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Fatty Acyl-CoA Binding Domain of the Transcription Factor FadR

1998; Elsevier BV; Volume: 273; Issue: 50 Linguagem: Inglês

10.1074/jbc.273.50.33652

1083-351X

Concetta Dirusso, Vadim Tsvetnitsky, Peter Højrup, Jens Knudsen,

Microbial Metabolic Engineering and Bioproduction

The Escherichia colitranscription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRΔ1–167, with a palmitoyl-CoA analogue, 9-p-azidophenoxy[9-3H]nonanoic acid-CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187–195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRΔ1–167 for acyl-CoA. The binding was characterized by a large negative ΔH 0, −16 to −20 kcal mol−1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, K d of 45 and 63 nm and 68 and 59 nm, respectively. The K d for palmitoyl-CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding. The Escherichia colitranscription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRΔ1–167, with a palmitoyl-CoA analogue, 9-p-azidophenoxy[9-3H]nonanoic acid-CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187–195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRΔ1–167 for acyl-CoA. The binding was characterized by a large negative ΔH 0, −16 to −20 kcal mol−1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, K d of 45 and 63 nm and 68 and 59 nm, respectively. The K d for palmitoyl-CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding. Long chain acyl-coenzyme A thioesters (LCACoA) 1The abbreviations used are: LCACoA, long chain acyl-coenzyme A thioesters; HPLC, high pressure liquid chromatography; ES-MS, electrospray-mass spectrometry; [9-3H]APNA-CoA, 9-p-azidophenoxy[9-3H]nonanoyl-CoA; ITC, isothermal titration calorimetry. are critical intermediates in cellular metabolism. These activated fatty acids are required for the biosynthesis of higher lipids, the acylation of proteins, and catabolism of fatty acids (1Faergeman N.J. Knudsen J. Biochem. J. 1997; 323: 1-12Crossref PubMed Scopus (587) Google Scholar). Acyl-CoA plays a critical role in mitochondrial energy metabolism and in certain pathological responses related to disease. LCACoA are important regulatory ligands for a number of enzymes including, for example, acetyl-CoA carboxylase, citrate synthase, glucokinase, the mitochondrial adenine nucleotide translocase, and the uncoupling protein. LCACoA have also been implicated as effectors of vesicular transport and fusion. A number of studies have been directed at elucidating a role for acyl-CoA compounds in the regulation of the activity of transcription factors. However, there has been only indirect evidence that acyl-CoA compounds regulate gene activity of eucaryotes at the level of transcription (2Choi J.Y. Stukey J. Hwang S.Y. Martin C.E. J. Biol. Chem. 1996; 271: 3581-3589Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 3Kamiryo T. Parthasarathy S. Numa S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 386-390Crossref PubMed Scopus (61) Google Scholar). To date, the Escherichia coli FadR protein is the only transcription factor for which there is substantial and convincing evidence that direct binding of LCACoA to the protein prevents DNA binding, transcription activation, and repression (4DiRusso C.C. Vanderhoek J.Y. Frontiers in Bioactive Lipids. Plenum Press, New York1996: 15-22Crossref Google Scholar, 5DiRusso C.C. Heimert T.L. Metzger A.K. J. Biol. Chem. 1992; 267: 8685-8691Abstract Full Text PDF PubMed Google Scholar, 6DiRusso C.C. Metzger A.K. Heimert T.L. Mol. Microbiol. 1993; 7: 311-322Crossref PubMed Scopus (73) Google Scholar, 7Henry M.F. Cronan Jr., J.E. Cell. 1992; 70: 671-679Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 8Raman N. DiRusso C.C. J. Biol. Chem. 1995; 270: 1092-1097Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). FadR is a 239-amino acid protein that regulates the transcription of many unlinked genes and operons encoding proteins required for fatty acid synthesis and degradation. Among the genes directly regulated by FadR are those encoding a specific membrane-associated fatty acid transport protein (FadL), acyl-CoA synthetase, all of the enzymes required for the β-oxidation of fatty acids, two enzymes essential for unsaturated fatty acid biosynthesis, and the repressor of the genes encoding the glyoxylate bypass genes, IclR. The effect of FadR on the level of transcription is caused by its direct binding to DNA in the promoter regions of FadR-responsive genes (5DiRusso C.C. Heimert T.L. Metzger A.K. J. Biol. Chem. 1992; 267: 8685-8691Abstract Full Text PDF PubMed Google Scholar, 6DiRusso C.C. Metzger A.K. Heimert T.L. Mol. Microbiol. 1993; 7: 311-322Crossref PubMed Scopus (73) Google Scholar). This binding in vitro is specifically prevented by long chain fatty acyl-CoA esters and not medium chain acyl-CoA esters or fatty acids (5DiRusso C.C. Heimert T.L. Metzger A.K. J. Biol. Chem. 1992; 267: 8685-8691Abstract Full Text PDF PubMed Google Scholar). The protein binds DNA as a dimer (9Raman N. Black P.N. DiRusso C.C. J. Biol. Chem. 1997; 272: 30645-30650Crossref PubMed Scopus (54) Google Scholar). Our interests lie in understanding the mechanism by which acyl-CoA controls FadR activity. Previous genetic and biochemical analyses have identified amino acid residues in the carboxyl terminus of FadR that are likely to constitute in part the acyl-CoA (CoA presumably) ligand-binding pocket (8Raman N. DiRusso C.C. J. Biol. Chem. 1995; 270: 1092-1097Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Raman N. Black P.N. DiRusso C.C. J. Biol. Chem. 1997; 272: 30645-30650Crossref PubMed Scopus (54) Google Scholar). In the present work, we have further localized the acyl-CoA binding domain by deletion and affinity labeling. Additionally, we have used isothermal titration microcalorimetry to assess acyl-CoA binding to the full-length protein, a His-tagged derivative and an amino-terminal deletion protein. Together these studies contribute to our understanding of FadR-DNA and FadR-acyl-CoA interactions and help to further define the region of FadR involved in forming the acyl-CoA binding pocket. E. coli strains LS1155, fadR fadB-lacZ, and LS1348, fadR fabA-lacZ, were used to assess FadR function in vivo as described previously (6DiRusso C.C. Metzger A.K. Heimert T.L. Mol. Microbiol. 1993; 7: 311-322Crossref PubMed Scopus (73) Google Scholar). BL21(λDE3)/pLysS was used as host for overexpression of FadR proteins (10Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). Plasmid pCD129 was used for the production of full-length wild-type FadR as previously detailed (5DiRusso C.C. Heimert T.L. Metzger A.K. J. Biol. Chem. 1992; 267: 8685-8691Abstract Full Text PDF PubMed Google Scholar). Plasmid pCD307-6 encoding His6-FadR was constructed as follows. An NdeI site was generated at the initiating methionine codon of the wild-type FadR gene by site directed mutagenesis of pCD152 to generate pCD306-6 using the Altered Sites System of Promega as described previously (8Raman N. DiRusso C.C. J. Biol. Chem. 1995; 270: 1092-1097Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). An NdeI–BamHI fragment from pCD306-6 containing the complete coding sequence of FadR was cloned into the T7 RNA polymerase-responsive expression plasmid pET15b (Novagen) such that the coding sequence of FadR was fused in frame at the amino terminus to the His tag in the vector to generate pCD307-6 encoding His6-FadR. Plasmid pCD307-3 encoding FadRΔ1–167 was constructed in a similar manner, but in this case an NcoI site was constructed in pCD152 at Met168 in wild-type FadR to generate pCD306–3. The NcoI–BamHI fragment encoding amino acid residues 168–239 was subcloned from pCD306–3 into the expression plasmid pET3a to generate pCD307–3. All constructions were verified by restriction enzyme analysis and DNA sequencing using promoter T7- and FadR-specific oligonucleotides as primers. Wild type FadR, its truncated mutant FadRΔ1–167, and full-length soluble His6-FadR were overexpressed in E. coliBL21(λDE3)/pLysS harboring the appropriate plasmid using T7 polymerase expression system. Cultures (4 liters) were grown in an Amersham Pharmacia Biotech fermenter with automatic pH control at 37 °C in the previously described medium (11Mandrup S. Højrup P. Kristiansen K. Knudsen J. Biochem. J. 1991; 276: 817-823Crossref PubMed Scopus (57) Google Scholar) containing ampicillin (100 μg/ml) and chloramphenicol (15 μg/ml) untilA 600 was about 4. T7 RNA polymerase was induced by the addition of isopropyl thio-β-d-galactopyranoside to 0.4 mm. Growth continued for 3–4 h, and cells were harvested by centrifugation. For isolation of full-length, native FadR encoded within pCD129, bacterial cells were disrupted in a French press in 300 ml of 20 mm Tris-HCl buffer (pH 8.0), 1 mmdithiothreitol, 1 mm EDTA (TDE buffer), and the homogenate was centrifuged at 70,000 × g for 20 min. The FadR protein was subsequently recovered from inclusion bodies as follows. The pellet was dissolved in the same buffer containing 6 murea, which was then removed by dialysis against TDE buffer containing 20% (v/v) glycerol. A part of the protein that precipitated during urea removal was redissolved and redialyzed. Soluble protein in TDE buffer containing 20% (v/v) glycerol was applied to a Q-Sepharose Fast Flow column preequilibrated with the same buffer, and the protein was eluted with a linear gradient of KCl (to 0.5 m). Fractions containing the protein of interest were pooled and dialyzed against 20 mm ammonium acetate buffer (pH 5.5) containing 1 mm dithiothreitol, 20% (v/v) glycerol. This was applied to an S-Sepharose high performance column, and the protein was eluted with a linear gradient of 1 m KCl in the same buffer at pH 6.5. FadR-containing fractions were pooled and dialyzed against 20 mm Tris-HCl buffer (pH 8.0), 20% (v/v) glycerol. Protein was concentrated by binding to a 5-ml Q-Sepharose High Trap column and eluting with a linear gradient of 0.4 m KCl. Final purification was achieved by gel filtration chromatography on a Sephacryl S-200 HR column developed with 20 mm Tris-HCl buffer (pH 8.0), 200 mm KCl containing 20% (v/v) glycerol. Protein was concentrated by ultrafiltration using a Centriprep 10 concentrator and stored at −20 °C. Protein purity and identity was confirmed by electrospray mass spectrometry (ES-MS) and N-terminal sequencing. For isolation of FadRΔ1–167 encoded in pCD307–3, bacterial cells were disrupted in a French press in 300 ml of TDE buffer, and the homogenate was separated by centrifugation as above. FadRΔ1–167 protein was recovered from inclusion bodies as follows. The pellet was dissolved in the same buffer containing 6 m urea and purified to apparent homogeneity by single step reverse-phase HPLC on a Nucleosil RP18 (230 × 150-mm) column. Prior to injection, the sample was supplemented with trifluoroacetic acid to 0.1%. The column was equilibrated with 0.1% trifluoroacetic acid in water, and protein was eluted with a linear gradient of 96% ethanol containing 0.08% trifluoroacetic acid (0–100% in 50 min). Fractions containing pure protein, as evidenced by the appearance of a single protein band upon 15% SDS-polyacrylamide gel electrophoresis, were freeze-dried. Lyophilized protein was redissolved in 20 mm ammonium acetate buffer (pH 6.0), 1 mm dithiothreitol and stored at −20 °C. Protein purity and identity were further confirmed by ES-MS and N-terminal sequencing. For isolation of His6-FadR encoded within pCD307-6, bacterial cells were disrupted in 200 ml of the binding buffer (50 mm potassium phosphate buffer (pH 8.0), 300 mmNaCl, 1 mm phenylmethylsulfonyl fluoride, 10% glycerol). Most of the target protein (∼90%) was in the soluble fraction after centrifugation. The crude protein preparation was incubated at 4 °C for 2 h with 15 ml of a 50% slurry of nickel-nitrilotriacetic acid-agarose (Qiagen), which had been prewashed several times with the binding buffer. The slurry was then packed into a column and subsequently washed with 20 column volumes of the binding buffer to elute unbound proteins. The target protein was then eluted with 250 mm imidazole in the binding buffer. Due to a high binding capacity of nickel-nitrilotriacetic acid-agarose, eluted protein was so concentrated that some precipitation occurred but disappeared upon dilution. Imidazole was removed by dialysis against the binding buffer. Protein was essentially pure (initiating Met lost) as confirmed by HPLC, ES-MS, and N-terminal sequencing. The preparation was stored at −20 degrees C. Buffer exchange for isothermal titration calorimetry (ITC) experiments was later done using a 5-ml fast desalting High Trap column. The binding of purified His6-FadR to the fadBpromoter in vitro was tested using protein-DNA gel shift assays. A 159-base pair fragment containing the fadBpromoter was amplified by thermocycling using the oligonucleotides BFW (5′-TGATTTCTGCCGAGCGTG-3′) and BRE (5′-AGTCAAGGTACAGGGTGTC-3′) as primers and pCD154 (5DiRusso C.C. Heimert T.L. Metzger A.K. J. Biol. Chem. 1992; 267: 8685-8691Abstract Full Text PDF PubMed Google Scholar) as template. Reactions were cycled 35 times at 94 °C for 1 min; 36 °C for 1 min; and 72 °C for 1 min. The fragment of interest was gel-purified and end-labeled using polynucleotide kinase and [γ-32P]ATP (3000 Ci/mmol). Conditions for gel shifts were as described previously (5DiRusso C.C. Heimert T.L. Metzger A.K. J. Biol. Chem. 1992; 267: 8685-8691Abstract Full Text PDF PubMed Google Scholar). In vivo activity of His6-FadR was assessed by measuring β-galactosidase activities in transformants of strain LS1155 (fadR fadB-lacZ) to test repression and LS1348 (fadR fabA-lacZ) to test activation as described (6DiRusso C.C. Metzger A.K. Heimert T.L. Mol. Microbiol. 1993; 7: 311-322Crossref PubMed Scopus (73) Google Scholar). The plasmids used to transform LS1155 and LS1348 to test FadR activity included pCD129, which encodes wild-type FadR under its native promoter as a positive control; pET15b, the His6 vector, a negative control; pGP1-2, which encodes T7-RNA polymerase under the control of an isopropyl thio-β-d-galactopyranoside-inducible promoter; and pCD307-6, which encodes His6-FadR under the control of a T7-RNA polymerase-responsive promoter. Oleic acid (4 mmol) was dissolved in 40 ml of dioxane, and then 4 ml of 1 m NaOH and 3.2 ml of 2% OsO4 were added. To this mixture, NaIO4(8 g) was added slowly over 5–10 min with stirring, which was continued for 18 h. The solvent was removed by evaporation, and the residue dissolved in water, made alkaline with 2 mNa2CO3, and extracted three times with petroleum spirits (40–60 °C). The aqueous phase was acidified with 4 m HCl and extracted three times with petroleum spirits (40–60 °C)/diethyl ether (1:1). The solvent was evaporated with a stream of nitrogen, and the product 9-oxononanoic acid was dissolved in petroleum spirits/diethyl ether (4:1) and purified on a silica gel column. The yield was 121 mg (21%). The 9-oxononanoic acid was dissolved in 8 ml of absolute ethanol titrated to pH 12 with 2m NaOH and reduced with NaBH4 in two steps. NaBH4 (1.9 g) was dissolved in 1 ml of absolute ethanol and mixed with 750 mCi of [3H]NaBH4 (specific activity 20–40 Ci/mmol, Amersham, UK) dissolved in 1 ml of ethanol. This solution was added slowly over 30 min to the 9-oxononanoic acid solution under continuous stirring. The stirring was continued for 90 min, an additional 5.9 mg of NaBH4 was added, and stirring continued for another 3 h. The solvent was removed with a stream of nitrogen; 10 ml of water was added; the solution was acidified with HCl; and the 9-hydroxy[9-3H]nonanoic acid was extracted with diethyl ether. The ether phase was dried over MgSO4for 6 h. The yield was 131 mg. The 9-hydroxy[9-3H]nonanoic acid was methylated with diazomethane (12Christie W.W. Lipid Analysis. 2nd Ed. Pergamon Press, New York1982: 54-55Google Scholar). The methylated product was dried carefully with benzene and dissolved in 1 ml of dry pyridine and placed on ice; 315 mg of dry tosylchloride was added, and the reaction was allowed to continue overnight with stirring. The reaction mixture was acidified with 2 ml of 1 m HCl and extracted three times with 10 ml of diethyl ether. The combined extracts were washed twice with 5 ml of 1 m HCl and dried with MgSO4; the solvent was evaporated with nitrogen; and the product was dissolved in petroleum spirit and purified on a silica gel column. The yield was 132 mg of 9-tosyl[9-3H]nonanoic acid methyl ester. All the following synthetic steps were carried out under dim light.p-Azidophenol (111 mg) was dissolved in 1 ml of ethanol, 44 mg of sodium methoxide was added, and then the solvent was evaporated in a vacuum centrifuge over night. The dry residue was dissolved in 1.5 ml of dry hexamethyl formamide and added to the dried 9-tosyl[9-3H]nonanoic acid methyl ester. The reaction was allowed to run for 5 h at room temperature with stirring. The product 9-p-azidophenoxy[9-3H]nonanoic acid methyl ester was purified on a silica gel column. The fractions containing the product were pooled, and the solvent was evaporated with nitrogen. The dry residue was dissolved in 7 ml of NaOH, and the methyl ester was extracted with diethyl ether. The yield was 77 mg. The 9-p-azidophenoxy[9-3H]nonanoic acid methyl ester was dissolved in 3 ml of ethanol and hydrolyzed with 1 ml of 1m HCl for 6 h at room temperature, 2 ml of water was added, and the free acid was extracted with diethyl ether. The ether phase was dried with MgSO4, the solvent was evaporated with nitrogen, and the product was redissolved in petroleum spirits. Purity was evaluated by thin layer chromatography on silica gel 60 HP-TLC (using petroleum spirit (40–60 °C)/diethyl ether, 1:1). The specific radioactivity was determined to be 870 Ci/mol using the value of 10600 m−1 cm−1 for the molecular extinction coefficient at 257 nm. 9-p-Azidophenoxy[9-3H]nonanoyl-CoA was synthesized as described previously (13Rosendal J. Ertbjerg P. Knudsen J. Biochem. J. 1993; 290: 321-326Crossref PubMed Scopus (135) Google Scholar). All experiments with light-sensitive reagents were performed in dim light. For photoaffinity labeling experiments, FadR or its truncated derivative FadRΔ1–167 was incubated for 3–5 min on ice with [9-3H]APNA-CoA (873 Ci/M) at a molar ratio of 1:1.25 in a final volume of 100 μl in TBE buffer (pH 8.0) followed by UV illumination (300 nm) for 30 s. For competition experiments, a 5-fold excess of palmitoyl-CoA over photoaffinity ligand was included in the reaction. Ten microliters of the reaction mixture were subjected to 15% SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Blue, soaked in Amplify reagent, dried, and fluorographed. For peptide mapping, radiolabeled protein was HPLC-purified from nonreacted [9-3H]APNA-CoA on a Dynasphere column (8 × 60 mm) with a 2-propanol linear gradient. This column material was found to provide superior recovery of both protein and very hydrophobic peptides. Label incorporation efficiency was estimated to be 15%. Photoaffinity-labeled protein was dissolved in 50 mm Tris (pH 7.4), 6 m urea and digested with trypsin (2%, w/w) after dilution to bring urea concentration to ∼0.5 m. Peptides were resolved on a Dynasphere column preequilibrated with 0.1% trifluoroacetic acid and developed with a linear gradient of acetonitrile containing 0.08% trifluoroacetic acid (0–70% in 40 min). Poorly resolved peaks containing most of the radioactivity were rechromatographed on the same column using a shallower gradient, and individual peptides were collected and sequenced. Calorimetric measurements were carried out using an OMEGA titration microcalorimeter from MicroCal, Inc. (Northampton, MA). This instrument has been described in detail by Wiseman et al. (14Wiseman T. Williston S. Brandts J.F. Lin L. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2438) Google Scholar). The reference cell was filled with water containing 0.02% sodium azide. The calorimeter was electrically calibrated at each temperature. All solutions used for the experiments were thoroughly degassed by stirring under vacuum. If necessary, protein solutions were spun for several minutes in a bench top centrifuge to remove any visible particles. The concentration of the protein was estimated spectrophotometrically at 280 nm using ε = 33060m−1 cm−1 for FadR and His6-FadR and 12,210 m−1cm−1 for FadRΔ1–167 and was approximately 0.03 mm. Protein under study in appropriate buffer was placed in the sample cell, and a ligand (dissolved in the same buffer as the protein) was drawn into the injection syringe, which was then mounted into a stepper motor for delivery into the sample cell. The syringe with stirrer paddle was rotated at 400 rpm during the experiment to assure immediate mixing. Experiments were performed at a temperature of 27 or 31 °C. The concentration of the ligand, about 0.5 mm, was chosen to ensure full saturation well before final injection. Appropriate blank runs were conducted and subtracted from the corresponding data. The peaks of the thermograms obtained in this manner were integrated using the ORIGIN software supplied with the instrument. A nonlinear regression fitting to the isotherm was done using the CALREG (version 3.0) program (15Sigurskjold B.W. Berland C.R. Svensson B. Biochemistry. 1994; 33: 10191-10199Crossref PubMed Scopus (84) Google Scholar). The fitting procedure yields the binding constant of the ligand K a, the heat of binding H, and the concentration of the binding sites (stoichiometry) N. Electrospray mass spectra of the proteins were recorded on a Vestec instrument (Vestec Corp., Houston, TX). Buffers and salts were removed from the purified proteins by HPLC. The sample in aqueous buffer was loaded onto a 60 × 8-mm Dynasphere column and then stepwise eluted with a mixture of 90% acetonitrile, 10% water, and 0.08% trifluoroacetic acid. The sample was dried and brought to a final concentration of ∼20 pmol/μl with methanol/water (1:1, v/v) containing 1% acetic acid. The sample was introduced into the mass spectrometer by infusion with a syringe pump with a flow rate of 0.3 μl/min. Spectra were acquired in the positive ion mode at 10 s/scan and mass window of m/z 600–1500 using Teknivent Vector 2 data system. The molecular mass of the protein was calculated by weighted averaging as described by Mann et al. (16Mann M. Meng C.K. Fenn J.B. Anal. Chem. 1989; 61: 1702-1708Crossref Scopus (543) Google Scholar). The spectrometer was independently calibrated using myoglobin. Purified proteins and radiolabeled peptides were sequenced on a Knauer 910 pulsed liquid sequencer with chemicals and program as recommended by the manufacturer. Samples of 20 μl of the amino acid phenylthiohydantoin derivatives were used for amino acid identification on a Knauer on-line HPLC 64 using a 250 × 4-mm Lichrosphere 100 C-18 (5-mm particle size) column and a gradient of acetonitrile in 50 mm sodium acetate buffer, pH 5.2, as described by the manufacturer. All protein purification was done on an FPLC system (Amersham Pharmacia Biotech) with columns supplied by the same manufacturer. HPLC separations were performed using the Kontron system with a gradient former equipped with a double wavelength detector (used at 216 and 280 nm). A Knauer 8 × 60-mm column was packed with Dynasphere PD-102-RE monosized particles (from Dyna Particles, Lillestrøm, Norway). Nucleosil ODS, 10-μm particle size, 30-μm pore size, was from Machery Nagel (Duren, Germany). Propan-2-ol, acetonitrile (both HPLC grade), and trifluoroacetic acid (trifluoroacetic acid, gas phase sequenator grade) were from Rathburn (Walkerburn, Scotland). l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (EC 3.4.21.4) was from Sigma. Fluorographic reagent Amplify was from Amersham. Other reagents were obtained from commercial sources and were of reagent grade. Water was of Milli-Q quality. All acyl-CoA esters were synthesized from corresponding fatty acids as described by Sanchez et al.(17), and their purity was checked by HPLC on a Nucleosil ODS column. Restriction enzymes, T4 DNA ligase, T4 DNA polymerase, and Sequenase version 2.0 were purchased from U.S. Biochemical Corp. [α-35S]dATP and [γ-32P]ATP were purchased from NEN Life Science Products. In previous work, we identified by random and site-directed mutagenesis a region in FadR including amino acids 216–228 of the 239-amino acid protein that was specifically required for acyl-CoA binding (8Raman N. DiRusso C.C. J. Biol. Chem. 1995; 270: 1092-1097Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This led us to hypothesize that the acyl-CoA binding domain was located toward the carboxyl terminus of the protein and that the acyl-CoA binding domain might be structurally and functionally separable from the amino-terminal DNA binding domain of FadR (9Raman N. Black P.N. DiRusso C.C. J. Biol. Chem. 1997; 272: 30645-30650Crossref PubMed Scopus (54) Google Scholar, 18DiRusso C.C. Nucleic Acids Res. 1988; 16: 7995-8009Crossref PubMed Scopus (24) Google Scholar). Therefore, to further localize and analyze the acyl-CoA binding domain, we have constructed pCD307-3, which overexpresses a protein made up of amino acids 168–239. Amino acid 168 was chosen, since it is an internal methionine that would make a convenient site for translational initiation (18DiRusso C.C. Nucleic Acids Res. 1988; 16: 7995-8009Crossref PubMed Scopus (24) Google Scholar). The protein encoded within pCD307–3, called FadRΔ1–167, has been purified to apparent homogeneity as evidenced by SDS-polyacrylamide gel electrophoresis and ES-MS (data not shown). Dimerization of a portion of the purified FadRΔ1–167 protein was observed when the protein was analyzed by ES-MS, and dimerization could be prevented by dithiothreitol treatment. Partial N-acetylation was also suggested by ES-MS. Both full-length FadR and FadRΔ1–167 were photoaffinity labeled under identical conditions with [9-3H]APNA-CoA. [9-3H]APNA-CoA was previously shown to mimic palmitoyl-CoA in binding to acyl-CoA-binding protein, and the labeled peptides identified in that study were later shown by NMR to indeed be involved in acyl-CoA binding (19Kragelund B.B. Andersen K.V. Madsen J.C. Knudsen J. Poulsen F.M. J. Mol. Biol. 1993; 230: 1260-7719Crossref PubMed Scopus (123) Google Scholar). In the present work, we were able to show that [9-3H]APNA-CoA can photoaffinity-label FadR, and this cross-linking was prevented by an excess of palmitoyl-CoA (Fig. 1). No labeling of the protein was detected when it was incubated with [9-3H]APNA-CoA without subsequent illumination. Similar labeling experiments in the presence of delipidated bovine serum albumin (which binds acyl-CoA with a K d of ∼0.5 μm) showed that FadR could be labeled in the presence of a 2-fold excess of bovine serum albumin and therefore that FadR competes effectively with bovine serum albumin for the [9-3H]APNA-CoA (data not shown). The radiolabeled proteins were digested with trypsin in order to identify peptides cross-linked to [9-3H]APNA-CoA. Since the affinity label is at the ϖ-end of the acyl chain, the amino acid residues within a labeled tryptic peptide are expected to be part of a hydrophobic pocket within FadR that binds long chain acyl-CoA. Additionally, some of the amino acid residues within the labeled peptide may make specific contacts with the acyl chain. A tryptic map of full-length FadR photo-cross-linked to [9-3H]APNA-CoA is shown in Fig. 2. The peptides generated by digestion with trypsin were separated by HPLC on a Dynasphere column preequilibrated with 0.1% trifluoroacetic acid and developed with a linear gradient of acetonitrile with 0.08% trifluoroacetic acid (0–70% in 40 min) (Fig.3 A). The major amount of radioactivity was eluted in