THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Purification of Receptor Protein Trg by Exploiting Property a Common to Chemotactic Transducersof Escherichia coZi*
(Received for publication, February 22, 1989)

Gregory G. Burrows, MarciaE. Newcomer$ and GeraldL. Hazelbauerl

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

The methyl-acceptingchemotactic transducers of membrane is spanned by a hydrophobic segment near the Escherichia coli were found to bind strongly to Ciba- amino terminusand anothernear residue 200 of these 60-kDa cron blue-Sepharose. Among potential elutants tested, proteins, creating two domains, one in the periplasm and the only S-adenosylmethionine moderate concentrations other in thecytoplasm. The periplasmic domain contains few at and NaCl at concentrations greater than 1.5 M caused conserved residues and recognizes specific ligands characterdissociation of these detergent-solubilized transmemistic of each particular transducer. The cytoplasmic domain brane proteins from the dye. Release by S-adenosyl- contains many conserved residues; 158 of 314 are identical for methionine may be a generalized effectrather than the all fourtransducers. That domain performs the common result of a specific binding site for that compound on functions of excitation by affecting the phosphorylation retransducers. A truncated trg gene was created that actions that begin with CheA (Hess et al., 1987; 1988) and coded forthecarboxyl-terminalthree-fifths of the adaptation by altering theextent of modification of the transducer, which constitutes the cytoplasmic domain common to all four transducers E. coli.This domain methyl-accepting glutamyl residues, 4-6 depending on the in bound to Cibacron blue-Sepharose and was eluted ain transducer(Kehry and Dahlquist, 1982; Tenvilliger et a!., pattern similar to that exhibited by intact Trg, indi- 1986; Nowlinet al., 1987), that are modified to create carboxyl methyl esters. cating that interaction with the dye occurred in this This laboratory has been interested in Trg, which mediates conserved domain. Adherence to Cibacron blue and elution by high salt formed the core of an efficient taxis toward galactose and ribose by recognizing ligand-ocpurification scheme, developed for Trg butapplicable cupied glactose- and ribose-binding proteins and thus is a to all transducers in E. coli and perhaps to methyl- primary receptor protein for a particularconformational state accepting chemotaxis proteins inother species. Deter- of those two polypeptides. Full understanding of the mechamination of the amino acid sequenceat the beginning nisms by which this or any polypeptide receptor functions of purified Trg confirmed that it contained a longer will require extensive characterization of the purified receptor hydrophilic segment at its amino terminus than other protein. We have developed an effective purification procetransducers of E. coli. The initialmethionine of Trg is dure for Trg, based primarily on a specific binding property neither cleaved nor modified, in contrast to the Tar common to the conserved cytoplasmic domain of all chemotransducer in which the amino terminus was found tactic transducers. previously to be blocked. Circular dichroic measurements of purified Trg indicated that the secondary EXPERIMENTALPROCEDURES structural organization the protein predominantly of is Bacterial Strains-All strains were derivatives of E. coli K12. a-helix. CP334, CP361, and CP553 are derivatives of OW1 (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-directed mutagenesis was used to change the sequence TAAACC, beginning at position -25 relative to the initiation codon of trg, to GAATTC, creating an EcoRI recognition site in the 2.2-kb BglIIHincII fragment,containing trg and incorporated into Ml3mpll between the BamHI and SmaI sites. The 1.9-kb EcoRI fragment from the 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. The ladq gene was introduced into pBB7by isolating the 1.6kb 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 fragment to the blunt-ended plasmid. The resulting 8.1-kb plasmid was named 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 Trg

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 intracellular signalling, and mediate sensory adaptation (for a recent review see Hazelbauer, 1988). The deduced amino acid sequences of these proteins are related and suggest a model for disposition of the transducer across the cytoplasmic membrane (Boyd et al., 1983; Krikos et al., 1983; Russo and Koshland, 1983; Bollinger et al., 1984). In the model, the
*This workwas supported by Grant GM29963 and protein sequences were determined using an instrument purchased through Shared Instrumentation Grant RR02677, both 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 aduertisemenf in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Current address: Dept. of Biochemistry, Vanderbilt University, Nashville, T N 37232. Recipient of an American Cancer Society FacultyResearch Award.

The abbreviations used are: kb, kilobase; AdoMet, S-adenosylmethionine;bisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecy1 sulfate; and TLCK, A-p-tosyl-L-lysinechloromethyl ketone.

17309

17310

Purification of Chemotactic Transducer a

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 a total volume that would completely fill an integral number of tubes for the vertical rotor, see below). Typically 25-30 mg of membrane protein was obtained per g of cell paste. A portion of the suspended membrane that contained 400 mg of protein was immediately processed for solubilization and the remainder stored a t -70 "Cfor later use. A solution of 15% (w/v) octyl glucoside was added to suspended pGBl membrane to a concentration of 1.25%. After 10 min on ice the suspension was centrifuged for 34 min at 50,000 rpm in a Beckman VTi65 rotor. The orange-colored supernatant was applied immediately to a 2.6 X IO-cm column of blue-Sepharose CL-GB previously equilibrated with column buffer and connected to a pump. The column was developeda t a flow rate of 1ml/min with a linear gradient of NaCl (0-2 M) in 10 column volumes (500 ml) of column buffer, followedby100 ml of column buffer containing 2 M NaC1. 24-ml fractions were collected and 15 pl of each was analyzed by SDSpolyacrylamide gel electrophoresis. Trg was present in fractions conFIG. 1. Map of pGB1. The position of genetic units and recog- taining 1.6-2.0 M NaCl. At this point, Trg is stable for several days nition sites for selected restriction endonucleases are indicated for at 4 "C or can be stored frozen at -20 "C for extended periods. this 8140-base pair plasmid. Abbreviations are as follows: A, AflII; Anion-exchange Chromatography-Trg-containing fractions from Ap, ApaI; E, EcoRI; H, HindIII; Hp, HpaI; M, MluI; N, NdeI; R, the previous step were pooled, concentrated to 4 0 ml by filtration RsrII; S, Sack Sc, Scal; Sm, SmaI; Sn, SnaI; St, StuI;X, XmaIII. through a YM-100 Diaflo filter (Amicon Corp.), and dialyzed against 100 volumes of Mono Q column buffer for 24 h. The dialysate was was inserted between the NcoI and PstIsites of a modified pKK233- diluted with an equal volume of column buffer and pumped at a flow 2 (Pharmacia), placing that coding sequence under the control of the rate of 1 ml/min onto a Mono Q HR 5/5 anion exchange column "trc" promotor, by annealing and ligating the complimentary NcoI (Pharmacia)fitted to a high performance liquid chromatography sites, filling in the exposed ends with T4 DNA polymerase, and apparatus (LKB).The column was washed with 5 ml of column buffer ligating the resulting blunt ends. The pKK233-2 had been modified and then developed at 0.5 ml/min with a linear gradient of NaCl (0previously by joining the SalI-BamHI fragment containing lacP from 300 mM) in 20 ml of the same buffer. Fractions were examined for the presence of Trg by SDS-polyacrylamide gel electrophoresis. ElupGBl to pKK233-2 cleaved with the same enzymes. Chemi~ak"L-[methyl-~H]Methionine Ci/mmol) and [35S]me- tion of the protein occurred at NaCl concentrations centered around (12 thionine (1000 Ci/mmol) were from Du Pont-New England Nuclear. 200 mM. Radiolabeled Cell Extracts-Cells from logarithmic phase cultures Octyl-P-glucopyranoside (octyl glucoside) was from Boehringer (Hazelbauer and Engstrom, Mannheim. Blue-Sepharose CL-GB was from Pharmacia. DNase I, were labeled with ~-[~~S]methionine in bisTris, isopropyl-P-D-thiogalactoside, pepstatin A, leupeptin, p-hy- 1981) or ~-[methyl-~H]methionine the absence of protein synthesis droxymercuribenzoate, phenylmethylsulfonyl fluoride (PMSF), and (Engstrom and Hazelbauer, 1980) as described, removed from the Nu-p-tosyl-L-lysinechloromethyl ketone (TLCK) were from Sigma; labeling medium by centrifugation, and suspended in ice-cold lysis 1,lO-phenanthroline was from Aldrich. S-Adenosylmethionine was buffer without DNase at 0.8 mg cell protein/ml (equivalent to apused as the p-toluenesulfonate salt (Sigma). Enzymes and material proximately 2.5 X lo9 cells/ml). Lysozyme was added to 80 pg/ml. for manipulation of DNA were fromNew England Biolabs, Bethesda The suspension was frozen in a -70 "C freezer, thawed at room temperature, submitted thefreeze-thaw cycle again, and then to made Research Laboratories, and Boehringer Mannheim. Buffers and Solutions-Buffers were adjusted to the indicated pH 1.25% for octyl glucoside. After 10 min on ice, DNase and MgCh were at room temperature. The lysis and solubilization buffers were pat- added to 20 pg/ml and 5 mM, respectively, to reduce viscosity, and ternedafter those used by Foster et al. (1985). The lysis buffer after 10 min EDTA was added to restore the free EDTA concentration consisted of: 0.1 M sodium phosphate, pH 7.2, 0.3 m p-hydroxyme- to 5 mM. The suspension was centrifuged 20 min at 40,000 rpm in a M curibenzoate, 2 m PMSF, 0.5 pg/ml DNase, and a stabilization 42.2 rotor and thesupernatant applied to blue-Sepharose. M Analysis of Interaction with Blue-Sepharose-50 pl of swelled resin mixture of 10% (w/v) glycerol, 5 m EDTA, 5 m 1,lO-phenanthroM M line, 0.5 m TLCK, 1 p~ leupeptin, and 1 p M pepstatin A. Phenan- was placed in a 1-ml plastic syringe a t 4 "C and washed with 1 ml of M throline was added to the buffer during preparation from a 1 M analytical column buffer. The detergent-solubilized extract from apsolution in ethanol. Because of the short lifetime of PMSF inwater, proximately 10' cells, or the detergent solution of several micrograms the compound was added from a 0.1 M solution in ethanol to thecell of partially purified Trg was layered on the resin, allowed to flow in, suspension in lysis buffer made without PMSF immediately before and incubated 10 min. The column was then eluted sequentially with passage through the French press. Solubilization buffer consisted of: 0.5 ml of analytical column buffer (pooled with the volume passed 50 m Tris-HC1, pH 7.4, and the stabilization mixture. Column from the column at sample application), 1 mlof the same buffer M buffers contained 10% (w/v) glycerol, 1.25% octyl glucoside, and 50 containing 0.2 M NaC1, 1 ml of buffer, 0.5 ml of buffer containing a M m Tris-HC1, pH 7.3, for blue-Sepharose or 50 m sodium phos- potential elutant, and 0.5 ml of buffer containing 2M NaCl. Material M phate, pH 7.5, for the Mono Q columns. For some analytical columns eluted by each solution was precipitated with 5% trichloroacetic acid, of blue-Sepharose, Tris was replaced by bisTris-HC1, pH 6.7, and 5 rinsed with acetone, mixed with electrophoresis sample buffer, boiled m MgCl, was present when nucleotides and their derivatives were for 2 min, and applied to a polyacrylamide gel. M Determination of Amino-terminal Sequences-The procedures were tested for ability to elute transducers from the resin. Preparation of Cells and Membrane"HB1032 was grownovernight essentially those described by Hunkapiller et al. (1983) and by LeGendre and Matsudaira (1988).Approximately 150 pg of purified Trg in 1 liter Luria broth containing ampicillin (50 pg/ml) and that M culture (OD I 3) was used to inoculate 50 liters of the same medium in double strength sample buffer containing 0.1 m thioglycolate was in a fermentor (Labline Bioengineering Co., modelLP351-75). At OD applied to a 12-cm-widesample well in a0.5 mmSDS-polyacrylamide = 0.5, isopropyl-0-D-thiogalactoside was added to 1 mM. At 4 h (OD (10%)gel, the lower portion of which had been allowed to polymerize over 15 h and the upper portion of which had polymerized 2 h. Before I 6) cells were harvested by centrifugation and stored a t -70 "C. 40 g of cell paste suspended in 160 ml of ice-cold lysis buffer were thawed sample application, the gel was prerun for 45 min at 150 V using on ice. All subsequent manipulations were a t 4 "C. Cells werebroken running buffer containing 50 p~ glutathione in the upper chamber M with a French pressure cell equipped with a rapid fill kit (FA-073 and then that buffer replaced with fresh buffer containing 0.1 m SLM-Aminco, Urbana, IL) by two passages at 20,000 p.s.i. in 40-ml thioglycolate. After electrophoresis, protein was transferred to a portions. Unbroken cells and debris were removed by two 20-min polyvinylidene difluoride membrane (Immobilon-P, Millipore COW.) M centrifugations at 10,000 rpm in a Sorvall SS-34 rotor. The super- using an electroblotting apparatus filled with a solution of 16 m M natant was centrifuged for 190 min at 50,000 rpm in a Beckman 60Ti Tris, pH 8.3, 120 m glycine, 20% methanol, and 0.025% SDS by application of 30 V for approximately 14 h. The membrane was rotor and theresulting membrane pellets stored at -70 "C. stained for 1 min in methanol-acetic acid-water (5:1:4) containing Solubilization and Cibacron Chromatography-Membrane Blue from 40 g frozen cell paste was suspended in 10 ml of solubilization 0.05% Coomassie Brilliant Blue and destained for approximately 15

Purification of a Chemotactic Transducer

min in the same solvents (45:745). Sections of membrane containing the desired material (intact or fragmented Trg) were cut out, rinsed extensively in H20 to remove any glycine retained from the transfer buffer, and stored at -20 "C in capped tubes. Membrane containing protein to be analyzed was choppedto small pieces with a razor blade, and the sequence of amino acids beginning at the amino terminus was determinedusing an Applied Biosystems 470A protein sequencer. Other Methods-Protein content was determined using the bicinchoninic acid protein assay (Pierce Chemical Co.) with bovine serum albumin as the standard. SDS-polyacrylamide gel electrophoresis, autoradiography,and fluorography were described previously as (Harayama et d., 1982).

17311

and methyl esterase. Fig. 2B shows an example of the second type of experiment in which [35S]methionine-labeled Tsr, resolved as a distinct band among the array of proteins in the extract, is retained in a column of blue-Sepharose (compare lunes 1 and 2 ) and released by 2 M NaCl (lane 5). We did not test specifically for interaction of Tap with Cibacron blue, but the common behavior of all [3H]methyl-labeled species in experiments like the one illustrated in Fig.2A led us to believe that Tap has the same property in this regard as the other transducer proteins. Partially purified Trg was used to test the ability of relevant RESULTS small molecules to release the transducer from Cibacron blue. In those experiments an excess of protein was applied to the Transducer Proteins Interact with Cibacron Blue-We resin to insure saturation with transducerand thus maximum found that the transducer proteins of E. coli bound strongly to Cibacron blue F3G-A immobilized on Sepharose CL-GB. sensitivity to elution by the test compound. An example is Binding persisted after extensive washing with buffer con- shown in Fig. 2C. Lane 1 contained 20% of the amount of taining 0.2 M NaC1. Transducers could be released from the sample applied to the resin, lane 2 materialnotretained resin by 2 M NaCl or by S-adenosylmethionine (AdoMet) at (including approximately 20% of the Trg applied), and lanes concentrations of 10-40 mM, but not by several other relevant 3-6 material eluted by buffer containing 0, 0.1, 0.4, and 2 M small molecules (Table I). Fig. 2 provides representative ex- NaCl, respectively. Elution of a small amount of Trg by 0.4 amples of experimental results that illustrate this phenome- M NaCl appeared to be an artifact of step gradients, since in ofNaC1, Trg was not non. In each panel the electrophoretic position of the trans- elutionwithacontinuousgradient ducer protein or proteins is indicated by an arrow. As shown released until the NaCl concentration was over 1.5 M. The in Fig. 2A, the entirearray of [3H]methyl-labeledtransducers only eluents that effectively released Trg from Cibacron blue including various electrophoretic forms of Tsr, Tar, Tap, and were salt at high concentrations and AdoMet (Table I). ExTrg was retained by Cibacron blue-Sepharose when chemo- periments with [3H]methyl-labeled cells revealed that other tactically wild-type cells were broken by treatment with ly- transducers were also eluted by AdoMet. As noted previously sozyme and EDTA, the broken cells treated with octyl glu- (Hoffman,1986), the proportion of authentic AdoMet in coside, and thedetergent-soluble extract applied to a column commercially available preparations varied substantially. of the resin, compare lune 1 (soluble extract) with lanes 2 and This plus the instability of the compound resulted in variable 3 (column eluate inlow salt). Application of buffer containing elution of transducers by solutions of AdoMet that should 30 m AdoMet released most of the radiolabeled transducers have been at the same concentrations. In some experiments M (lane 4) and a buffer with 2 M NaCl eluted the remainder 10 mM was sufficient to elute most retained transducer; in (lane 5). Since all electrophoretic forms of [3H]methyl-labeled other experiments the apparent concentration required was transducers exhibited a similar pattern of retention and elu- as high as 40 mM. In any case, the effect was specific for the tion, it appeared that all four of the transducer proteins were chemical nature of AdoMet, since Trg was not eluted by a bound and released. Interaction with Cibacron blue and elu- higher concentration (200 mM)of NaCl nor by equivalent tion by AdoMet and by 2 M NaCl were demonstrated individ- concentrations of analogs like S-adenosylhomocysteine or ually for [3H]methyl-labeled Tsr and Tar by using mutant methionine-S-methyl sulfonium chloride, which like AdoMet cells deleted of all but the specific transducer gene, for t3%] is cationic. However, AdoMet released not only transducers, methionine-labeled Tsr and Trg cells with chromosomal but essentially all proteinsretained by the resin. General using deletions in all transducer genes but harboring a multicopy elution by AdoMet of proteins bound to blue-Sepharose has plasmid carrying the specific transducer gene, and for unmod- been noted by others (Borczuk et al., 1987). This phenomenon ified Trg using plasmid-containing cells that also contained means that elution by AdoMet does not necessarily reflect deletions in genes for the transducers, the methyl transferase, the existence of a specific binding site for the molecule on the transducers. TABLEI We found that thecytoplasmic domain of Trg, independent Release of Trg from Cibacron blue-Sephnrose of the rest of the transducer, interacted with Cibacron blue. For mixtures of elutants, each compoundwas at the indicated A truncated trg was constructed that coded for the entire concentration. cytoplasmic domain with the exception of the 2 arginines that Concen- Release of mark the end of the second membrane-spanning region and Elutant tration Trg the substitution of methionine for isoleucine at thefollowing mM position. The product of this genewas retained by blueNaCl 200 Sepharose and released by 2 M but not by 200 m NaC1, M NaCl 2000 exhibiting the same pattern as the intact transducer (Fig. ATP 25 2 D ) . Thus, theinteraction of transducers with Cibacron blue ATP + GTP 25 occurs at least through the cytoplasmic domain, presumably GTP + CTP + UTP 9 cAMP 5 via a region of conserved structure. In the experiment shown cAMP + cGMP 25 in Fig. 20, the cells used had alow content of the cytoplasmic NAD+ + NADP+ 25 domain, and thus, itwas necessary to utilize immunoblotting S-Adenosylmethionine 10 to detect the Trg polypeptide among other proteins. Not all S-Adenosylhomocysteine 25 the cytoplasmic domain was retained by blue-Sepharose. We S-Adenosylethionine 25 Methionine do not know the reason for this, but it could reflect the 25 Adenosine 25 presence of many other proteins that interacted with the resin Methionine-S-methyl sulfonium chloride 100 and thus saturated it a proportion of the Trg polypeptide or Folinic acid 42 incapable of binding, perhaps because it was not in the native

17312

Purification of a Chemotactic Transducer

A
1 2 3 4 5

61-

I+
C

97.

5561-

42-

5 5-

9 7,

6 1.

+
3629-

55.
42.

+
f

36.

29-

FIG.2. 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 analyzed by 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-labeledtransducers. 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 O w l . Material M eluted by two washes with 10 volumes of buffer is shown in lanes 2 and 3, by 30 m 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-labeled Tsr. 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 volumes of 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 m NaCl in lane 4, by 400 mM NaCl in lane 5, and by 2 M NaCl in lane 6. D, M cytoplasmic domain of Trg. Analysis was with a 12% polyacrylamide gel and immunoblotting with anti-Trg serum and a peroxidase-linked second antibody. The sample applied to the resin (lane 1 ) was the soluble fraction of a detergent extract of lysed cells of HB1033, a strain that deletions in the chromosomal genes for the modification had enzymes and the transducers but harbored pDD2 which carried the fragment of trg coding for the cytoplasmic domain. Lane 1 represents 37.5% of the material analyzed in the 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 of buffer is shown in lanes 3 and 4 and by 2 M NaCl in lane 5. The heavy and light arrows indicate the position of intact cytoplasmic domain and a smaller proteolytic fragment, respectively.
state. The domain isunstable in uiuo, disappearing rapidly as the result of proteolysis. A distinct fragment of the cytoplasmic domain (indicated by the thin arrow in Fig. 20), presumably the result of proteolysis, was observed in cells containing the truncated trg. It is interesting that thisshortenedform, approximately 7 kDa smaller thantheintact cytoplasmic domain, did not interact with Cibacron blue. Purification of Trg-We utilized the interaction of transducers with Cibacron blue to devise an efficient purification for the Trgprotein. In combination with amplification of the cellular content of Trg by appropriate genetic constructs, the fractionation steps yielded approximately 5 mg of pure Trg protein from 1 liter of cell culture by manipulations that can be completed in a few days. The procedure is described in detail under Experimental Procedures. The protein content and yields at various steps in the purification are illustrated

Purification of a Chemotactic Transducer

in Fig. 3 and Table 11. The result was a protein preparation in which over 98% of the material is intact Trg. In lane 9 of the gel shown in Fig. 3, a sample of this purified material was applied in excess to illustrate that the major contaminants appeared asa series of bands migrating justbelow intact Trg andasdistinctbandsatapparent molecular weights of 116,000, 36,000, and 29,000. All these electrophoretic species were recognized by anti-Trg antibody in immunoblots and thus are likely to be forms of Trg. The 116-kDa band may correspond to a small proportion of dimeric Trg that persists in the conditions electrophoresis, perhaps held together by of a disulfide bond between the single cysteines present on each molecule. The first 10 residues of the 29-kDa polypeptide corresponded to the amino-terminal sequence of Trg (see below) and thus that polypeptide must be an amino-terminal fragment of the protein. We expect that the and 29-kDa 36species are fragmentsof Trg created a proteolytic cut near by

17313

4 2 3 4 5 6 7 8 9
11697 -

61

5542-

36-

29-

FIG. 3. Purification of Trg. The figure isanSDS-polyacrylamide (12%)gel stained with Coomassie Brilliant Blue. Positions are indicated with a value in kilodaltons for the proteins listed in the legend to Fig. 2 plus 8-galactosidase (116). Samples for lanes 1 and for 3-7 represented material from approximately equivalent amounts of starting material. Cells of HB1032, induced for production of Trg (lane 1 ), were broken with a French pressure cell and centrifuged at low speed to separate unbroken cells (lane 2, material from 10-fold more starting material) and a clarified supernatant (lane 3 ) . The supernatant was centrifuged at high force to separate soluble material (lane 4 ) from membrane (lane 5 ) . Pelleted material was treated with detergent and centrifuged at high force to separate solubilized protein (lane 6) from material that remained particulate (lane 7). Trg was separated from other solubilized proteins by elution from Cibacron blue-Sepharose (lane 8 ) and then from a Mono Q anion exchange column (lane 9 ) .

the middle of the polypeptide chain, analogous the to cleavage documented for T a r (Mobray et al., 1985). We presume that the bands justbelow intact Trg are the result proteolysis of at the ends the protein, again also of observed for Tar (Foster et al., 1985). a copy of trg contain Since strains with single chromosomal only approximately150 Trg proteins per cell, the first step in purification was to amplify the cellular content of this transducer. The trg gene was placed under the control of the tac promotor in a multicopy plasmid that also carried the lacIq gene (see Experimental Procedures and 1).Addition of Fig. 1 mM IPTG to a growing culture of cells harboringthis a of plasmid resulted in cellular content Trg 500 times greater than for cells with a normal copy of trg on the chromosome. T o avoid heterogeneity of covalent modification of Trg, the host cells contained deletions in the chromosomal genes for the methyl transferase and demethylase. T o avoid possible contamination with other transducers, the host chromosome also carried deletions in tsr, trg, tar, and tup. Cells induced for Trg were broken in a French press and the membranes separated from soluble material by ultracentrifugation. Cells or membrane could be stored a t -70C for several months without apparent effect on later purification of Trg. As reported for T a r by Foster et al. (1985), Trg was exquisitely sensitive to proteolysis from the moment the membranes were solubilized by detergent, and thus, we followed the lead of those authors by including an array of proteinase inhibitors in thesolubilization buffer. Trg was effectively solubilized by octyl glucoside, resulting in substantial purification from the membrane proteins that remained insoluble (Fig. 3 compare lane 6, detergent-solubilized protein, to lane 7, insoluble material). It should be noted that two major proteins occupied the position of Trg on thegel pattern shown in Fig. 3, lanes 1-7. One was Trg and the was aprotein of approximately other 60 kDa inducedby cellularstress. Our procedure for level high expression of trg under the control of the tac promotor also resulted in increased production of this second protein. We suspect it corresponds to GroEL (Tilly et al., 1985). In any case thetwo proteins were separated by the column fractionations, but comigration meant that yield of Trg at earlier the steps could not be estimated by comparing the intensity of stained bands. Immediately following detergent treatment, the solubilized protein was applied to a column of bluea gradient of NaCl, Sepharose and subsequently eluted in appearing in fractions containing1.6-2 M salt. The resulting preparation of Trg was substantially purified (approximately 80% intact Trg) and was not prone to the degradation observed in the unfractionated material. However, manipulations of this preparation to concentrate and to the salt reduce concentration usually resulted in substantial precipitation of protein and thus loss of Trg (Table 11). We have not yet discovered conditions that avoid these losses. In any case, essentially all remaining contaminants unrelated Trg to could be removed by an ion-exchange column. Preparations of Trg like that shown in Fig. 3, lane 9,were used for preliminary characterization of the purified protein. Properties of Purified Trg-The amino acid composition determinedexperimentallyforthepurifiedproteincorresponded well to the amino acid composition predicted from the nucleotide sequence of trg (Fig. 4). The first 19 residues 10 residues of the 29-kDa fragment, of intact Trg and the first identified by automated aminoacid sequencing, corresponded in both cases to the amino-terminal sequence deduced from the nucleotide sequence of trg (Bollinger et al., 1984). These correspondences indicate that the purified protein was Trg trg (Bollinger and that the postulated translation start site in

17314

Purification of a Chemotactic Transducer

TABLE I1
Purification of Trg receptor protein
Fraction Total protein mg
Trg"

Yield for step

%

Recovery of Trg relative to membrane

Purification factor
~~

-fold

Membraneb Octyl glucoside extract Cibacron blue pool Concentrated Cibacron blue pool Cibacron Dialyzed blue pool Mono Q pool 4.9

5.3'

166 106 22 16' 8.5' 4.9

21.4 17.2 17.2 12.8'

80
100

74 41 92

100 80 80 60 25 23

1.0 1.25 60 . 5.0 4.8 7.8

a peroxidase-linked second antibody. Determined by quantitative immunoblotting with anti-Trg serum and *The starting material represents membrane obtainedfrom 1 L of a culture of cells induced for expression of Trg asdescribed under "Experimental Procedures." Precipitation of protein during this step resulted in of protein. loss
90

AMINO ACID RESIDUE

FIG. 4. Amino acid analysis of purified Trg protein. 80 fig of purified Trg was hydrolyzed in anevacuated sealed ampule with double-distilled 6 N HCI for 24 h at 110 "C. After removal of solvents by vacuum evaporation, the hydrolysate was dissolved in 0.2 N sodium citrate, p H 2.2, and analyzed by the method of Spackman et al.
(1958) using a Beckman 121 MB Automatic Amino Acid Analyzer equipped with a Hewlett Packard Integrator (HP3396A). The open bars indicate average values for duplicate hydrolysates. The dark bars indicate amino acid composition deduced from the gene sequence (Bollinger et QL, 1984) with previously noted corrections (Nowlin et aL, 1987, 1988).

AL

contrasts with Tar, in which the amino terminus appears to be 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 ligand-occupied galactose-binding protein. Characterization of that interaction will be the subject of a separate report. The secondary structure content the Trgtransducer was of investigated by obtaining the circular dichroic spectrum of detergent solubilized purified protein (Fig. 5). The data indicated thatTrg hasa high (>80%) helical content, some random coil, and essentially no detectable @-sheet.The spectrum closely resembles that obtained for Tar (Foster et al., 1985), an observation consistent with a common structure for the two transducer proteins.
DISCUSSION

WAVELENGTH (nm)

FIG. 5. Circular dichroic spectrum of purified Trg protein. Spectra were recorded at 20 "C with a Jasco J-40A spectropolarimeter
using 1-mm cells in a nitrogen-flushed chamber. The instrument was calibrated with 10-camphorsulfonic acid. Purified Trg protein, a t 90 pg/ml as determined by the bicinchoninic acid assay, was analyzed in 50 mM Tris, pH7.2,1.25% octyl glucoside. The datawere corrected using a sample without protein and were expressed as mean residue ellipticity (degree cm2 dm").

et al., 1984) is correct. This is of interest since the deduced precede sequence of the extreme amino terminus Trg differs of from the other transducers in that 16 rather than 6 residues precede the hydrophobic membrane-spanning segment. The initial methionine of Trg is neither cleaved nor modified. This

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

Purification of a Chemotactic Transducer

17315

nantly those that contain or arethought to contain the the sequences of the periplasmic domains (Park and Hazelspecific dinucleotide fold domain (Rossman et al., 1975) char- bauer, 1986;Wolff and Parkinson, 1988,Lee et al., 1988), acteristic of manyproteins that bind nucleotides such as implying a common placement of the binding sites and thus NAD+ and ATP. However, some proteins that bind toCiba- of structural units. These and other suggestions about the cron blue have no known affinity for nucleotides or related structural organization of transducers can now be explored molecules and apparently do not contain a dinucleotide fold using purified protein obtained by the procedures we have (Stellwagen, 1977). In general, the former class of proteins described here. remain bound to a Cibacron blue resinin moderate salt Acknowledgments-We thank John Bollinger for suggesting the concentrations butare released by high salt and the specific by ligand for which the binding site is designed (see the booklet use of Cibacron blue-Sepharose and for constructiong pBB7, David Dutton for construction of pDD2, Gerhard Munske for determining Affinity Chromatography published by Pharmacia). In con- amino acid sequences, and Chris Russell for supplying pCR44. trast, members of thelatter class, with the exception of REFERENCES albumin which is known for its ability to bind manydyes, are released by moderate salt concentrations(Easterday and Ames, P., and Parkinson, J. S. (1988) Cell 55,817-826 Easterday, 1974). The transducers were released from Ciba- Bollinger, J., Park, C., Harayama, S., and Hazelbauer, G. L. (1984) Proc. Nutl. Acud. Sci. U. S. A. 8 1 , 3287-3291 cron blue only by quite high concentrations of NaCl (in fact, this is a primary basis for the extensive purification of Trg Borczuk, A., Stock, A., and Stock, J. (1987) J. Bacterwl. 169,32953300 accomplished by the Cibacron blue column), and thus, these Boyd, A., Kendall, L., and Simon, M. I. (1983) Nature 301,623-626 proteins may belong to thegroup of polypeptides that bind to Easterday, R. L., and Easterday, I. M. (1974) in Immobilized Biochemthe dye because of a dinucleotide fold, perhaps designed for icals and Affinity Chromatography (Dunlap, R. B., ed) pp. 123-134, binding a specific small molecule. However,among the potenPlenum Publishing Corp. tial ligands tested only AdoMet caused release and itappears Engstrom, P., and Hazelbauer, G. L. (1980) Cell 2 0 , 165-171 that thismolecule causes generalized release of proteins from Foster, D. L., Mowbray, S. L., Jap, B. K., and Koshland, D. E., Jr. (1985) J. BWl. Chem. 2 6 0 11706-11710 Cibacron blue (Borczuk et al., 1987). Attempts to detect direct Harayama, S., Engstrom, P., ,Wolf-Watz, H., Iino, T., and Hazelbauer, binding of radiolabeled AdoMet to purified Trg have been G. L. (1982) J. Bacteriol. 152,372-383 unsuccessful. In any case, binding to Cibacron blue occurs at Hazelbauer, G. L., and Engstrom, P. (1981) J. Bucteriol. 1 4 5 , 35-42 minimum through the cytoplasmic domain of the transducers Hazelbauer, G. L. (1988) Can. J. Microbwl. 34,466-474 and thus is likely to involve some of the many residues Hess, J. F.,Oosawa,K., Matsumura, P., and Simon, M. I. (1987) Proc. Natl. Acud. Sci. U. S. A. 84, 7609-7613 conserved in this domain among the four transducers. The Hess, J. F., Oosawa, K., Kaplan, N., and Simon, M. 1. (1988) Cell 5 3 , truncated form of the Trg cytoplasmic domain, with an ap79-87 parent molecular weight approximately 7000 less than the Hoffman, J. L. (1986) Biochemistry 25,4444-4449 intact domain did not bind Cibacron blue, a behavior also Hunkapiller, M. W., Lujan, E., Ostrander, F., and Hood, L. E. (1983) Methods Enzymol. 9 1 , 227-236 noted for a fragment of the Tarcytoplasmic domain which is missing 50 amino-terminal residues of that domain (Kaplan Kaplan, N., and Simon, M.I. (1988) J.Bucteriol. 170,5134-5140 Kehry, M. R., and Dahlquist, F. W. (1982) J.Biol. Chem. 257,10378and Simon, 1988). Thus,it appears that the polypeptide 10386 segment immediately following the second membrane span- Krikos, A., Mutoh, N., Boyd, A., and Simon, M. I. (1983) Cell 3 3 , ning-region is crucial for Cibacron blue binding by transducers 615-622 although this does not necessarily mean that thebinding site Lee, L., Mizuro, T., and Imae, Y. (1988) J.Bacteriol. 170,4769-4774 itself occurs in this region. The function of this segment is LeGendre, N., and Matsudaira, P. (1988) Biotechniques 6 , 154-156 D. L.. . , not well defined but the occurrence of mutational substitu- Mowbrav,iS.oL.,i Foster. 7 1 8 and Koshland. D. E.., Jr. (1985) J. Biol. chem- s , 1n-i1 tions that lock the transducer in a particular signaling state Nowlin. D. M.. Nettleton. D. 0.. Ordal. G. W.. and Hazelbauer. G. L. led Ames and Parkinson (1988) to suggest that the segment (1985) J. Bucteriol. 1 6 3 , 2621266 is involved in the functional linkage between ligand binding Nowlin, D. M., Bollinger, J., and Hazelbauer, G. L. (1987) J. Bwl. Chem. 262,6039-6045 and intracellular excitation. Nowlin, D. M., Bollinger, J., and Hazelbauer, G. L. (1988) Proteins The striking similarity of the circular dichroic spectra of 3,102-112 Tar (Foster et al., 1985) and Trg (Fig. 5) suggests a very Ordal, G., and Adler, J. (1974) J. Bucteriol. 117, 509-516 similar content of secondary structural units in the proteins Park, C., and Hazelbauer, G. L. (1986) J. Bucteriol. 168, 1378-1383 and may well reflect a similar three-dimensional structure. Rossman, M. G., Liljas, A., Branden, C. I., and Banaszak, L. J. (1975) in The Enzymes (Boyer, P. D., ed) Vol. XI, 3rd Ed., pp. 62-94, Such similarities would be predicted from the conserved priAcademic Press, New York mary sequences of the respective cytoplasmic domains, but Russo, A. F., and Koshland, D. E., Jr. (1983) Science 220,1016-1020 not from the primary sequences of the two periplasmic do- Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. mains, in which the number of identical residues is not 30,1190-1206 significantly greater than chance. However, recent genetic Stellwagen, E. (1977) Acc. Chem. Res. 1 0 , 92-98 data imply that the periplasmic domains of Tsr, Tar, and TrgTerwilliger, T. C., Wang, J. Y . and Koshland, D. E., Jr. (1986) J. Bwl. Chem. 2 6 1 , 10814-10820 have similar organizations, even in the absence of significant Tilly, K., Chandrasekhar, G. N., Zylicz, M., and Georgopoulous, C. identities in primary sequence between Trg and the other P. (1985) in Microbiology 1985 (Leive, L., ed) pp. 322-326, ASM, two. Mutational substitutions that affect ligand binding in Washington, D. C. the three transducers cluster in two similar positions along Wolff, C., and Parkinson, J. S. (1988) J. Bucteriol. 170,4509-4515