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THE OURNALF BIOLOGICALHEMISTRY0 1989 by The A merican Society for Biochem istry and M olecular Biology, Inc.

Vol. 264, No . 29, Issue of October 15,pp. 17309-17315,1989Printed in U.. .

Purification of Receptor Protein Trg by Exploiting PropertyCommon to Chemotactic Transducersof Escherichia coZi*

(Received for publication, February 22, 1989)

Gregory G. Burrow s, MarciaE . Newcomer$ and GeraldL. Hazelbauerl

From the BiochemistrylBiophysics Program, Washington State University, Pullman, Washington 99164-4660

The methyl-acceptingchemotactic transducers ofEscherichia coli were found to bind strongly to Ciba-cron blue-Sepharose. Among potential elutants tested,only S-adenosylmethionine t moderate concentrationsand NaCl at concentrations greater than1.5 M causeddissociation of these detergent-solubilized transmem-brane prote ins from the dye. Release by S-adenosyl-methionine may be a generalized effect athe r than theresult of a specific binding site for tha t compound ontransducers. A truncated trg gene was created that

coded for hecarboxyl-terminal hree-fifths of thetransducer, which constitutes the cytoplasmic domaincommon to all four transducers in. coli.This domainbound to Cibacron blue-Sepharose and was eluted inpattern similar to that exhibited by intact Trg, indi-cating that interaction with the dye occurred in thisconserved domain. Adherence to Cibacron blue andelution by high salt formed the core of an efficientpurification scheme, developed for Trg butapplicableto all transducers in E. coli and perhap s to methyl-accepting chemotaxis proteins inother species. Deter-mination of the amino acid sequence t the beginningof purified Trg confirmed that it contained a longerhydrophilic segment at its amino terminus than othertransducers of E. coli. The initialmethionine of Trg is

neither cleaved nor modified, in cont rast to the Tartransducer in which the amino terminus was foundpreviously to be blocked. Circular dichroic measure-ments of purified Tr g indicated that the secondarystructural organization f the proteins predominantlya-helix.

Sensory transducer proteins are centralcomponents in the

chemotactic machinery of Escherichia coli. The four proteins

Tsr, Tar, Trg, and Tap recognize tactic stimuli, transduce

information across the cytoplasmic membrane, initiate int ra-

cellular signalling, and mediate sensory adaptation (for a

recent review see Hazelbauer, 1988). The deduced amino acidsequences of these proteins are related and suggest a model

for disposition of the transducer across the cytoplasmic mem-

brane (Boyd et al., 1983; Krikos et al., 1983; Russo and

Koshland, 1983; Bollinger et al., 1984). In he model, the

*T hi s work was supported by G rant GM29963 and protein se-quences were determined using an nstrument purchased hroughShared Instrumenta tion Grant RR02677, both from the National

Institutes of Health. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must

therefore be hereby marked “aduertisemenf” in accordance with 18U.S.C. Section 1734 solely to indicate thi s fact.

Nashville, T N 37232.$ Curren t address: Dept. of Biochemistry, Vanderbilt University,

§Recipient of an American Cancer Society FacultyResearch

Award.

membrane is spanned by a hydrophobic segment near the

amino terminusand another ear residue 200 of these 60-kDa

proteins, creating two domains, one in the periplasm and the

other in thecytoplasm. The periplasmic domain contains few

conserved residues and recognizes specific ligands character-

istic of each particular transducer. The cytoplasmic domain

contains many conserved residues; 158 of 314 are identical for

all four ransducers. That domain performs the common

functions of excitation by affecting the phosphorylation re-

actions that begin with CheA (Hess e t al., 1987; 1988) and

adaptat ion by altering theextent of modification of the

methyl-accepting glutamyl residues, 4-6 depending on the

transducer(Kehry and Dahlquist, 1982; Tenvilliger et a!.,

1986; Nowlinet al., 1987), that are modified to create carboxyl

methyl esters.This laboratory has been interested in Trg, which mediates

taxis toward galactose and ribose by recognizing ligand-oc-

cupied glactose- and ribose-binding proteins and thus is a

primary receptor protein for a particularconformational state

of those two polypeptides. Full understanding of the mecha-

nisms by which this or any polypeptide receptor functions

will require extensive characterization of the purified receptor

protein. We have developed an effective purification proce-

dure for Trg, based primarily on a specific binding property

common to the conserved cytoplasmic domain of all chemo-tactic transducers.

E X P E R I M E N T A LP R O C E D U R E S

Bacterial Strains-All strains were derivatives of E. coli K12.

CP334, CP361, an d CP553 are derivatives of OW 1 (Ordal and Adler,

1974) that each contain Atrg-100, zdb::Tn5 and in addition contain

respectively A(tar-tap)5201; Atsr-7028; or A(tar-tap-cheR-cheB)2234,

Atsr-7028. HB1032 isCP553/pCB1 and HB1033 is CP553/pDD2.

The plasmid pGBl was constructed as follows. Oligonucleotide-di-

rected mutagenesis was used to change the sequence TAAACC, be-ginning at position -25 relative to the initiation codon of trg, toGAATTC, creating an EcoRI recognition site in the 2.2-kb’ BglII-

HincII fragment,containing trg and incorporated into Ml3m pll

between the BamHI and SmaIites. Th e 1.9-kb EcoRI fragment fromthe replicative form of this bacteriophage DNA was introduced into

the EcoRI site of pKK223-3 (Pharmacia LKB Biotechnology Inc.),

placing trg under the control of the “tac” promotor, and creating

pBB7. Th e ladqgene was introduced into pBB7by isolating the 1.6-

kb EcoRI-PuuI fragment from pCR44 (from ChrisRussell, University

of Oregon), cleaving pBB7 with SphI, treating both fragment and

cleaved plasmid with S1 nuclease, and joining the blunt-ended frag-

ment to the blunt-ended plasmid. The resulting 8.1-kb plasmid wasnamed pGBl (Fig. 1). The 7.8-kb plasmid pDD2 was constructed as

follows. Oligonucleotide-directed mutagenesis was used to change the

sequence GGATCG beginning with nucleotide 671 of trg to CCATGG,creating an NcoI recognition site. The 1.2-kb NcoI-EcoRI fragment

containing the codons for the carboxyl-terminal 312 residues of Tr g

’The abbreviations used are: kb, kilobase; AdoMet, S-adenosyl-

methionine;bisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)-

methane; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dode-

cy1 sulfate; and TLCK ,A’”-p-tosyl-L-lysine chloromethyl ketone.

17309

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17310 Purification of Chemotactic Transducer

p G B l

FIG. 1. Map of pGB1. The position of genetic units and recog-nition sites for selected restriction endonucleases are indicated forthis 8140-base pair plasmid. Abbreviations are as follows: A, AflII;

Ap, ApaI; E, EcoRI; H, HindIII; Hp, HpaI; M, MluI; N, NdeI; R,RsrII; S,Sack Sc, Scal; Sm, SmaI;Sn, SnaI; St, StuI;X, XmaIII.

was inserted between the NcoI and PstI ites of a modified pKK233-

2 (Pharmacia), placing tha t coding sequence under the control of the"trc" promotor, by annealing and ligating the complimentary NcoIsites, filling in the exposed ends with T4 DNA polymerase, andligating the resulting blunt ends. The pKK233-2 had been modifiedpreviously by joining the SalI-BamHI fragment containing lacP frompGBl topKK233-2 cleaved with the same enzymes.

Chemi~ak"L-[methyl-~H]Methionine12 Ci/mmol) and [35S]me-thionine (1000 Ci/mmol) were from Du Pont-New England Nuclear.Octyl-P-glucopyranoside (octyl glucoside)was from BoehringerMannheim. Blue-Sepharose CL-GB was from Pharmacia. DNase I,bisTris, isopropyl-P-D-thiogalactoside, epstatin A, leupeptin, p-hy-droxymercuribenzoate, phenylmethylsulfonyl fluoride (PMSF), andNu-p-tosyl-L-lysinechloromethyl ketone (TLCK) were from Sigma;1,lO-phenanthroline was from Aldrich. S-Adenosylmethionine wasused as the p-toluenesulfonate salt (Sigma). Enzymes and materialfor manipulation of DNA were fromNew England Biolabs, Bethesda

Research Laboratories, and Boehringer Mannheim.Buffers and Solutions-Buffers were adjusted to the indicated pHat room temperature. The lysis and solubilization buffers were pat-ternedafter those used by Foster et al. (1985). The lysis bufferconsisted of: 0.1 M sodium phosphate, pH 7.2, 0.3 mM p-hydroxyme-curibenzoate, 2 mM PMSF, 0.5 pg/ml DNase, and a stabilizationmixture of 10% (w/v) glycerol, 5 mM EDTA, 5 mM 1,lO-phenanthro-line, 0.5 mM TLCK, 1 p~ leupeptin, and 1 p M pepstatin A. Phenan-throline was added to he buffer during preparation from a 1 M

solution in ethanol. Because of the short lifetime of PMSF inwater,

the compound was added from a 0.1 M solution in ethanol to thecellsuspension in lysis buffer made without PMSF immediately beforepassage through the French press. Solubilization buffer consisted of:50 mM Tris-HC1, pH 7.4, and he stabilization mixture. Columnbuffers contained 10% (w/v) glycerol, 1.25% octyl glucoside, and 50mM Tris-HC1, pH 7.3, for blue-Sepharose or 50 mM sodium phos-

phate, pH 7.5, for the Mono Q columns. For some analytical columns

of blue-Sepharose, Tris was replaced by bisTris-HC1, pH 6.7, and 5mM MgCl, was present when nucleotides and their derivatives weretested for ability to elute transducers from the resin.

Preparation of Cells and Membrane"HB1032 was grown vernightin 1 liter Luria broth containing ampicillin (50 pg/ml) and hatculture (O D I ) was used to inoculate 50 liters of the same mediumin a fermentor (Labline Bioengineering Co., modelLP351-75).At OD= 0.5, isopropyl-0-D-thiogalactoside as added to 1mM. At 4 h (ODI ) cells were harvested by centrifugation and stored a t -70 "C. 40g of cell paste suspended in 160 ml of ice-cold lysis buffer were thawedon ice. All subsequent manipulations were a t 4 "C. Cells werebrokenwith a French pressure cell equipped with a rapid fill kit (FA-073SLM-Aminco, Urbana, IL) by two passages a t 20,000 p.s.i. in 40-mlportions. Unbroken cells and debris were removed by wo 20-mincentrifugations at 10,000 rpm in a Sorvall SS-34 rotor. The super-natant was centrifuged for 190 min at 50,000 rpm in a Beckman 60Tirotor and the esulting membrane pellets stored at -70 "C.

Solubilization and CibacronBlueChromatography-Membranefrom 40 g frozen cell paste was suspended in 10 ml of solubilization

buffer using a syringe with an 18-gauge and then a 22-gauge needle.Protein concentration was determined and the suspension diluted

with the same buffer to 12-15 mg protein/ml (adjusted to produce atotal volume that would completely fill an integral number of tubesfor the vertical rotor, see below). Typically 25-30 mg of membrane

protein was obtained per g of cell paste. A portion of the suspendedmembrane that contained 400 mg of protein was immediately proc-essed for solubilization and the remainder stored a t -70 "C or later

use. A solution of 15% (w/v) octyl glucoside was added to suspended

membrane to a concentration of 1.25%. After 10 min on ice thesuspension was centrifuged for 34 min at 50,000 rpm in a BeckmanVTi65 rotor. The orange-colored supernatant was applied immedi-ately to a 2.6 X IO-cm column of blue-Sepharose CL-GB previouslyequilibrated with column buffer and connected to a pump. Thecolumn was developed t a flow rate of 1ml/min with a linear gradientof NaCl (0-2 M) in 10 column volumes (500 ml) of column buffer,followed by 100 ml of column buffer containing 2 M NaC1. 24-mlfractions were collected and 15 pl of each was analyzed by SDS-polyacrylamide gel electrophoresis. Trg was present in fractions con-taining 1.6-2.0 M NaCl. A t this point, Trg is stable for several daysat 4 "C or can be stored frozen at -20 "C for extended periods.

Anion-exchange Chromatography-Trg-containing fractions fromthe previous step were pooled, concentrated to 4 0 ml by filtrationthrough a YM-100 Diaflo filter (Amicon Corp.), and dialyzed against

100 volumes of Mono Q column buffer for 24 h. The dialysate wasdiluted with an equal volume of column buffer and pumped at a flow

rate of 1 ml/min onto a Mono Q HR 5/5 anion exchange column(Pharmacia) itted to a high performance liquid chromatographyapparatus (LKB).The column was washed with 5 ml of column bufferand then developed at 0.5 ml/min with a linear gradient of NaCl (0-300 mM) in 20 ml of the same buffer. Fractions were examined forthe presence of Trg by SDS-polyacrylamide gel electrophoresis. Elu-tion of the protein occurred at NaCl concentrations centered around

200 mM.Radiolabeled Cell Extracts-Cells from logarithmic phase cultures

were labeled with ~-[~~S]methionineHazelbauer and Engstrom,1981) or ~-[ methyl-~H]methioninen the absence of protein synthesis(Engstrom and Hazelbauer, 1980) as described, removed from thelabeling medium by centrifugation, and suspended in ice-cold lysisbuffer without DNase at 0.8 mg cell protein/ml (equivalent to ap-proximately 2.5 X lo 9 cells/ml). Lysozyme was added to 80 pg/ml.The suspension was frozen in a -70 "C freezer, thawed at room

temperature, submitted o the reeze-thaw cycle again, and thenmade1.25% for octyl glucoside. After 10 min on ice, DNase and MgCh wereadded to 20 pg/ml and 5 mM, respectively, to reduce viscosity, andafter 10 min EDTA was added to restore the free EDTA concentrationto 5 mM. The suspension was centrifuged 20 min a t 40,000 rpm in a42.2 rotor and thesupernatant applied to blue-Sepharose.

Analysis of Interaction with Blue-Sepharose-50 pl of swelled resinwas placed in a 1-ml plastic syringe at 4 "C and washed with 1ml ofanalytical column buffer. The detergent-solubilized extract from ap-proximately 10' cells, or the detergent solution of several microgramsof partially purified Trg was layered on the resin, allowed to flow in,and incubated 10 min. The column was then eluted sequentially with0.5 ml of analytical column buffer (pooled with the volume passed

from the column at sample application), 1 mlof the same buffercontaining 0.2 M NaC1, 1 ml of buffer, 0.5 ml of buffer containing apotential elutant, and 0.5 ml of buffer containing 2M NaCl. Materialeluted by each solution was precipitated with 5% trichloroacetic acid,

rinsed with acetone, mixed with electrophoresis sample buffer, boiledfor 2 min, and applied to a polyacrylamide gel.

Determination of Amino-terminal Sequences-The procedures wereessentially those described by Hunkapiller et al. (1983) and by Le-Gendre and Matsudaira (1988).Approximately 150 pg of purified Trgin double strength sample buffer containing 0.1 mM thioglycolate wasapplied to a 12-cm-widesample well in a0.5 mmSDS-polyacrylamide(10%)gel, the lower portion of which had been allowed to polymerizeover 15 h and the upper portion of which had polymerized 2 h. Beforesample application, the gel was prerun for 45 min at 150 V usingrunning buffer containing 50 p~ glutathione in the upper chamberand then that buffer replaced with fresh buffer containing 0.1 mMthioglycolate. After electrophoresis, protein was transferred to apolyvinylidene difluoride membrane (Immobilon-P, Millipore COW.)using an electroblotting apparatus filled with a solution of 16 mMTris, pH 8.3, 120 mM glycine, 20% methanol, and 0.025% SDS byapplication of 30 V for approximately 14 h. The membrane was

stained for 1 min in methanol-acetic acid-water (5:1:4) containing0.05% Coomassie Brilliant Blue and destained for approximately 15

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Purification of a Chemotacticransducer 17311

min in the same solvents (45:745).Sections of membrane containingthe desired material (intact or fragmented Trg) were cut out, rinsedextensively in H 2 0 to remove any glycine retained from the transferbuffer, and stored at -20 "C in capped tubes. Membrane containingprotein to be analyzed was chopped o small pieces with a razor blade,and the sequence of amino acids beginning at the amino terminuswas determined using an Applied Biosystems 470A protein sequencer.

Other Methods-Protein content was determined using the bicin-choninic acid protein assay (Pierce Chemical Co.) with bovine serum

albumin as the standard. SDS-polyacrylamide gel electrophoresis,autoradiography,and fluorography were s described previously Har-ayama et d., 1982).

RESULTS

Transducer Proteins nteract wit h Cibacron Blue-Wefound that the transducer proteins of E . coli bound stronglyto Cibacron blue F3G-A immobilized on Sepharose CL-GB.Binding persisted after extensive washing with buffer con-taining 0.2 M NaC1. Transducers could be released from theresin by 2 M NaCl or by S-adenosylmethionine (AdoMet) atconcentrations of 10-40 mM, but not by several other relevantsmall molecules (Table I). Fig. 2 provides representative ex-amples of experimental results that illustrate this phenome-non. In each panel the electrophoretic position of the trans-ducer protein or proteins is indicated by an arrow. As shownin Fig. 2 A , the entirearray of [3H]methyl-labeled ransducersincluding various electrophoretic forms of Tsr, Tar, Tap, andTrg was retained by Cibacron blue-Sepharose when chemo-tactically wild-type cells were broken by treatment with ly-sozyme and EDTA, the broken cells treated with octyl glu-coside, and thedetergent-soluble extract applied to a columnof the resin, compare lune 1 (soluble extract) with lanes 2 and3 (column eluate in ow salt). Application of buffer containing30 mM AdoMet released most of the radiolabeled transducers(lane 4) nd a buffer with 2 M NaCl eluted the remainder(lane5). Since all electrophoretic forms of [3H]methyl-labeledtransducers exhibited a similar pattern of retention and elu-tion, it appeared that all four of the transducer proteins were

bound and released. Interaction with Cibacron blue and elu-tion by AdoMet and by 2 M NaCl were demonstrated individ-ually for [3H]methyl-labeled Tsr and Tar by using mutantcells deleted of all but the specific transducer gene, for t3%]methionine-labeled Tsr and Trgsing cells with chromosomaldeletions in all transducer genes but harboring a multicopyplasmid carrying the specific transducer gene, and for unmod-ified Trg using plasmid-containing cells tha t also containeddeletions in genes for the transducers, the methyl transferase,

TABLE

Release of Trg f rom Cibacron blue-Sephnrose

For mixtures of elutants, each compoundwas at the indicatedconcentration.

Elutant Concen- Release oftration Trg

NaClNaClATPATP + GTPGTP + CTP +UTPcAMPcAMP + cGMPNAD++ NADP+S-AdenosylmethionineS-AdenosylhomocysteineS-AdenosylethionineMethionineAdenosine

Methionine-S-methyl sulfonium chloride

mM

2002000

25

2595

25

25

10

25

25

25

25

100Folinic acid 42 -

and methyl esterase. Fig. 2B shows an example of the secondtype of experiment in which [35S]methionine-labeled Tsr,resolved as a distinct band mong the array of proteins in theextract, is retained in a column of blue-Sepharose (comparelunes 1 and 2 ) and released by 2 M NaCl (lane 5). We did nottest specifically for interaction of Tap with Cibacron blue, butthe common behavior of all [3H]methyl-labeled species inexperiments like the one illustrated in Fig.2A led us to believe

that Tap has the same property in this regard as the othertransducer proteins.

Partially purified Trg was used to test thebility of relevantsmall molecules to release the transducer from Cibacron blue.In those experiments an excess of protein was applied to theresin to insure saturation with transducerand thusmaximumsensitivity to elution by the test compound. An example isshown in Fig. 2C. Lane 1 contained 20% of the amount ofsample applied to he resin, lane 2 materialnot etained(including approximately 20% of the Trg applied), and lanes3-6 material eluted by buffer containing 0, 0.1, 0.4, and 2 M

NaCl, respectively. Elution of a small amount of Trg by 0.4

M NaCl appeared to be an artifact of step gradients, since inelution with a continuous gradient ofNaC1, Trg was not

released until the NaCl concentration was over 1.5 M. Theonly eluents tha t effectively released Trg from Cibacron bluewere salt a t high concentrations and AdoMet (Table I). Ex-periments with [3H]methyl-labeled cells revealed that othertransducers were also eluted by AdoMet. As noted previously(Hoffman,1986), the proportion of authentic AdoMet incommercially available preparations varied substantially.This plus the instability of the compound resulted in variableelution of transducers by solutions of AdoMet that shouldhave been a t the same concentrations. In some experiments10 mM was sufficient to elute most retained transducer; inother experiments the apparent concentration required wasas high as 40 mM. In any case, the effect was specific for thechemical nature of AdoMet, since Trg was not eluted by a

higher concentration (200 mM)of NaCl nor by equivalentconcentrations of analogs like S-adenosylhomocysteine ormethionine-S-methyl sulfonium chloride, which like AdoMetis cationic. However, AdoMet released not only transducers,but essentially all proteinsretained by the resin. Generalelution by AdoMet of proteins bound to blue-Sepharose hasbeen noted by others (Borczuket al., 1987). This phenomenonmeans that elution by AdoMet does not necessarily reflectthe existence of a specific binding site for the molecule on thetransducers.

We found that the ytoplasmic domain of Trg, independentof the rest of the transducer, interacted with Cibacron blue.A truncated trg was constructed t hat coded for the entirecytoplasmic domain with the exception of the 2 arginines tha t

mark the end of the second membrane-spanning region andthe substitution of methionine for isoleucine at the ollowingposition. The product of this genewas retained by blue-Sepharose and released by 2 M but not by 200 mM NaC1,exhibiting the same pattern as the intact transducer (Fig.2 D ) .Thus, the nteraction of transducers with Cibacron blueoccurs at least through the cytoplasmic domain, presumablyvia a region of conserved structure. In the experiment shownin Fig. 20, the cells used had a ow content of the cytoplasmicdomain, and thus, itwas necessary to utilize immunoblottingto detect the Trg polypeptide among other proteins. Not allthe cytoplasmic domain was retained by blue-Sepharose. Wedo not know the reason for this, but i t could reflect thepresence of many other proteins that interacted with the resin

and thus saturated it r a proportion of the Trg polypeptideincapable of binding, perhaps because it was not in the native

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17312 Purification of a Chemotactic Tran sducer

1 2 3 4 5

A

61-

55-

42-

C

9 7,

6 1.

55.

42.

36.

29 -

B

I+97.

61-

5 5-

D

+

36-

29-

+

f

FIG..

Interaction of transducers with Cibacron blue-Sepharose.Detergent-solubilized protein was

applied to a column of blue-Sepharose and washed sequentially with several solutions. Eluted materialwas analyzedby SDS-polyacrylamide gel electrophoresis and an appropriate means of detecting the transducers. Positions of

standard proteins are indicated in each panel with a value of molecular mass in kilodaltons: 97, phosphorylase b;

61, n-amylase; 55, glutamic dehydrogenase; 42, actin; 36, glyceraldehyde-3-phosphate dehydrogenase; and 29,carbonic anhydrase. A , [3H]methyl-labeled ransducers. Analysis with a 9% polyacrylamide gel and fluorography.

The sample (lane 1 ) was 6 X lo7 [3H]methyl-labeled cells of the chemotactically wild-type strain Owl . Materialeluted by two washes with 10 volumes of buffer is shown in lanes 2 and 3, by 30 mM AdoMet in lane 4 and by 2 M

NaCl in lane 5.The bar and arrow indicate the positions of the various electrophoretic forms of the four transducers,which in the chemotactically wild-type cell are predominantly forms of Tsr and Tar. B , [35S]methionine-labeledTsr . Analysis was with an 11% polyacrylamide gel containing 25% the usual amount of bisacrylamide. The sample(lane 1 ) was 10’ [35S]methionine-labeledcells of strain HB897 which contains deletions in all chromosomal

transducer genes but harbors a multicopy plasmid carrying tsr. Material eluted by three washes, with 10 volumesof buffer is shown in lanes 2, 3, and 4 and by 2 M NaCl in lane 5. The arrow indicates the position of Tsr. C,partially purified Trg. Analysis was with a 12% polyacrylamide gel and staining by Coomassie Brilliant Blue. 20%of the amount applied to the column is shown in lane 1. Material eluted by two washes with 10 volumes of buffer

is shown in lanes 2 and 3, by 10 mM NaCl in lane 4, by 400 mM NaCl in lane 5, nd by 2 M NaCl in lane 6.D,

cytoplasmic domain of Trg. Analysis was with a 12% polyacrylamide gel and immunoblotting with anti-Trg serumand a peroxidase-linked second antibody. The sample applied to the resin (lane 1 ) was the soluble fraction of adetergent extract of lysed cells of HB1033, a st rain tha t ad deletions in the chromosomal genes for the modificationenzymes and the transducers but harbored pDD2 which carried the fragment of trg coding for the cytoplasmicdomain. Lane 1 represents 37.5% of the material analyzed in t he following lanes (equivalent to 1.2 X 10’ cells).

Material that did not adhere to the resin is shown in lane 2. Material eluted by two washes with 6 volumes ofbuffer is shown in lanes 3 and 4 and by 2 M NaCl in lane 5. The heavy and light arrows indicate the position ofintact cytoplasmic domain and a smaller proteolytic fragment, respectively.

state. The domain isunstable in uiuo, disappearing rapidly asthe result of proteolysis. A distinct fragment of the cyto-plasmic domain (indicated by the thin arrow in Fig. 20),presumably the result of proteolysis, was observed in cellscontaining the truncated trg. It is interesting that thisshort-

ened orm, approximately 7 kDa smaller than he ntactcytoplasmic domain, did not interact with Cibacron blue.

Purification of Trg-We utilized the interaction of trans-

ducers with Cibacron blue to devise an efficient purificationfor the Trgprotein. In combination with amplification of thecellular content of Trg by appropriate genetic constructs, thefractionation steps yielded approximately 5 mg of pure Trgprotein from 1 iter of cell culture by manipulations that can

be completed in a few days. The procedure is described indetail under “Experimental Procedures.’’ The protein contentand yields at various steps in the purification are illustrated

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Purification of a Chemotactic Transducer 17313

in Fig. 3 and Table 11. The result was a protein preparationin which over 98% of the material is intact Trg. In ane 9 ofth e gel shown in Fig. 3, a sam ple of this purified m aterial wasapplied in excess to i l lustrate that the major contam inantsappeared asa series of bands migrating justbelow inta ct Trga ndasdis t inc tb a n d sa tapparent molecularweights of116,000, 36,000, an d 29,000. All th ese electro pho retic specieswere recognized by anti-Trg antibody in imm unoblots andthus are l ikely to be forms of Trg. The 116-kDa band m aycorrespond to a small proportion of dimeric Trg that p ersistsin the con dition s f electrophoresis, perhap s held together bya disulfide bond between th e single cysteines present on eachmolecule. The first 10 residues of the 29 -kD a polypeptidecorresponded to he amin o-term inal sequence of Trg (see

below) an d thu s tha t olypeptide must be an am ino-terminalfragment of the protein . W e e xpec t tha t th e 6- and 2 9-kDaspecies are f ragments f Trg created y a proteolytic cut near

4 2 3 4 5 6 7 8 9

116-

97-

61-55-

42-

36-

29 -

FIG. 3. Puri f i ca t io n of Trg. The figure isanSDS-polyacryl-amide (12%)gel stained with Coomassie Brilliant Blue. Positions areindicated with a value in kilodaltons for the proteins listed in thelegend to Fig. 2 plus 8-galactosidase (116). Samples for lanes 1 andfor 3-7 represented material from approximately equivalent amountsof starting material. Cells of HB1032, induced for production of Trg(lane 1 ), were broken with a French pressure cell and centrifuged atlow speed to separate unbroken cells (lane 2, material from 10-foldmore starting material) and a clarified supernatant (lane 3 ) . Thesupernatant was centrifuged at high force to separate soluble mater ial(lane4 ) rom membrane (lane 5 ) .Pelleted material was treated withdetergent and centrifuged at high force to separ ate solubilized protein(lane 6) from material that remained particulate ( lane 7) . Trg was

separated from othe r solubilized proteins by elution from Cibacronblue-Sepharose (lane 8 ) and then from a Mono Q anion exchangecolumn ( lane9) .

th e middle of th e polypeptide chain, analogo us o th e leavagedocumented for Ta r (Mobray e t al., 1985) . We presume thatt he ba nds j us tbelow inta ct Trg are the resu lt f proteolysisa t t h e e n d sf the protein, again alsobserved for Tar (Fo steret al., 1985).

Since strains with single chromosomal opy of tr g containonly approximately150 Trg protein s per ell , the f irst step inpurif ication was to amplify theellular content of this trans -

ducer . Th e tr g gene was placed under the control of the tacpromotor in a multicopy plasmid th at also carr ied the lacIqgene (see “Experimental Procedures” andig . 1).Addition of1 mM I PT G o a growing cultu re of cells harbo ring hisplasmid resulted in cellular content f Tr g 500 t imes greaterthan for cells with a normal copy of tr g o n t h e chromosome.T o avoid heterog eneity of covalent modification of Trg , thehost cells contained delet ions in the chrom osom al genes forthe methyl transferase and demethy lase. T o avoid possiblecontamination with other transducers, the host chromosomealso carried deletions in tsr, trg, tar, a n d tup. Cells inducedfor Trg were broken in a French press and the mem branessepar ated from soluble material by ultracentrifugation . Cellsor mem brane could be stored a t -70°C for several monthswith out apparen t effect on later purification of Trg. As re -por ted for T ar by Foste r e t al. (1985), T rg was exquisitelysensit ive to roteolysis f rom the mo men t the mem brane seresolubilized by deterg ent, and thus , we followed the lead ofthose autho rs by in cluding an array of proteinase inhibitorsin t hesolubilization b uffer. T rg was effectively solub ilized byoctyl glucoside, resulting in substan tial pu rification from th emem brane proteins that remain ed insoluble (Fig. 3 comparelane 6, detergent-solubilized protein, to lane 7, insoluble m a-ter ial) . I t should be noted that two m ajor proteins occupiedthe pos i t ion of Trg on thegel pattern shown in Fig. 3, lanes1-7.O ne w a s T r g a nd t he o the ras a rotein of approxim ately60 kDa induced y cellularstress. Our procedure forigh levelexpression of tr g under the cont rol of the ta c promotor alsoresulted in increased production of this second protein. Wesuspect i t corresponds to GroEL (Til ly e t al., 1985). In an ycase the wo proteins were separated by the colum n fraction-a t ions , but comigra t ion meant tha t theield of Trg at earliersteps could not be est imated by com paring the intensity ofstained bands. Imm ediately following detergent reatment,the solubil ized protein was applied to a column of blue-Sepharose and subseq uently eluted in a gradient of NaCl,appearing in f ractions co ntaining .6-2 M salt . The result ingpreparation of T rg was s ubstantial ly purif ied (approximately80% in tac t Trg) an d was not prone to the degrada t ion ob-served in the unfractionated mater ial . However , manipula-t ions of th is prepara t ion to concent ra te and toeduce the sal t

concentrat ion usually resulted in substantial precipitat ion fprote in and thus loss of Trg (Table 11). We have not ye tdiscovered con ditio ns tha t avoid these losses. In an y case,essentially all remaining contaminants unrelatedo Trg ouldbe removed by an ion-exchange column. Preparations of Trglike that shown in Fig. 3, lane 9,were used for preliminarycharacterization of th e purified p rotein.

Properties of Purified Trg-The amin o acid compositiondeterminedexperimentally or hepurif iedproteincorre-sponded well to the am ino acid composit ion predicted fromthe nucleotide sequence of tr g (Fig. 4) . The f irst 19 residuesof in tac t Trg and the f i r s t0 residues of the 29-kDa fragm ent,identified by auto ma ted amin o cid sequencing, correspon dedin b oth cases to the amin o-term inal sequence deduced from

th e nucleotide sequence of tr g (Bollinger e t al., 1984). Thes ecorresponden ces indicate that the purif ied protein was Tr gand tha t the pos tula ted t r ans la t ion s ta r t s i te inrg (Bollinger

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17314 Purification of a Chemotactic Transducer

TABLE1

Purification of Tr g receptor protein

FractionTotal

Trg"Yield

Recovery of

protein for step Trg relativet o membrane

Purificationfactor

mg~~

%

Membraneb 166 21.4 100Octyl glucoside extract 106 17.2 80 80

Cibacron blue pool 22 17.2100

8016'oncentrated Cibacronlue pool 12.8' 74 60Dialyzedibacron blue pool 8.5'.3' 41Mono Q pool

25

-fold

1.01.25

6.05.0

4.9.9 92 23 7.84.8

*The starting material represents membrane obtained rom 1 L of a culture of cells induced for expression ofDetermined by quantitative immunoblotting with anti-Trg serum andperoxidase-linked second antibody.

Trg asdescribed under "Experimental Procedures."Precipitation of protein during this step resulted inoss of protein.

90 I

AMINO ACID RESIDUE

FIG.4. Amino acid analysis of purified Trg protein. 80 fig of purified Trg was hydrolyzed in anevacuatedsealed ampule with double-distilled 6 N HCI for 24 h at 110 "C. After removal of solvents by vacuum evapora tion,the hydrolysate was dissolved in 0.2 N sodium citrat e, pH 2.2, and analyzed by the method of Spackman et al.(1958) using a Beckman 121 MB Automat ic Amino Acid Analyzer equipped with a Hewle tt Packard Integrator(HP3396A). Th e open bars indicate average values fo r duplicate hydrolysates. The dark bars indicate amino acidcomposition deduced from the gene sequence (Bollinger e t QL, 1984) with previously noted corrections (Nowlin etaL, 1987, 1988).

A L I

WAVELENGTH (nm)

FIG. 5. Circular dichroic spectrum of purified Trg protein.Spectra were recorded at 20 "C with a Jasco J-40A spectropolarimeterusing 1-mm cells in a nitrogen-flushed chamber . The instrumentascalibrated with 10-camphorsulfonic acid. Purified Trg protein, a t 90pg/ml as determined by the bicinchoninic acid assay, was analyzedin 50 mM Tris , pH .2,1.25% octyl glucoside. The datawere correctedusing a sample without protein and were expressed as mean residueellipticity (degree cm2 dm").

et al., 1984) is correct. This is of interest since the deducedprecede sequence of the extreme amino terminusf Trg differs

from the other transducers in that 16 rather than 6 residues

precede the hydrophobic membrane-spanning segment. Theinitial methionine of Trg is neither cleaved nor modified. This

contrasts with Tar, in which the amino terminus appears tobe blocked (Mowbray et al., 1985). Preparations of purified

Trg were relatively stable. In 50 mM Tris,pH 7.3, 1.25% octyl

glucoside, degradation occurred with a tlh of 12 h at room

temperature. The purified transducer was found to bind li-

gand-occupied galactose-binding protein. Characterization of

that interaction will be the subject of a separate report.

The secondary structure content f the Trg ransducer was

investigated by obtaining the circular dichroic spectrum of

detergent solubilized purified protein (Fig. 5). The data indi-

cated thatTrg hasa high (>80%) helical content, some

random coil, and essentially no detectable @-sheet.The spec-

trum closely resembles that obtained for Tar (Foster et al.,

1985), an observation consistent with a common structure for

the two transducer proteins.

DISCUSSION

We exploited the strong interaction of Trg with Cibacron

blue to devise a rapid and efficient purification procedure for

this transmembrane receptor protein. The same strategy will

be useful for purification of other transducer proteins of E.coli since they also bind effectively to the dye. It could be

applicable to purification of methyl-accepting chemotactic

proteins observed in other bacterial species, if the homology

between thoseproteins and he transducers from E . coli(Nowlin et al., 1985) includes conservation of the feature that

we have detected by interaction with Cibacron blue.

We have no decisive information about the functional sig-

nificance of the ability of transducers to bind Cibacron blue.Many proteins are known to interact with this dye, predomi-

8/3/2019 89 JBC_264_17309


Purification of a Chemotactic T r a n s d u c e r 17315

nantly those that contain or are hought to contain the

specific dinucleotide fold domain (Rossman et al., 1975) char-

acteristic of manyproteins that bind nucleotides such as

NAD+ and ATP. However, some proteins that bind toCiba-

cron blue have no known affinity for nucleotides or related

molecules and apparently do not contain a dinucleotide fold

(Stellwagen, 1977). In general, the former class of proteins

remain bound to a Cibacron blue resin n moderate salt

concentrations but re released by high salt and y the specificligand for which the binding site is designed (see the booklet

“Affinity Chromatography” published by Pharmacia). In con-

trast, members of the atter class, with the exception of

albumin which is known for its ability to bind manydyes, arereleased by moderate salt concentrations Easterday and

Easterday, 1974). The transducers were released from Ciba-

cron blue only by quite high concentrations of NaCl ( in fact,

this is a primary basis for the extensive purification of Trg

accomplished by the Cibacron blue column), and thus, these

proteins may belong to thegroup of polypeptides that bind t o

the dye because of a dinucleotide fold, perhaps designed for

binding a specific small molecule. However,among the poten-

tial ligands tested only AdoMet caused release and itappears

that thismolecule causes generalized release of proteins fromCibacron blue (Borczuket al., 1987). Attempts to detect direct

binding of radiolabeled AdoMet to purified Trg have been

unsuccessful. In any case, binding to Cibacron blue occurs at

minimum through the cytoplasmic domain of the transducers

and hus is likely to involve some of the many residues

conserved in this domain among the four transducers. The

truncated form of the Trg cytoplasmic domain, with an ap-

parent molecular weight approximately 7000 less than the

intact domain did not bind Cibacron blue, a behavior also

noted for a fragment of the Tarcytoplasmic domain which ismissing 50 amino-terminal residues of that domain (Kaplan

and Simon, 1988). Thus , t appears tha t he polypeptide

segment immediately following the second membrane span-

ning-region is crucial for Cibacron blue binding by transducersalthough this does not necessarily mean that thebinding site

itself occurs in this region. The function of this segment isnot well defined but the occurrence of mutational substitu-

tions that lock the transducer in a particular signaling state

led Ames and Parkinson (1988) to suggest tha t the segment

is involved in the functional linkage between ligand binding

and intracellular excitation.

The striking similarity of the circular dichroic spectra of

Tar (Foster et al., 1985) and Trg (Fig. 5) suggests a very

similar content of secondary structural units in the proteins

and may well reflect a similar three-dimensional structure.

Such similarities would be predicted from the conserved pri-

mary sequences of the respective cytoplasmic domains, but

not from the primary sequences of the two periplasmic do-mains, in which the number of identical residues is not

significantly greater than chance. However, recent genetic

data imply that the eriplasmic domains of Tsr, Tar, and Trg

have similar organizations, even in the absence of significant

identities in primary sequence between Trg and the other

two. Mutational substitutions that affect ligand binding inthe three transducers cluster in two similar positions along

the sequences of the periplasmic domains (Park and Hazel-

bauer, 1986;Wolff and Parkinson, 1988,Lee et al., 1988),

implying a common placement of the binding sites and thus

of structural units. These and other suggestions about the

structural organization of transducers can now be explored

using purified protein obtained by the procedures we have

described here.

Acknowledgments-We thank John Bollinger for suggesting theuse of Cibacron blue-Sepharose and for constructiong pBB7, DavidDutton for construction of pDD2, Gerhard Munske for determiningamino acid sequences, and Chris Russell for supplying pCR44.

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