)h;tg:c. nlcrrr~)r,lnr*. II f(Jllows that IA1 (IOCJS not rfvrclll~ I)f’nct rilte t hf. I’hitgf* 111f’111 hrane. It should he noted that the I’I<l I)S.4 c1’111d nrat t)fa laheled with EAI either u hvn Iof~atf~~ ~$lrhlrl thf* L 1rlon I~II,I not shown) or when cxp~‘srd to the rf~agf~nt 111 ~1 I’tlrltifd, protein-free state (Fig IO)

(‘ro.\s-lrnklrz~ of limm /‘rotr~lrl.\ 10 I’l:‘ .A\ fl~~f~r~~~f~tl Xit’o\f.. one ot’ the derlvatlves of I’I< formfd tn rf~;ic3ion \\it h I<XI \\a~ tentatively identilied 21s .V-I’E rth\l ;tf ~~~IIIII~,IIc ( I’ III 1’1:: :{.

IF being phoq)hatdylet hanol). ‘I‘hl, f~~~~~l’or~ntl 15 <III IIIIIdrw.

ter and hence can react utt h aIlk nfa;rrt)\ I’rlm;lr\ an1IIIf’. .,I( h

as that of’s second molef~ule of I’K. or it nfaart’\ prc’tf,ln c~n~lno group. The I)rotluct of this react Ion L\IIIII(I t’c ;I n11’1~1f~1f~ 01 I’E: cross-linked either to ;I ~c~ond molf~ulf~ o! I’I-1 or to protein. Thus, ICAI can act 215 ‘I monotunf~t IOI~~II f‘ro\\ Ilnkln:: agent, the product I)e~nt: a dl+~r~)~tltrltf~fl anl~d~nf~ (I’etc-r\ ,111fl Richards, 1977). \‘lrlc’n protf~lns III (.IIJW proxlmlt\ 11) t hc* head group of I’E were dent Itid to t hc*lr at)ll1t\ III 1~ f’ro,y linked to “I’-1aheled matt~rlal t)\ I<:11 I’hage \lnlforn1l\ Ii)- heled with “I’, were trratcd with ICXI. purllid 1n ~cIo(~t~ sedimentation, solubillzed. and elec.1 roI)hored ( E‘I~:. I I I FILER proteins (I?, I’!). the protein l)c~t~\vc~n I’9 and I’IO. 1’11’. iIn[l 1’13) contained “I’ after trfvitni~nt \rlth K.41. 11~1 111 tht a )sence of EAI treatment t hfarf> U;I~ no Id’f*lln:: S~rlc~ phcpi- I pholipld anti I)SA are the onI\ ‘I’ I~llIf~lf~tl lIhil)Jt’ f‘l’llll~lJIlf~Ilt~

mcl since DNA does not rf’af’t K II h .lf’fst IIIII~XI~=. (WC ,lt)o\ VI, the labeled I)antls must hf. due to rcaf t 1011 1’1 I he I)rotf*lns v, 11 h B I’E derivative. ‘I-he ht rlkln:: rc+ult R:I\ t hiit I he ma,,,r f ;11)\1fl protein I’:! was not crash-linked to 1’1’

It should be noted that In thfA r~ntrf~;~tf~d f ant rc’l\ \+f’ f 1’111fl have detected a protf~ln prfvnt 111 !i\f* f OI)IV”L lrlon f I’I~I;III~ ing one molecule 01’c~oValentl~ I)orlIlcl I~hO~I’hiltf~‘~~r~‘tf~lll i11ol ecule. Since no proteln5 uf’rffi Iil~)Vled ( Fig. I Il. I’]{ 1 tlcbf*s iir~t contain I)h~‘sI)h~‘I)rc’telnh. It 4iould. honf3f’r. t)fd r~~tfd t h;it I,hosphor~latIorl of protein 1 ~rr’uld nl’t ha\fa t)fvn df~tf*f td due to the background 111 I hl\ rf’glon 1’1 t hv 21.1 ,~ntl t IIf* y(‘girf II L of the protan.

(‘rowl~nk~rf~ o/ L’rrrori I’rr~lc’lft\ to I’~i~~r~~lr~~trfl~l:‘l~i # r111 I’eriodate cleavage t)rtween t hf. L ~cin;il h\ tlrl~x~ I groIiI’5 of I’(; with periodate lorms a ‘?I’-l;rl,~lrti ald~tl\d,~ H hlf,h (‘;,I1 I hffill l’orm B Schiff*5 base \vIt h a IIc;IrlIv I’rlm;lr\ :tmInf~ ‘I‘hca Sc~h111’~ base can he reduced to iIn iini~w In .SaHII, ‘I’hv rf*iif,t 1011 15 specific IO I’(; SII~W onI\ this ~)hosl)holll)ld hiI:. LIVII~~II h\ drox>l groups.

Imidoesters as Structural Probes of Phage PR4 667

P1

P2

P3 P5

P7

I P 9

bh

B' C' D A B C I "9

. . *L

-M 4

\

A B C

FIG. 11. Cross-linking of PR4 proteins to phospholipid. h f t panel, cross-linking of proteins to PI?. :'2P,-laheled phage (4 .8 X IO" particles/ml; 50% infectious) were treated with nonradioactive EA1 (25 mM for 150 min) and sedimented on a sucrose gradient. Two peak fractions were pooled, the phage proteins were precipitated with 5% trichloroacetic acid, solubilized, electrophoresed in the SDS system, and autoradiographed. Lane A, phage labeled with ["CIEAI, as de- scribed in Fig. 8 (these phage were not "'P-labeled); lane R, "P- labeled phage treated with nonradioactive EAI; lane C, the same amount of radioactivity as in lane R hut with no imidoester treatment; lanr D, same as law C but 3-fold more radioactivity was loaded. Lanrs E', C', and D' are a 3 to &fold darker exposure of the same ( R , C, and D, respectively) gel. Right panel, cross-linking of proteins to PG. The phage were labeled with 32P-labeled glycerol %phosphate by infecting strain TD6 growing in modified medium LPAL (Davis, et al., 1982) lacking valine but containing 90 mg/liter each of L- leucine and L-isoleucine. The medium also contained 1 mM phosphate and 15 PM "P-labeled sn-glycerol3-phosphate (140 Ci/mol). Purified phage (7 X 10"' ml) were treated with 5 mg/ml of periodate as described under "Experimental Procedures." The samples were then lyophilized, solubilized, and electrophoresed on a 15% polyacrylamide gel containing 8 M urea (Davis et a/., 1982). After staining and destaining (to locate the virion proteins and molecular weight stand- ards) the gel was then dried and autoradiographed. Lane A, phage treated with periodate and reduced with NaBH,; lane R, phage treated only with NaRH.; lane C, untreated phage. The arrows mark the position of the stained molecular weight standards: 0, ovalbumin; C, carhonic anhydrase; M, myoglobin; and L, lysozyme.

using 10-fold more radioactivity and a 100-fold higher con- centration of periodate was performed (Fig. 11). In this ex- periment, only 0.5% of the phage remained infective but again protein P2 was heavily labeled. Other proteins, either P5 or P7 (which electrophoresed as a doublet in this system) and several of the small proteins were cross-linked to "'P-contain- ing material (Fig. 11). Although some label was found in small proteins, the diffuse bands did not allow identification. In these experiments, the low concentration of phage precluded random cross-linking of PC to proteins from any disrupted virions (if present).

DISCUSSION

Our experiments clearly show that the virion of PR4 con- tains two classes of PE molecules as defined by reactivity

with EA1 and IAI. The most straightforward interpretation of the two classes is that they represent the PE molecules of the two leaflets of the bilayer (Roseman et al., 1975; Whiteley and Berg, 1974; Rothman and Kennedy, 1977). The PE mol- ecules in the outer leaflet are immediately accessible to EA1 and react very quickly with the reagent. These molecules also are accessible to the nonpenetrating reagent, IAI. Our data indicate that approximately half of the PI? molecules reside in the outer leaflet. The molecules of the inner leaflet would be expected to react slowly with EA1 and he inaccessible to IAI, and our data show that approximately half of the PE molecules have such properties. The rate-limiting step of the reaction between EA1 and the slow reacting component of PE seems likely to be diffusion of the reagent across the phage membrane. I t is possible that the chemical environment, rather than the physical location, of these PE molecules is responsible for the slow reaction with EA1 and the lack of reaction with IAI. However, such environmental differences seem most unlikely to affect such a large fraction of the PE molecules especially since the two imidoesters have very sim- ilar reactivities. In addition, the inaccessibility of protein P5A to IAI provided independent evidence that IAI was unable to penetrate the PR4 membrane.

The organization of the phage proteins in relation to the phospholipids as demonstrated by imidoester reactivity is straightforward. The phage lipids are structured into a lipid bilayer with about half of the PE on each leaflet. Since all of the proteins except protein 5A can he labeled with IAI, at least a portion of each protein (except P5A) is exposed on the external face of the lipid bilayer. P5A, the protein covalently linked to the DNA molecule, is enclosed within the lipid bilayer. It should be noted that our conclusion that the phos- pholipids are structured into a bilayer is consistent with the cooperative nature of the melting behavior of the phage lipids (Muller and Cronan, 1983) and with the internally located concave and convex fracture faces observed by freeze fracture electron microscopy (Lundstrom rt al., 1979).

Our data complement those of Ramford and Mindich (1982) who examined the structure of the virion of phage PRDI, a close relative of PR4, by fractionating the phage after treat- ment with 2 M guanidine hydrochloride. This treatment re- moves the major capsid protein and a protein analogous to P3 of PR4. Thus, these two proteins are not integral mem- brane proteins. The remaining proteins are found in a mem- brane vesicle and thus, by this criterion, these proteins are components of (or are located within) the vesicle. Protease treatment of these DNA-less vesicles results in attack of all vesicle proteins except the protein analogous to PS of PR4. Since the vesicles (as visualized by electron microscopy) ap- pear permeable to the external milieu, proteolytic attack probably indicates only that the attacked proteins are incom- pletely protected by the membrane. I t should be noted that none of the proteins in the intact virion are attacked by protease (Bamford and Mindich, 1982). A comparison of these results with our imidoester data indicates that most of the PR4 proteins are in intimate contact with the lipid bilayer and protrude from the external face of the hilayer. However, our finding that only a subset of the virion proteins can he cross-linked to phospholipid indicates that membrane asso- ciation is not of itself sufficient to allow cross-linking of a protein to phospholipid.

Our most unexpected result was the selective cross-linking of the virion proteins to the phage phospholipids. Although both of the cross-linking reactions used would he classified as zero length reactions and involve attack on protein amino groups, the major capsid protein of the phage (P2) cross-

668 Imidoesters as Structural Probes of Phage PR4

linked only to the derivative of phosphatidylglycerol; no cross- linking to the PE derivative was detected. Since this protein makes up the external surface of the phage, its internal surface should be in close juxtaposition to the polar head groups of the external phospholipid leaflet of the bilayer. How then can cross-linking only be observed to PG? PE is present in the outer leaflet as shown by its rapid reaction with EA1 and accessibility to IAI but does not cross-link to P2. It, therefore, seems likely that the phospholipid annulus surrounding that portion of P2 (or a P2 oligomer) contacting the lipid bilayer, contains PG but not PE. The postulated ability of this protein to interact specifically with PG seems likely to be involved in the relative enrichment of the phage membrane in PG as compared to the host membranes. This specific affinity for PG cannot be attributed to the net negative charge of the phospholipid (Muller and Cronan, 1983) and seems likely to involve a protein-lipid interaction specific to a given phos- pholipid such as those seen in lipid activation of various enzymes.

Acknowledgments-We thank Professors Bob Gennis and Steve Sligar for helpful discussions.

REFERENCES Bamford, D. H., and Mindich, L. (1982) J. Virol. 4 4 , 1031-1038 Bamford, D., Rouhiainen, L., Takkinen, K., and Soderlund, H. (1981)

J. Gen. Virol. 57, 365-373

Bamford, D. H., McGraw, T., MacKenzie, G., and Mindich, L. (1983)

Bradley, D. E., and Rutherford, E. L. (1975) Can. J. Microbiol. 2 1 ,

Browne, D. T., and Kent, S. B. H. (1975) Biochem. Biophys. Res.

Davis, T. N. (1983) Ph. D. thesis, Yale University Davis, T. N., and Cronan, J. E., Jr. (1983) Virology 126,600-613 Davis, T. N., Muller, E. D. and Cronan, J. E., Jr. (1982) Virology

Hand, E. S., and Jencks, W. P. (1962) J. Am. Chern. SOC. 8 4 , 3505- 3514

Hunter, M. J., and Ludwig, M. L. (1962) J. Am. Chem. Soc. 84,3491- 3504

Lundstrom, K. H., Bamford, D. H., Palva, E. T., and Lounatmaa, K. (1979) J. Gen. Virol. 43, 583-592

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Muller, E. D., and Cronan, J. E., Jr. (1983) J. Mol. Bwl. 165, 109- 124

Peters, K., and Richards, F. M. (1977) Annu. Reu. Biochem. 46,523- 551

Roseman, M., Litman, B. J., and Thompson, T. E. (1975) Biochern-

Rothman, J. E., and Kennedy, E. P. (1977) J. Mol. Biol. 110, 603-

Whiteley, N. M., and Berg, H. C. (1974) J. Mol. Bwl. 87,541-561

J. Virol. 47,311-316

152-163

Commun. 67,126-132

120,287-306

k t t y 14,4826-4830

618

Imidoesters os Structural Probes of Phage PR4 ~ ~ ~ ~ l e ~ ~ t a l mterisl To: An Alkyl Imidaze Labeling Study of the Organization of

PhoepholipldS and P ro te ins i n the Lipid-Containing Bacteriophage PR4. Trimha Ne11 Dsvie and John E. Cronan, J r .

EXPERIMENTAL PROCEDURES

chased from ARershan. The e t h y l and i s e t h i a n y l a c e t i m i d a t e s were purchased from AldriCh and Sigma Chemical Companies. r eapec f ive ly .

[ I-14CIEthyI acerimidate and [l-14CliseLhionyl acetimidate were pur-

Phage Pur i f i ca t ion .

Cronan, 1983). Phage were routinely propagated on Salmonella typhimurium S t r a i n Phage PR4, plasmid plM2 and strain TU6 were descr ibed previously (Davis and

LT2 carrying plasmid pLH2. for prepa ra t ion of "on-radioactive phage the host was

g r a m i n medium 2 x YF (Miller, 1972). For 32P-labeling of phage, the host wag grown in medivm LPC (Davis e t a l . , 1982) except as noted. Buffer PB Was a 50 mn sodium phosphate buffer. pH 8 . 0 , containing 0.1 N NaC1. Phage were propagated and t i r e r e d 88 descr ibed previously and pu r i f i ed by a modif icat ion Of the Prace- dure described previovsly (Davis et 2.1.. 1982) . The following procedure Was used for the rap id p repara t ion of concentrated phage stocks. Lyseces were f i r s t c l ea red by cen r r i fuga r i an at 7000 g at L0'C and then polyethyelene glycol and NaCl were added Lo t h e f i n a l cancenfrsCions of 8% and 0.5 H. r e s p e c t l v e l y ~ The phage p ~ e c i p i c a t e d In cen t r i fuge battles f o r 4-5 hours at 4°C and were then eel- lecced by lar speed cencrifugarion at 1000 g without braking che cotor. The phage ( i n about 702 y i e l d ) were reeuspended in l i50 th the volume of PB b u f f e r . The phage (3-4 ml Yere loaded per tube) were eedimented On 5-2OZ ( V / V ) 9UCrDBe

rotor. The phage were removed from the gradients using a Pasteur plperte char g rad ien t s i n PB a t 20' f o r 50 mi" BL 82,700 g (25,000 rpm) using P Beckman SU28

had been d r a m out end curved. The phage stocks were then dialyzed overnight af

o r i g i n a l infected phage were recovered. The overall yields decreased by CWO LO

15-2O'C in PB buf fe r w i th one change of buffer. Approximately 30% of the

t h r e e - f o l d i f t h e d i a l y s i s s t e p was omit ted. A t t h i s stage, the concentration Of the phage par t ic les was est imated from the absorbance at 280 nm a f t e r s u i t a b l e

d i l u t i o n ( 2 0 t o 40-fold) i n P B . A concentration of loL1 pa r t i c l ea /ml in a

phage were loaded on 35% ( u l v ) C s C l g rad ien t8 ( 4 - 5 x IOL2 p a r t i c l e s l g r a d i e n t )

cn pa th leng th had an absorbance of 0.05 . For t he f i na l step of t h e p u r i f i c a t i o n ,

and i sopycn ic cen t r i fuga t ion was performed in a Beckman Ti 70.1 rotor at 83,500 g (35.000 rpm) f o r 20 hours st IO'. The phage were removed from the g rad ien ts

at 8'. Ten to twenty percent of the PPU in the c leared c rude lysare were (about 0 .5 mi per gradienr) with a syr inge , d ia lyzed i n PB aC 15-20', and s t o r e d

composiLione as phage purified by the procedure deacribed previously (Davis. recovered. Phage p u r i f i e d by t h i s method had the same pro te in and phoephalipid

1983).

Lipid Analyses.

Lip id exrractions were performed a6 descr ibed (Davis et .?I.. 1982). Phos- pho l ip ids were sepa ra t ed by one-dimensional thin layer chromatography. The l i p i d r e s idue was resuspended I n chloroformlmerhanal (2:I) and sported on heat aeci- vated (0.5-1 hour a t 100°) s i l i c a g e l G places (250 or 500 u thick, AnalCech). The m l v e n r mysten ( f reshly prepared for each p l a r e ) was chloroforn/methanol l - a c e t i c a c i d (b5 :25 :8 . "1" ) . JLP-Labeled PE was prepared from 5. Lyphimurium by 8 ~ r a p i n g t h e a p p r o p r i a t e area of s i l ica ge l from the p l a t e , and ex t r ac t ing t he

CHC13 under N2 then sonicated (Branson W-310 with microtip. 70% outpur) i n 50 l i p i d . The PE was deposi ted on the sur face of a glass Lube by evaporat ion of the

mH Sodium phosphate buffer (pH 8.4) containing 0.1 M NaCl fo r two mi".

Storage and TILrat ion of Imidoesrers.

Drierice. Evezy month the reagents Were assayed by ritracion co determine the decompoeition of Lbe reagent. The pK, of EA1 i s 7.3 (Whiteley and Berg, 1974)

and since che Products of decompositian, ammonia, acetamide, and aeefamidine a l l have pK.'a greater than 9 .2 , it was poss ib l e L O s p e c i f i c a l l y t i t r a t e t h e

m g l m l ) vas r a i sed LO pH 8.3 with N NaOH. Then s e v e r a l q q af i d d o e s t e r

i d d o e s t e r . The pH of a Solufion of phenol red (IO ml of approximately 0.1

were added. The volume of N NaOH requi red to return the pH to R.3 was used t o ralculafe che mol of t i t r a t a b l e graupa per g of reagent . L A 1 VBB assayed in a s i m i l a r oanner . except t h a t s i n c e i t s pK, is 6.1 (Uhiceley and Berg.

1974). it was CiicaCed co pH 7.8. A f t e r f i v e roonchs ef storage, only 5% o f che EA1 and 1OX Of Lhe IAI had decomposed.

Preparat ion of lmidoesrer S m c k Solutions.

The maisLure sensitive reagents. EA1 and IAI. were s to red a t -20" Over

Mill igram quenflries of nonradioact ive EA1 and I A I were dissolved 30 6

NaOH (EAI) or 1.15 N NaOH ( IAI ) . The concentration of t i t r a t a b l e group& (pK, before Y B ~ by add i t ion of 0.25 M sodium phosphace buffer containing e i t h e r 1 N

solved in absolure e thanol . d iv ided into a l iqua t s , d r i ed unde r nitrogen and 7 ) was 1.0 M. The f i n a l pH of the solution was 8.5. [l-L4CIEAI was d i s -

s t o r e d a t -20' over Drierice unCil mse (wirh in severa l h ) . Since IAI is insolu-

b l e i n o r g a n i c ~ o l v e n t b , [ l - L 4 C l I A I was dissolved i n absolute erhano1/100 mH sodium carbonate, pH 10.5, ( R : 2 ) immediately before uee.

Rate of Hydrolysis of Imidoesters.

The h a l f - l i f e af EA1 vas determined by the merhod of Hunter and Ludwig (1962). Since IAI 1 s i n so lub le i n organic so lven t s , its ha l f - l i f e cou ld not be determined by t h i s method. IAI was d i s s o l v e d ( f i n a l concentration 25 O M ) in 50 UH sodium phaaphate buffer, pH 8.4. containing 0.1 M NaCl. E v e n f i v e min, a sample vas removed. the pH of the sample was lowered LO pH 8.0 and che sample was t i t r a t e d w i t h 0.1 N HCI t o pH 5 using methyl red (8 !Jg/nl) aa sn indicator . This p rocedure t i t r a t ed the phosphate buffer as well 88 the IAI. To correct f a r t h e

decomposed ( s i x h a f t e r t h e starc of the experiment). phosphate buf fe r . a sample of the solution w a s t i t r a t e d a f t e r 99% of t h e I A I had

Reaciiona with Excess h i d o e s t e r .

such as PE and an imidoerrer Can be descr ibed by the fol lowing equat ion:

l n [ : ] - - k t b

I n the presence of excess imidoesrer , the reaction between an amino compound

where a is t h e t o t a l eoneenLrarion of PE at a given L i m e , A is t h e i n i t i a l Con-

centration of P E , h is the on cent ration of imidoesfer averaged over the interval berween each p a i r Of da t a po in t s , k is the rate eonsCant which in chis case depends on the pH of the reaction mixture (Davia, 1983). Thus if i ) t h e imidoea- ter is i n great excess over the amine, i i) the average comencra t ion of imidoes- ~ e r is constant over the time i n t e r v a l between data points (and the data Points ere evenly spaced), and i i i) the pH of the reaction mixcure is held constant, then the disappearance of t he PE w i l l f o l l o w f i r s t o r d e r kinetics. I f a l l the PE molecules have Che same r e a c t i v i t y , a p lo t of t he f r ac t ion o f PE remaining O n a

i n t e r c e p t on che ord ina te will he 1.0. logari thmic s c a l e ( o r d i n a t e ) verens fine will be l i n e a r w i t h a slope of -kb. The

added t o p u r i f i e d phage ( 1 x mol amino &rovps per p a r t i c l e ) . Sample*

TO meet t he above Condi t ions a t l eas t a 50-fold exce88 af imidoerter Vas

f o r e i t h e r l l p i d s n a l y e i s or t i ter of in fecLiu i ty were removed every 30 min to determine the excent of the reaction. Fresh imidoester was then added co replace the imidoesrcr that had hydrolysed to return the imidoester eoncentrar ion LO Its i n i t i a l l e v e l . The concentrat ions at rime t and L + 30 min were equal (within

exposed to the same average COncenrlaLion of imidoes t e r bu t fo r d i f f e ren t l eng ths 10%) f o r a l l valve8 of t . Thus samples removed at 30 min i n t e r v a l s had been

which could react with the imidoesrer thua giving a fur rher decrease in concen- of time, A possible eOmplieation of hydrolysis was the product ion of ammonia

when the EA1 concentration was derermined every 15 min chroughont the course of cration. No correcrion was made fo r t he lose of imidoesrer to ammonia, since

an experiment done as descr ibed above (but in the absence of phage), the rate of CAI decomposition increased (10% during the experiment (data not shown). The pH of rhe reaction mixture was maintained between 8.4 and 8.6 by the addicion of H C 1

ua8 C O D S T ~ T . The samples removed f o r l i p i d m a l y s i 8 were added LO I d i spe r s ion whenever the pH reached 8.5 and chus che pH averaged over each 30 mi" i n t e r v a l

of EAI-treated &. carrier phosphal ipide in 15 mn Trie-HC1, pH 8.0. The carrier phosphol ipids Yere ex t r ac t ed from cells which had been Created with I O dl

were exr rac t ed and sepa ra t ed hy thin layer chromatography as described above. E A 1 f o r IO mi" in PB. When a l l che samples had been taken. The phosphol ipids

Labeling withJl'C1 Imidoebcers

proceinr in the v i r i o n . I n must experiments phage samples in P B yere expoeed t o

low cuncentraciom (1-6 mH) o f t h e [ 1 4 C l i m i d o e s r e r s ( s p e c i f i c a c ~ i v i t y 57.5- 58.1 CiImoI) by a s i n g l e a d d i t i o n of the reagent . After IO-b0 m i " at room temp- e r a t u r e ( 2 2 - 2 5 " ) ( the r ime course of rhe reaction we8 governed by the rate of

of sodium glycylglycine buffer (pH 9.0) t o quench the reaction. Al te rna t ive ly . i d d o e s t e r h y d r o l y s i s ) . t h e react ion was ha l t ed by add i t ion of a 100-fold excess

the reaction was quenched by p r e c i p i t a t i o n w i c h t r i c h l o r a c e t i c e c l d . The t r i c h -

descr ibed (Davis and Cronan, 19831 whereas the glycyglycine-quenehed samples were l o r o a c e r i c a c i d p r e c i p i t a t e s were prepared for e lec t rophores i s a6 previously

supplemented with one t en th volume of 0.5 H Sodium borate buffer and m i ~ r o ~ o e ~ a l nuclease (Sigma, 280 U / m l j vas added as ind ica ted . Af te r 2 h a t 37". the sample8 were supplenenzed with 20 mn EDTA and one-tench volume of a solution of IO-fold concentrated Tris-borate gel buffet containins 1% SDS. 50% g lyce ro l , and 0.1%

each tube folloued by b o i l i n g f o r 5 min. The semples (0.05 m l ) were rhen loaded each of the tracking dyes (bromophenol blue and. xylene cyanale FF) was added co

into t h e s l o t s of an agarose gel .

Per iodate Treatment Of

The IL4CIirnldoesrers were primarily used t o probe the arrangement of

_______ Purified 32P-labeled phage yere dialyzed asainst 50 mn sodium phosphate

b u f f e r , pH 5.5, c o n r l i n i n l IO mM NgS04. Phage were mixed w i t h sodium metaperi-

pH of the s o l u t i o n vas then ra ised to pH 8.0 with 0.1 H N a B q . After I 5 rnin at o d a r e s r i r r e d f o r 15 minures in the dark , and rhen puc on ice f a r 45-60 n i n . The

room temperature . U . 2 H NaBH4 in 0.15 H sodium ba ra t e bu f fe r , pH 8.0. was added

t o glve a f i n a l Concentration of 20 m n NaBHb. The phage were incubated for

30-4U min on i ce and then prepared for gel eleccrophaiesis.

EleCLrophoresis

SDS-polyacrylamide electrophoresis was done on 13.17% gradient ((ele and

f luorography BF previously descr ibed (Devis e t al., 1982). t he p ro t e ins on these qe16 Were v i sua l i zed by auroradiography or quanfitarive

0.75% agarose With 0.05 nl sample wel l s and an e lec t rophores i s buf fer (pH 8.3) of 89 dl T r i s containing 89 024 bor ic ac id , 2.5 mM disodivm EDTA, and 0.1% SUS (Hrmi- atis e t al.. 1982).

Velocity Sedimentation Analysis.

A g a r o ~ e gel e lec t rophores i s was done "wing a mini-gel ( I O x 1 x 0 . 6 cn) of

Imidaester-treated 3ZP-Labeled phage preparations were sedinented on 5.20% ( u l v ) l i n e a r s~crose grad ien t s made up in PB conraining 10 nu Iris-HC1

at the bottom of each grad ien t . The g rad ien t s were centr i fuged i n a neckman SY41 ( f i n a l pH 8.01. A cushion (0.3 rol) of bo% sucrose in PB lacking N s C l was placed

rotor a t 76,400 g (25,000 rpm) f o r 51 mln a t 20'. Eleven drop f r ac t ions were collected. AliquoCs of 0.05 m l were mixed with Yarer (U .2 ml) and PCS ( 4 mlj and counted.

Table 1

Characiecirar ion af Products of the Reactioii becueen €41 end PE.

Sonicated disperaions of 32P-labeled PB were prepared i n either PB ad- jue t ed t o pH 8.4 or 50 d sodium carbonate buf fer (pH 10.0) conteinlng 0.1 M

were incubated a t 25' with per iodic addi t ion of HC1 t o maintain the pH within NaC1. One addirlon of E A 1 (20 f i n a l c o n c e n t r a t i o n ) waa made and the eaoples

0.02 u n i t s of t h e i n i t i a l value. After 30 m i " t he reactions were terminaced and t h e l i p i d e x t r a c t e d and Chromafographed. The a m o n i ~ concentration was 0.1 H.

-

Calcu la t ion of t he f r ac t ion o f PE a c c e s s i b l e f o r reaction with 1AI. _ _ _ ~ _ _ ~ ~

k i n e t i c s were l a c xn the e?merimenfs of Fig. b and thus the decreasing slop2 of the curves rime s t rongly sugges t* tha t not a l l of the PE is ava i l ab le fo r rea~tion r i c h IAI. Assuming t h a t a l l t he PE has the s a w i n t r i n b i c chemical reactivity, the proport ion of PE access ib l e to IAI wab r a l cu la red .

The three condi t ions llecessary f u r r e a c t i o n t o follow paeudo-f i rs t order

I" 15 - -1 = I"(1 - 91 - *bi

where a is the c~n~enfraflon of PE at a given time, A is t h e i n i t i a l concenlra- t i o n of PE, b is the coneen~rarion of imidoesrer averaged over the 30 nin berveen each sampling. k 19 Lhe rate Constant and a(-) i s Lhe concentrat ion of amine remaining at I n f i n i t e time. A p lo t of 5 -a(..) on a l oga r i thmic s ca l e

A A

A A

should be a s t r a i g h t l i n e u i r h t h e i n t e r c e p t sf I - a ( t h e f r a c t i o n of amine

A

ava i l ab le for reaction with the imidoearer j . The value of a(..) giving the best

s t r a i g h t l i n e (by leas t squares ana lys i s ) wi th the cor rec t in te rcepc YBb de te r - mined by trial and elror for each of rhe IAI da ta sets in Figure 6. Far the d a t a from the lover concentration of IAI, values of a((3 of 0.48 t o 0.50 gave

A

the bea r s t r a igh t lines ( r 2 = 0.996 t o 0.993) wi th in te rcepts of 0.49 (the

670 Imidoesters a? Structural Probes of Phage PR4

Solvent Iron1

Unknom

NH I I

A) CH3-C,

OCHzCH,

E A 1

+

11 R2 NH,

1

NHR,

Ip 'NHR~

P

Imidoesters m Structural Probes of Phage PR4 67 1

*Slot 8.0 Untreated t Phage ' 6 I

8.0 % t

6.0 5 4.0 9 3 1

*DNA

m a 1 2 1 2 3 4 5 6

3.0 %

6

2,0uL 5 IO 15 2 0 25

P12 7 2

I A !

['4C]EAI I hen