Molecular Properties of Acyl Carrier Protein Derivatives*

T H EJ O U R N A L

OF

BIOLOGICAL CHEMISTRY

Vol. 256, No. 6, Igsue of March 25. pp. 2669-2674, 1981 Prrnted in U.S.A.

Molecular Propertiesof Acyl Carrier Protein Derivatives* (Received for publication, June 24, 1980, and in revised form, October 23, 1980)

Charles 0. Rock From the Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101

John E. Cronan, Jr. From the Department of Microbiology, University of Illinois, Urbana, Illinois61801

Ian M. Armitage From the Departmentof Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510

Acyl carrier protein (ACPSH) functions as the acyl as thioestersof the only sulfhydryl group of the protein. This carrier in fatty acid biosynthesis. The acylmoieties are sulfhydryl is the terminal portionof the 4’-phosphopantethebound to the sole sulfhydryl of the protein located on ine prosthetic group attached to serine-36 of ACPSH via a the 4‘-phosphopantetheine prosthetic group. Disulfide- phosphodiester linkage (4). NativeACPSHhas a high cylinked dimers of ACPSH were formed by the reaction helical content and is reversibly denatured by high pH ( 5 , 6). of ACPSH withacyl-ACPorthemixeddisulfideof Recent experimentalfindings suggest that the fatty acid moiACPSH and thionitrobenzoate. The formationof ACP ety of native acyl-ACP interacts with the protein. The ”F dimers was established by electrophoresis, gel filtra- NMR spectrum of 6,6-difluoro-CI4:o-ACPshows the two fluotion, and sedimentation equilibrium. ACP purified from rines to be nonequivalent giving rise to an AB quartet indicstationary phase Escherichiacoli B cells was found to ative of a substantial degree of immobilization (7), however, exist primarily as a mixed disulfide with glutathione. the ”F NMR spectrum of 13,13-difluoro-C14.,,-ACPgave a This species was identifiedby gel electrophoresis single resonance indicative of relatively unhindered rotation amino acidanalysis and 31PNMR spectroscopy. A nondenaturing gel electrophoresis system was developed at carbon-13 ( 7 ) . Fatty acid-protein interaction is also sugthat allows the comparison of the effects of various gested by the finding that acyl-ACP is more stable to pH( 5 ) . Amodelfor the protein and sulfhydryl modifications on the stability of induced denaturationthanACPSH secondary structure of ACP has been proposed and a cluster the ACP protein moietyto pH-induced denaturation.In general, attachmentofhydrophilicligands to the of hydrophobic residues implicated inthe formation of a fatty sulfhydryl of ACPSH resulted in less stable protein acid-binding domain ( 5 ) . Several investigators(8,9) have reportedthat purified ACP a hydrophobic structures whereas thepresenceof gives rise to two bands on nativegel electrophoresis. In these thioester resulted in stabilization of the protein conformation. The less stable ACP structures were found to reports, theslower migratingband was converted to the faster have 31PNMR chemical shifts displaced downfield from migrating component by treatment with reducing agents. On ACPSH andthe morestable acyl-ACP derivatives were the basis of this result, both laboratories concluded that the found to have chemical shifts displaced upfield from slower migrating ACP band was a dimeric species of ACPSH ACPSH. cross-linked through thesingle sulfhydryl groupof the protein. We have recently reported( 5 ) ,that thedimerization of E . coli .~ ACPSH does notreadily occur in solutions storedat alkaline pH, and the separation between the two ACP species observed ACPSH’ functionsas the acyl carrier forde nouo fatty acid by Etemadi and Josse (8) seems too small to be consistent biosynthesis (1)and as an acyl donor in glycerolipid synthesis (2, 3) in Escherichia coli. ACPSH is an acidic protein of with the presence of ACP dimers. Recently, we have developed an improved method for the purification of ACP (10). molecularweight of 8847 (4), and exists in solution as an asymmetric monomer(5). Acyl moieties are bound to ACPSH Nondenaturing gel electrophoresis of ACP purified by this method in the absence of reducing agents revealed the presence of several ACP species. This result hasled us to charac* This research was supported by National Institutes of Health terize the chemical modifications of ACP that give rise to Grant AI 15650and by ALSAC.The costs of publication of this article these multiple forms and to explore the molecular basis for were defrayed in part by the payment of page charges. This article their resolution on native gels. musttherefore be hereby marked “adueriisement” in accordance ~

~~~

with 18 U.S.C. Section 1734 solely to indicate this fact. I The abbreviations used are: ACP, acyl carrier protein; ACPSH, holo-acyl carrier protein; acyl-ACP, acyl-acyl carrier protein; (ACPS)?, acyl carrier protein dimer linked through the prosthetic group sulfhydryls; apo-ACP, acyl carrier protein lacking the prosthetic group; ACPSSG, ACPSSCoA, and ACPS-TNB, ACPSHmixed disulfides with glutathione, CoASH, and thionitrobenzoate (TNB), respectively; (AC)~-ACPSAC, ACPSH with all five amino groups and the prosthetic group sulfhydryl acetylated; DTNB, 5,5”dithio-bis(2nitrobenzoic acid); SDS, sodium dodecyl sulfate; CHES, cyclohexylaminoethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; GSH, glutathione; R,, Stokes’ radius; bis-Tris, 2,2”bis(hydroxymethyl)-2,2,2”-nitriloethanol.

EXPERIMENTALPROCEDURES

Materials-Sodium dodecyl sulfate, ammonium persulfate, Tris, glycine, Aff-Gel 501, N,N-methylenebisacrylamide, acrylamide, N,N,N’,N’-tetramethylethylenediamine, Coomassie Blue R-250, and bromphenol blue were purchased from Bio-Rad Laboratories. Frozen E . coli B cells (late log) were obtained from Grain Processing Co. GSH, GSSG, CoASH, and dithiothreitol were purchased from P-L Biochemicals. PIPES and CHES were purchased from Calbiochem and guanidine HCI (ultrapure) was obtained from Schwarz/Mann. DTNB was a Sigma product. AII other chemicals were reagent grade or better.

2669

Properties of ACP Derivatives

2670

NativeGelElectrophoresis-Theseparating gel contained 20% (w/v) acrylamide,0.5% (w/v) N,N-methylenebisacrylamide, 0.10% (v/ v) N,N,N’,N’-tetramethylethyldiamine, 0.375 M Tris-HCI. pH9.0, and polymerization was accomplished by the addition of freshly prepared ammoniumpersulfatesolutionto a concentration of0.03% (w/v). Using thesamestocksolutions foraperiod of severaldays,the separations were found to steadily deteriorate and the culprit was identified as the separatinggel buffer. The pH of the buffer does not change, and we do not understand the exact nature of the problem. Consistent results were obtained when the separating gel buffer was freshly prepared. A stacking gel was poured over the separating gel and contained the same components except the acrylamide concentration was 5% and the Tris-HCI buffer was 0.125 M, pH 6.8. The running buffer consisted of 3.1 g of Tris and 14.4 g of glycine/liter. Samples containedglycerol and bromphenolblue and the pH adjusted to between 6.0 and 7.5. Gels were run at 25 mA until the tracking dye reached the gel bottom. Staining gels overnight in 50% methanol, 10% acetic acid, 0.1% Coomassie Blue R-250, and destaining in 10% methanol, 10%acetic acid. ACP is an acidic protein and stainspoorly using most protocols. Several combinations of methanol, acetic acid, and different stains were tried and the above procedure was determined empirically to be the most satisfactory. Preparation of ACP Derivatives-ACP was purified from E. coli B cells in the absenceof reducing agents using the 2-propanol method describedpreviously (IO). ACPSH mixed disulfideswith GSH and CoASH were prepared under similarconditions. The ACPSH sample was reduced with dithiothreitol and thereducing agent removed using a Sephadex G-25 column. Reaction mixtures contained ACPSH (1 pmol), sodium borate, pH 8.5(50 pmol), and either GSSG or CoFIG. 1. Nondenaturing gel electrophoresis of ACP derivaASSCoA (50 pmol) in a final volume of 1 ml. The reaction mixture was incubatedovernight a t room temperature, the low molecular tives. Lane 1, ACPSSCoA; lane 2, ACPS-TNB plus (ACPS),; lane weight components were resolved fromthe protein on a G-25 column. 3, (ACPS),; lane 4, ACPSSG; lane 5, ACP sample purified from E. ACI’S-TNB was prepared by reacting a reduced solution of ACPSH coli B cells in the absence of reducing agents; lane 6, apo-ACP; lane (1 pmol/ml)with 2 volumes of 10 mM DTNB in 50 mM Tris-HCI, pH 7, acyl-ACP; lane 8, ACPSH, lane 9, (Ac):,-ACPSAc, Electrophoretic 8.5, for 1 h a t room temperature. (Ach-ACPSAc was prepared by conditions were as described under “Experimental Procedures.” mixing 0.1 ml of reduced ACPSH (0.1 pmol) in 10 mM Tris-HCl, pH 8.5, with 0.1 ml of tetrahydrofuran. Acetic anhydride (IO pl) was added, the mixture allowed to stand atroom temperature for 30 min from standard ACPSH (Fig. 2). During the course of these and another 10 p1of acetic anhydride was added and the incubation studies, the‘”PNMR spectra were obtained on four different continued for another 30 min. (Ac)l-ACPAc was precipitated by the preparations of ACPfrom 0.5 kg of E . coli B cells. The addition of 0.4 ml of water and the derivatized protein collected by spectrum shownin Fig. 2A contained the most ACPSHfound centrifugation. The apo-ACP samplewas agift from Dr. MaryPolacco in the fourpurifications. In the other three instances, only the and was prepared by treating ACPSH with hydrofluoric acid (11). resonance corresponding to new the ACP species was observed When necessary, ACP derivatives were concentrated by loading the only as an unresolved shoulder sample ontoa 0.1-ml DEAE-cellulose column (DE-52, Whatman) and and the ACPSH peak occurred on the main peak. The spectrum in Fig. 2A was chosen to elution of the ACP with 0.5 M LiCl in the desired buffer. Acyl-ACP illustrate the chemical shift difference between thetwo peaks. was prepared by a slight modification of the previously published procedure (12). The modification was to substitute 20 mM bis-Tris- Corresponding with the resultsin the electrophoresisexperiHCl, pH 6.0, for the 20 mM Tris-HCI,pH 7.4. buffer in allthe ments, this resonancewas converted to the standard ACPSH chromatographic steps. This alteration was employed to reduce the position by dithiothreitol. SDS and SDS-ureagel electrophoformation of (ACPS)2by the reaction of ACPSH with acyl-ACP (see “Results”). (ACPS)r formation and thus lower acyl-ACP yields be- resis (5) of the new ACP form exhibiteda single band both in of dithiothreitol that migratedwith come a problem when the synthesisis scaled up from that previously the presence and absence molecular reported (12). [“CIACPSH and [,”H]ACPSH labeled in the prosthetic the ACPSH standard. These data show that the weight of the compound linked to ACPSH was small comgroup was preparedbiosynthetically as describedpreviously (5). ”P NMR--”’P NMRspectra wererecorded on an extensively pared to the molecular weight of ACP. This compound was modified Bruker HFX-90 single-coil pulsed Fourier transform spec- identified by dissolving 1pmol of ACP mixed disulfide in 1 ml trometeroperating a t 36.4 MHz. 1120 present in a3-mmco-axial of 20 mM Tris-HC1, pH 8.0, followed by reduction with 5 pmol capillary insert was used for the field frequency lock, and all spectra were obtained under conditionsof proton noise decoupling. Chemical of dithiothreitol. After 30 min at room temperature, the soshifts are reported in parts permillion relative to an external standardlution was titrated to pH 3.9 with acetic acid and the precipof 85% phosphoric acid run coincidentally with the sample. Sample itate allowed to flocculate. The ACP pellet was removed by volumes were 1.0 ml and ACP concentrations were 1 mM. centrifugation and an aliquot of the supernatant chromato-

graphedonanamino acidanalyzer.Asingle component eluting at the sameposition as the GSH standard (9 min) was Identification of ACPSSG-When ACP,isolatedin the detected. To further support the identity of this species as absence of reducing agents from E . coli B cells, was subjected ACPSSG, synthetic ACPSSG was prepared as described under“ExperimentalProcedures.”AuthenticACPSSG was tonative gel electrophoresis,themajorproteinbandwas on electrophoresis (Fig. found to migrate slower than the ACPSH standard (Fig. 1). found to have the same migration gel This new ACP species was found to be converted to a species 1) and an identical ‘”PNMR chemical shift (Fig. 2B) as the unknown ACP species extractedfrom the cells. migrating with standard ACPSH when the sample was reCharacterization of (ACPS)2”We have observed that hoduced with dithiothreitol prior toloading the gel. This result suggested that the new ACP species was a mixed disulfide mogeneous acyl-ACP (12) slowly decomposes ( t l r 2 z 2 years withanother molecule. The ‘”P NMRspectrum of ACP for CItin-ACP) when stored in 50 mM Tris-HCI, pH 7.4, at purified from E . coli B also revealed the presence of a new -20OC. Long chain-saturated (C14:oand ClwO) acyl-ACP have ACP speciesas shown by a resonanace shifted0.3 ppm upfield been found to be quite stable upon storage, whereas unsatuRESULTS

Properties of ACP Deriuatiues

1

A

thiols (14). At 37°C and pH 8.0, the reaction had an appreciable rate (Fig. 6) and is dependent on the concentration of ACPSH (Fig. 6). Thin-layer chromatography on Silica Gel G layers developed in hexane:diethyl ether:acetic acid (80:20:1, v/v), of samples from experiments as in Fig. 6 and [I4C]acylACPsamplesthathave significantly degraded onstorage revealed the presence of free fatty acids but no fatty aldehydes were detected. We do not understand the molecular details of this reaction, buta direct attackof ACPSH onacyl-ACP may be expected to produce a fatty aldehyde as a product. Since free fatty acid was the only product observed, it appears that hydrolysis of acyl-ACP preceedsdimerization,however, (ACPS)* is not found when ACPSH is treated similarly to a mixture of acyl-ACP andACPSH.This observation is of practical significance in storing acyl-ACPsince acyl-ACP preparations that are initially homogeneoushave muchlonger half-lives than if the acyl-ACP is contaminated with ACPSH. Although (ACPS), was not readily formed in ACPSH solutions (5), significant quantities of (ACPS), were formed when ACPSH was added to a mixed disulfide of ACP and another thiol. For example, when ACPSH was mixed with ACPSTNB in 50 mM Tris-HC1, pH 8.0 (ACPS):! was readily formed and could be assayed spectrophotometrically by observing the release of the TNB anion. (ACPS)s prepared by this method was found to have the same'"P NMR spectrum as the (ACPS),isolated from theacyl-ACP preparations (Fig. 5). (ACPS), was also formed when ACPSH was chromatographed on thiol affinity resins. When ACPSH was passed

!

B

C

I

I

2

I

I

0

2671

I

I

I

-2 A

PP* FIG. 2. 31P NMR spectra of ACPSH and ACPSSG. A, '"P NMR spectrum of an ACPsamplepurifiedfrom E. coli B cells in the absence of reducingagents; B, J I P NMR spectrum of synthetic ACpSSG; C,O'P NMR spectrum of ACPSH. Additionof dithiothreitol to the samples in A and B resulted in spectra having a chemical shift identical with that shown in spectrum in C .

rated acyl-ACPs and shorter chain lengths were found to be considerably lessstable. Surprisingly, the cleavage of the fatty acid did not result in the formationof ACPSH, but rather in a protein that hasa higher molecular weight. The properties of this ACP species were investigated by gel filtration chromatography (Fig. 3). The breakdown product of acyl-ACP was found to possess an R, appropriate for a dimeric species of ACP (Fig. 3). Addition of dithiothreitol to the sample resulted in anelution volume identical withmonomeric ACPSH (Fig. 3) and led us to conclude that 2 molecules of ACPSH had been cross-linked via the sulfhydryl group. SDS andSDS-urea gel electrophoresis (5) of (ACPS),samples showed a band having a molecular weight corresponding to twice the size of ACPSH in the absence of reducing agents and this bandwas converted to ACPSHby addition of dithiothreitol to the sample prior electrophoresis. to Similar results were obtained when the mobility of (ACPS)* wasexamined in the native gel electrophoresis system (Fig. 1).Definitive evidence for the existence of dimeric ACP wasprovidedby sedimentation equilibrium experiments (Fig. 4). In the absence of dithiothreitol, (ACPS), was found to possess a molecular weight twice that found in the presence of dithiothreitol (Fig. 4). (ACPS)z was also detected in acyl-ACP samples by virtue of its characteristic"P NMR chemical-shift (Fig. 5). As found in our other experiments, (ACPS), was converted to ACPSH by the addition of dithiothreitol to thesample. T h e degradation of acyl-ACP in the presence of ACPSH is surprising in light of the low reactivity of acyl-ACP to simple

I

FRACTION NUMBER

FIG. 3. Resolution of ACP monomers and dimers by gel filtration chromatography. A column (47 X 0.3 cm) of Bio-Gel P-100 (18 ml bed volume) was equilibrated with 10 mM bis-Tris-HC1, pH 6.5,0.25 M LiCI, and developed at a flow rate of 1.1 ml/h. The sample size in all cases was 100 pl and 325-pl fractions were collected. Panet A , a sample containing['4C](ACPS)e and ~ c ~ ~ - [ ' ~ C ] A ACPSH C P . and acyl-ACP have previously been shown in a similar solvent (5). Panel B, a samplecontainingpurified ['4C](ACPS)r.Panel C, a sample that had been reduced with dithiocontaining purified ['4C](ACPS)2 threitol prior to application to the column. In all cases, recovery of applied radioactivity was >956.

2672

Properties ofACP Derivatives -02t

1

-0.4

I

I

\

j '\ I

I

those of CoASH and were not affected by the addition of dithiothreitol to the ACPSSCoA sample. All acyl-ACP chain lengths examined possessed the same chemical shift (Table 11). In contrast tothe ACP disulfides, the acyl-ACP chemical shift was upfield from ACPSH and not affected by the addition of dithiothreitol. Structural Consequencesof Sulfiydryl Group and Protein

I

IO 20 MOLECULAR WEIGHT x

FIG. 4. Sedimentation equilibrium of (ACPS)2. The sedimentation experiment was performed by a modification (5) of the method of Bothwell et al. (13). The values plotted are the log of the fraction of protein remaining in the top 40% of the tube after centrifugation at about 68,000 rpm (14 p.s.i.)for 20 h uersus the molecular weight normalized to a partialspecific volumeof 0.730 ml/g. The buffer used was0.02 M LiCl in bis-Tris-HCl, pH 6.5. All samples contained 10 mg/ml of Dextran T-10 to stabilize against convention. Dithiothreitol (DTT) (when added) was present a t 20 mM. The ['4C](ACPS)2sample was the same as that shown in Fig. 3, B , and the distributions of the radiolabeled ACP species were determined by scintillation counting. The distributions of the standards were determined spectrophotometrically at 240 nm or 418 nm (cytochrome c ) . The F values in the (ACPS), experiments were obtained in the presence of an internal standard of cytochrome c (4.5 mg/ml). Essentially identical results were obtained without the internal standard. Standardproteins were ['HIACPSH (8,850; U = 0.731), horse heart cytochrome c (11,700; U = 0.728), sperm whale myoglobin (16,900, U = 0.741),and chymotrypinogen (25,700; U = 0.734). through an organomercurial agarose column (Affi-Gel 501), two fractions were obtained. One ACP fraction did not absorb to the column and the other was eluted with dithiothreitol. Examination of these two fractions inthe native gel electrophoresis system revealed that the fraction that did not bind I I I l l the fractionelutedwith to the column was (ACPS), and dithiothreitol was ACPSH. Rigorous exclusionof oxygen was 2 0 -2 not attempted and the oxidation of ACPSH to (ACPS), may PPm have been accomplished by the presence of metal ions other FIG. 5. 31P NMR spectra of acyl-ACP and (ACPS)2. A, "'P than mercury on the support. 31PNMR Investigations-ACPSH has been shown to be spectrum of C?;!l-ACP - ACP containing (ACPS)2contamination; B , reversibly denatured by high pH and temperature (5, 6) and 31P spectrum of (ACP% prepared by the DTNB method; C, "'P NMR spectrum of pure Cleo-ACP.Addition of dithiothreitol to the Ca" has been shown to stabilize the protein to these effects (ACPS)?samples resulted in a spectrumpossessing the same chemical (6, 15). Therefore, we have investigatedthe "'P NMR spectra shift as ACPSH, but did not affect the position of acyl-ACP. of ACPSH under these conditions to determine whether the phosphorus chemical shift is sensitive tothe conformational W I I state of ACPSH. The '"P chemical shift of ACPSH was not altered by changing the pH or by the presence or absence of dithiothreitol (Table I). Denaturation of ACP by heat was accompanied by a small but consistently observed downfield chemical shift (Table I). The addition of Ca2+ to ACPSH resulted inan upfield shiftin the '"P resonance (Table I). The small range of 3*Pchemical shifts observed in these studies suggests that the conformation of the phosphorus attached to 5 4 0 1 , , , serine-36wasnotdrasticallyalteredbymajorstructural s 2 4 6 8 changes in ACPSH. HOURS AT 37°C the '"P NMR spectraof ACPSH We have also investigated having a variety of sulfhydryl group modifications (Table11). FIG. 6 . Deacylation of acyl-ACP catalyzed by ACPSH. IncuAll ACP disulfides examined exhibited a downfield chemical bations contained 0.1 M Tris-HC1, pH 8.0, 5 mM dithiothreitol, 1.0 11).In all cases, addition p~ [1-'4C]-Cl,o-ACP(specific activity 50 Ci/moi) with no additions, shift as compared to ACPSH (Table of dithiothreitol to the sample resulted in the disappearance 0.4 m g / d of ACPSH, or 1.6 mg/ml of ACPSH. The capped Eppendorf tubes were incubated at 37°C and 5-pl samples were withdrawn of the ACP disulfide resonance and the appearance of the after the tubes were blended on a Vortex mixer and centrifuged for 2 ACPSH resonance at -0.01 ppm. ACPSSCoA exhibited two min on an Eppendorf microcentrifuge to sediment any water droplets additional resonances at +3.56 and -11.37 ppm arising from in the sides of the tubes. The amount of [l-I4C]acyl-ACPremaining the phosphates of the CoA moiety. These chemical shifts were was measured using the filter disc assay (18).

,

1

Properties Derivatives of ACP TABLE I Effect of solvent compositionand temperature on ACPSH 31P chemical shift

5.1 15

2673 DISCUSSION

ACP has been shown to undergo pH-induced denaturation manifested by a decrease in the a-helical content as measured Temperature pH" Additionsh Chemical shift by optical methods (6) and a dramatic increase in the R, of "C PPm the protein (5). Native gel electrophoresis at pH values above 28 3.4 8 M guanidine HCl -0.18 the onsetof the structural transition can be usedto determine -0.01 None 28 the effect of sulfhydryl and protein modification on the staNone +0.27 5.1 60 bilization of the native ACP conformation. ACP is a very -0.17 mM Cat+ 5.0 28 21 negativecharges and only 6 acidic proteinandcarries None -0.01 7.3 28 positive charges per mole at the pHof the separatinggel (4). None -0.01 9.4 28 This preponderance of negativelychargedresidues means 41 9.4 None +0.22 9.4 15 mM Ca2' -0.17 28 that modifications of the protein that add or subtract1 or 2 41 9.4 15 mM Ca2+ -0.11 negative charges/mol will have little effect on the velocity of 49 9.4 15 mM Ca2+ +0.09 migration during electrophoresis. Therefore, the molecular 28 9.4 30 mM Ca" -0.28 dimensions of the ACP species is the primary variable that Bufferswere: 25 mM sodium acetate, pH 5.1, 25 mM Tris-HCI, pH determines the migration of the ACP derivative in this gel 7.3, 25 m~ CHES, pH 9.4. (Fig. 1).Modifications that stabilize the native ACP 'AII spectra were fvst collected in the absence of dithiothreitol. system structure migrate faster in these gels than the ACPSH stanDithiothreitol (10 mM) was then added and a second spectrum collected. No alteration of the "P chemical shift occurred as a conse- dard and modifications that destabilize ACP migrate more slowly than ACPSH. The separations described in this report quence of dithiothreitol addition. were not observed when the native separating gel also conTABLEI1 tained urea (8 M ) , SDS or both, further supporting the conclu31 P chemical shift of ACP derivatives sion that conformationalisomers are being resolved. We have Sample" Chemical shift previously shown thatthe presenceof an acylgroup of ACPSH stabilizes the protein to pH-induced denaturation ppm ACPSH -0.01 and thatacyl-ACP has a smaller R, than ACPSH at elevated i0.24 (ACPSh pH (5). Accordingly, acyl-ACP was foundto migrate fasteron ACPSSG +0.30 native gels than ACPSH (Fig. 1).All acyl-ACP chain lengths ACPSSCoA' +0.18 examined (Cl0:,,to C,8,u)had an identical mobility in the native ACPS-TNB +0.07 gel electrophoresis system indicating that they all possessed Acyl-ACP' -0.58 the minimum structural featuresconferring maximum stabilAll samples were dissolved in 20 m~ PIPES, pH 6.8,0.2 M LiCL. ity to the protein moiety. All of the ACP-mixed disulfides * Two other resonances were observed at +3.56 and -11.37 ppm prepared in this report were found to destabilize the struct,ure corresponding to the "P resonances from the CoA moiety. 'Acyl-ACPsexaminedwere:C,.,.o-ACP;CIWJ-ACP; C%I-ACP; of ACP (Fig. 1).A model of ACP secondary structure has Cln.,,-ACP;C?iI-ACP;and C$.:-ACP. been proposed (5), and the stabilization induced by the acyl group hasbeen attributed to the interaction of the acyl moiety Modifications-We have previously shown that acyl-ACP is with hydrophobic amino acidresidues present in adjacent more stable to pH-induced denaturation than ACPSH (5). helical segments of the protein(5).If hydrophobic interactions This conclusion was based on the observation that at pH 9.4, are primarily responsible for the stabilization of acyl-ACP, acyl-ACP possesses a smaller lis than ACPSH and the two then the introductionof polar groups into this segmentof the forms can be separated a t this pHby gel filtration chromatog- molecule would be expected to destabilize thestructure. raphy (5).The nondenaturing gel electrophoresis experiment Therefore, the destabilization of ACP structure by charged is directly analogous to the gel fitration experiment due to ligandsbearing little resemblance to a fatty acid chain is the high pH (9.0) of the separatinggel, the sieving properties consistent with this model. of the acrylamidematrix, and the constantnegative charge of of The effects of protein modifications onthestability the ACP species. Therefore, the effect of various sulfhydryl ACPSH was also examined. Fully acetylated ACP has been groups, and protein modification on the R.,of ACP can be shown by spectral methods to be abnormally sensitive to pHcompared ona single gel. Accordingly, acyl-ACP was foundto induced denaturation (6). (AC)~-ACPSAC migrates in the namigrate faster than ACPin this gel system due to its smaller tive gel system just below (ACPS), (Fig. I), demonstrating R , (Fig. 1).In contrast, all the ACP mixed disulfides listed in that (Ac)$-ACPSAc has a large molecular radius consistent Table I1 were found to possess a larger R, than ACPSH (Fig. with this ACP derivative being in arandom coil configuration. 1). We conclude that derivatizing the sulfhydryl of ACPSH Removal of the prosthetic group caused a slight destabilizawith these reagents exerts a destabilizing influence on the tion of ACP structure. protein moiety. (ACPS)., was more retarded than any other Previous investigators have observed that ACP preparaACP form due to the doubling of the molecular weight (Fig. tions give rise to multiple bands on native gel electrophoresis 1). and that the slower migrating bands were converted to the The R, of apo-ACP was found to be greater than theR,of faster migratingband by addition of dithiothreitoltothe presence of the prosthetic sample prior to electrophoresis (8, 9). Since CoASH readily ACPSH (Fig. l), indicating that the group itself confers a degree of stability to the protein moiety. dimerizes in solution and the prostheticgroup of ACPSH has Acetylation of all the primary aminogroups of ACPSH with the same structure as CoASH, the notion that ACPSH also acetic anhydride has been reported to increase the sensitivitydimerizes in solution has been generally accepted ( 1 ) . Thereof the protein moiety to pH-induced denaturation and results fore, these workers (8,9) concluded that the slower migrating in a dramatic loss of secondary structure at pH 7.5 (6, 15). band was (ACPS)B and no further characterization of these When 8 M urea was included in the separating gel, all ACP ACP species was reported. Since our present data (Fig. 1) derivatives except (ACPS)2 were found to possess mobilities demonstrate that a variety of sulfhydrylmodifications of similar to that of (Ac)S-ACPSAc further supporting the con- ACPSH migrate more slowly than ACPSH in a native ge! clusion that (Ach-ACPSAc has a random coil structure atpH electrophoresis system similar to thatused by previous work9.0. ers (8, 9), the identification of these bands as (ACPS& must

2674

Properties of ACP Derivatives

be considered suspect. We have observed that ACP purified from E. coli B cells is primarily ACPSSG, and further that this ACP species migrates more slowly in native gel electrophoresis than ACPSH and is converted to ACPSH by reducing agents. A comparison of our gels to those shown by Etemadi and Josse (8) suggests that ACPSSG was present in their samples. Since GSHis the majorlow molecular weightsulfhydryl compound in E. coli (16), ACPSSG could arise by spontaneous nonenzymatic oxidation of ACPSH with GSSG. However, CoASH has been shown to be converted to CoASSG under anaerobic conditions in stationary phase E. coli (17). and this result suggests that ACPSSG may be formed under similar conditions to protect ACPSH from spurious oxidations. We arecurrently investigating theformation of ACPSSG in E. coli cultures under a variety of growth conditions to ascertain itsrole in ACP metabolism. The high nucleophilicityof the sulfhydryl of ACPSH makes theformation of (ACPS),rapidundercertain conditions. When ACPSHis reacted with ACPS-TNB,(ACPS):!is formed and can be monitored by release of TNB anion. Most surprisingly, acyl-ACP also appears to react with ACPSH to form (ACPE& (Fig. 5 ) . This rate is much slower than the above reactions and in this case, aspecific protein-protein interaction maybe involved since acyl-ACP is much less reactiveto simple thiols (Fig. 6; Refs. 12, 14). This observation is of practical significance in the preparation and storage of acylACP, and has resulted in the modification of the previous preparative method (see “Experimental Procedures”) by lowering the pH in the chromatography steps to 5.9 where acylACP hydrolysis and thus (ACPS)2formation is reduced. Although the ,”P chemical shifts reported in this paper (Tables I and 11) are significant for phosphate resonances, the exact nature of the structural alterations in the phosphate group responsible for the shiftsis not known. However, it can be concluded that the environmentof the phosphate group is altered as a consequence of sulfhydryl and protein modifications. Each electrophoretically distinct form of ACP was also found to possess a characteristic ,‘”Pchemical shift. In all

cases, protein modifications that resulted ina lessstable ACP structure were found to havechemical shifts displaced downfield from ACPSH andmodifications resulting in stabilization of ACP werefound to have chemical shifts upfield from ACPSH. Acknowledgments-We wish tothank PaulFletcherand Davis for performing the amino acid analysis.

Gary

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