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• W2037953375 abstract "Pheromone-binding proteins (PBPs), located in the sensillum lymph of pheromone-responsive antennal hairs, are thought to transport the hydrophobic pheromones to the chemosensory membranes of olfactory neurons. It is currently unclear what role PBPs may play in the recognition and discrimination of species-specific pheromones. We have investigated the binding properties and specificity of PBPs fromMamestra brassicae (MbraPBP1), Antheraea polyphemus (ApolPBP1), Bombyx mori (BmorPBP), and a hexa-mutant of MbraPBP1 (Mbra1-M6), mutated at residues of the internal cavity to mimic that of BmorPBP, using the fluorescence probe 1-aminoanthracene (AMA). AMA binds to MbraPBP1 and ApolPBP1, however, no binding was observed with either BmorPBP or Mbra1-M6. The latter result indicates that relatively limited modifications to the PBP cavity actually interfere with AMA binding, suggesting that AMA binds in the internal cavity. Several pheromones are able to displace AMA from the MbraPBP1- and ApolPBP1-binding sites, without, however, any evidence of specificity for their physiologically relevant pheromones. Moreover, some fatty acids are also able to compete with AMA binding. These findings bring into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal compounds. Pheromone-binding proteins (PBPs), located in the sensillum lymph of pheromone-responsive antennal hairs, are thought to transport the hydrophobic pheromones to the chemosensory membranes of olfactory neurons. It is currently unclear what role PBPs may play in the recognition and discrimination of species-specific pheromones. We have investigated the binding properties and specificity of PBPs fromMamestra brassicae (MbraPBP1), Antheraea polyphemus (ApolPBP1), Bombyx mori (BmorPBP), and a hexa-mutant of MbraPBP1 (Mbra1-M6), mutated at residues of the internal cavity to mimic that of BmorPBP, using the fluorescence probe 1-aminoanthracene (AMA). AMA binds to MbraPBP1 and ApolPBP1, however, no binding was observed with either BmorPBP or Mbra1-M6. The latter result indicates that relatively limited modifications to the PBP cavity actually interfere with AMA binding, suggesting that AMA binds in the internal cavity. Several pheromones are able to displace AMA from the MbraPBP1- and ApolPBP1-binding sites, without, however, any evidence of specificity for their physiologically relevant pheromones. Moreover, some fatty acids are also able to compete with AMA binding. These findings bring into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal compounds. pheromone-binding protein odorant-binding protein Mamestra brassicae Antheraea polyphemus Antheraea pernyi polymerase chain reaction 1-amino-anthracene cetyl alcohol (Z)-11-hexadecen-1-ol palmitic acid palmitoleic acid oleic acid (Z)-11-hexadecenal Z11-C16-Ald, (E6,Z11)-hexadecadienal (Z)-11-hexadecenyl-1-acetate Z11-C16-Ac, (E6,Z11)-hexadecadienyl-1-acetate Z9-C14-Ac, (E4,Z9)-tetradecadienyl-1-acetate polyacrylamide gel electrophoresis In the context of molecular recognition, the perception of pheromones by male moths is a system that shows both high specificity and significant affinity, resulting in mating within the same species and with the response being elicited by relatively low concentrations of pheromone. In a generally recognized scheme, the hydrophobic pheromone molecules enter the antennal sensilla (sensory hairs) through pores in the cuticle and traverse the aqueous sensillum lymph transported by low molecular mass (15–17 kDa) pheromone-binding proteins (PBPs),1 finally reaching receptors located in the dendritic membranes of the olfactory neurons, where they are recognized either free or in association with the PBP (1Vogt R.G. Riddiford L.M. Nature. 1981; 293: 161-163Crossref PubMed Scopus (893) Google Scholar, 2Vogt R.G. Riddiford L.M. Prestwich G.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8827-8831Crossref PubMed Scopus (286) Google Scholar, 3Ziegelberger G. Eur. J. Biochem. 1995; 232: 706-711Crossref PubMed Scopus (68) Google Scholar, 4Pelosi P. J. Neurobiol. 1996; 30: 3-19Crossref PubMed Scopus (203) Google Scholar, 5Kaissling K.E. Chem. Senses. 1996; 21: 257-268Crossref PubMed Scopus (109) Google Scholar).PBPs are present in the sensillum lymph at millimolar concentrations and were originally identified by their ability to bind radiolabeled compounds of the pheromone blend (1Vogt R.G. Riddiford L.M. Nature. 1981; 293: 161-163Crossref PubMed Scopus (893) Google Scholar) and by their N-terminal sequence (6Gyorgyi T.K. Roby-Shemkovitz A.J. Lerner M.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9851-9855Crossref PubMed Scopus (123) Google Scholar). The primary structures of PBPs from various moth species, established from molecular cloning (7Raming K. Krieger J. Breer H. FEBS Lett. 1989; 256: 215-218Crossref PubMed Scopus (98) Google Scholar, 8Prestwich G.D. Du G. LaForest S. Chem. Senses. 1995; 20: 461-469Crossref PubMed Scopus (115) Google Scholar, 9Krieger J. Raming K. Breer H. Biochim. Biophys. Acta. 1991; 1088: 277-284Crossref PubMed Scopus (94) Google Scholar, 10Krieger J. Ganssle H. Raming K. Breer H. Insect Biochem. Mol. Biol. 1993; 23: 449-456Crossref PubMed Scopus (119) Google Scholar, 11Krieger J. von Nickisch-Rosenegk E. Mameli M. Pelosi P. Breer H. Insect Biochem. Mol. Biol. 1996; 26: 297-307Crossref PubMed Scopus (187) Google Scholar, 12Maibeche-Coisne M. Jacquin-Joly E. Francois M.C. Nagnan-Le Meillour P. Insect Biochem. Mol. Biol. 1998; 28: 815-818Crossref PubMed Scopus (39) Google Scholar, 13Meritt T.J. Laforest S. Prestwich G.D. Quattro J.M. Vogt R.G. J. Mol. Evol. 1998; 46: 272-276Crossref PubMed Scopus (46) Google Scholar, 14Robertson H.M. Martos R. Sears C.R. Todres E.Z. Walden K.K. Nardi J.B. Insect Mol. Biol. 1999; 8: 501-518Crossref PubMed Scopus (205) Google Scholar), have indicated diversity among PBPs and their classification into a large family of insect pheromone/odorant-binding proteins, which also includes two classes of general odorant-binding proteins 1 and 2 (10Krieger J. Ganssle H. Raming K. Breer H. Insect Biochem. Mol. Biol. 1993; 23: 449-456Crossref PubMed Scopus (119) Google Scholar, 11Krieger J. von Nickisch-Rosenegk E. Mameli M. Pelosi P. Breer H. Insect Biochem. Mol. Biol. 1996; 26: 297-307Crossref PubMed Scopus (187) Google Scholar, 15Vogt R.G. Rybczynski R. Lerner M.R. J. Neurosci. 1991; 11: 2972-2984Crossref PubMed Google Scholar), antennal-binding protein Xs (11Krieger J. von Nickisch-Rosenegk E. Mameli M. Pelosi P. Breer H. Insect Biochem. Mol. Biol. 1996; 26: 297-307Crossref PubMed Scopus (187) Google Scholar, 14Robertson H.M. Martos R. Sears C.R. Todres E.Z. Walden K.K. Nardi J.B. Insect Mol. Biol. 1999; 8: 501-518Crossref PubMed Scopus (205) Google Scholar, 16Krieger J. Mameli M. Breer H. Invert Neurosci. 1997; 3: 137-144Crossref PubMed Scopus (32) Google Scholar), and antennal-binding proteins (14Robertson H.M. Martos R. Sears C.R. Todres E.Z. Walden K.K. Nardi J.B. Insect Mol. Biol. 1999; 8: 501-518Crossref PubMed Scopus (205) Google Scholar). Insect PBPs and OBPs are α-helical proteins (17Sandler B.H. Nikonova L. Leal W.S. Clardy J. Chem. Biol. 2000; 7: 143-151Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), thus completely different from mammalian OBPs which belong to the lipocalin superfamily and have a β-barrel fold (18Tegoni M. Ramoni R. Bignetti E. Spinelli S. Cambillau C. Nat. Struct. Biol. 1996; 3: 863-867Crossref PubMed Scopus (189) Google Scholar, 19Spinelli S. Ramoni R. Grolli S. Bonicel J. Cambillau C. Tegoni M. Biochemistry. 1998; 37: 7913-7918Crossref PubMed Scopus (106) Google Scholar, 20Tegoni M. Pelosi P. Vincent F. Spinelli S. Campanacci V. Grolli S. Ramoni R. Cambillau C. Biochim. Biophys. Acta. 2000; 1482: 229-240Crossref PubMed Scopus (205) Google Scholar).Although molecular cloning has rapidly increased the information available on moth PBPs primary structures, cloned genes have not greatly increased our knowledge of their binding affinities and specificities. Heterologously expressed PBPs from Antheraea polyphemus (ApolPBP1), Antheraea pernyi (AperPBP1 and -2), and Lymantria dispar (LdispPBP1) have already been used to develop quantitative binding assays with photoactivable derivatives of the pheromone or radioactive pheromones (8Prestwich G.D. Du G. LaForest S. Chem. Senses. 1995; 20: 461-469Crossref PubMed Scopus (115) Google Scholar, 21Plettner E. Lazar J. Prestwich E.G. Prestwich G.D. Biochemistry. 2000; 39: 8562-8953Crossref Scopus (165) Google Scholar, 22Krieger J. Raming K. Prestwich G.D. Frith D. Stabel S. Breer H. Eur. J. Biochem. 1992; 203: 161-166Crossref PubMed Scopus (29) Google Scholar, 23Prestwich G.D. Protein Sci. 1993; 2: 420-428Crossref PubMed Scopus (87) Google Scholar, 24Du G. Prestwich G.D. Biochemistry. 1995; 34: 8726-8732Crossref PubMed Scopus (174) Google Scholar). However, synthesis of photoactivable or radioactive pheromones is difficult and limits the candidate compounds tested, usually to the main components of pheromone blends identified in behavioral studies.In the present study, we have examined by fluorescence the binding affinity of 1-aminoanthracene (AMA) to recombinant PBPs of A. polyphemus (ApolPBP1), Mamestra brassicae (MbraPBP1),Bombyx mori PBP (BmorPBP), and a mutant of MbraPBP1 (Mbra1-M6) mutated at residues in the internal cavity to mimic the cavity found in B. mori PBP. The results show that AMA binds with high affinity to ApolPBP1 and MbraPBP1, but neither BmorPBP nor Mbra1-M6. The comparison of the binding behavior of MbraPBP1 and Mbra1-M6 indicates that AMA binds in the internal cavity of PBPs. The fluorescence of bound AMA was used to follow and quantify the binding of several pheromonal compounds, taking advantage of the ability of pheromones to displace AMA from the binding site. The dissociation constants estimated demonstrate a high affinity of MbraPBP1 and ApolPBP1 both for pheromones and fatty acids. The binding data reveal that the interaction of pheromones with binding sites of the two PBPs examined by AMA fluorescence shows little specificity.EXPERIMENTAL PROCEDURESPheromones and AnaloguesPheromones and analogues were purchased from Chemtech B.V. (The Netherlands) and Sigma, and stored as specified by the manufacturers. AMA was from Fluka. All alcoholic solutions were freshly prepared.Subcloning in pET22b(+) Vector and ExpressionEscherichia coli strain XL1-Blue was used for DNA subcloning and propagation of the recombinant plasmid. TheMbraPBP1 gene was amplified by PCR using pQE30/PBP1 as template (25Campanacci V. Longhi S. Nagnan-Le Meillour P. Cambillau C. Tegoni M. Eur. J. Biochem. 1999; 264: 707-716Crossref PubMed Scopus (41) Google Scholar) with the following primers: Mbra1-MscI 5′-CGAGTAAAGAACTGATCACG-3′; Mbra1-NotI 5′-CCGGGCGGCCGCCTACACGGCCGTCATGATCTC-3′. The amplified PCR fragment was purified and digested with MscI and NotI before being cloned between the same restriction sites of the pET22b(+) vector (Novagen). Recombinant MbraPBP1 was produced by growing E. coli BL21(DE3) transformed with the recombinant pET22b(+)/MbraPBP1 plasmid at 37 °C in LB medium supplemented with 50 μg/ml carbenicillin. Cultures were induced with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside, and the temperature was decreased to 28 °C when A600reached the value of 0.8.The gene of ApolPBP1 was amplified, subcloned, and expressed in a similar way. PCR was performed using the primers: ApolPBP1-MscI 5′-CTTCGCCAGAGATCATGAAGAAT-3′ and ApolPBP1-XhoI 5′-AGACACTCGAGCAATCTAAACTTCAGCTA-3′. AnApolPBP1 cDNA clone (Apo-3 (7Raming K. Krieger J. Breer H. FEBS Lett. 1989; 256: 215-218Crossref PubMed Scopus (98) Google Scholar)) was used as template. The fragment obtained by PCR was purified, digested by XhoI, and cloned into the MscI and XhoI restriction sites of the pET22b(+) vector. The recombinant pET22b(+)/ApolPBP1 plasmid was transformed into E. coli BL21(DE3) and expression was performed following the protocol of Wojtasek and Leal (26Wojtasek H. Leal W.S. J. Biol. Chem. 1999; 274: 30950-30956Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) by growing the cells without induction at 28 °C in LB medium supplemented with 50 μg/ml carbenicillin untilA600 reaches a value >2.5. The gene of BmorPBP was amplified, subcloned, and expressed as described by Wojtasek and Leal (26Wojtasek H. Leal W.S. J. Biol. Chem. 1999; 274: 30950-30956Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar).Site-directed Mutagenesis of MbraPBP1Six residues of MbraPBP1, Ile-5, Met-8, Ala-56, Leu-61, Ile-62, and Met-68 were replaced by Met, Leu, Ser, Met, Leu, and Leu, respectively, by PCR (Fig. 1). 5′ and 3′ regions of the cDNA of MbraPBP1 were amplified separately using pET22b(+)/MbraPBP1 as template and the following mutated primers (mutations are underlined, NruI site is in italic and encoded amino acids are indicated between parentheses).5′ RegionM6-NruI, 5′-CCGGTCGCGAGTAAAGAACTGATG(M5) ACGAAACTG(L8)AGTAGTGG-CTTCACGAAA-3′; M6-int-reverse, 5′-AGCCTTCCCGTGGTGGAG(L68)CTTCTGGTCTTCGCCCAG(L62)-CAT(M61)GTCCAGCTTGTTACT(S56)CATACACATCACCAT-3′.3′ RegionM6-int-forward, 5′-ATGGTGATGTGTATGAGT(S56)AACAAGCTGGACATG(M61)CTG(L62)-GGCGAAGACCAGAAGCTC(L68) CACCACGGGAAGGCT-3′. Mbra1-NotI, 5′-CCGGGCGGCCGCCTACACGGCCGTCATGATCTC-3′.After amplification and purification, the PCR fragments were used to reconstruct a single and continuous cDNA using M6-NruI and Mbra1-NotI primers. After purification and digestion by NruI and NotI, the mutated cDNA was cloned between the MscI andNotI restriction sites of the pET22b(+) vector (as described above) and the final construct was referred as pET22b(+)/Mbra1-M6. The mutant, Mbra1-M6, was produced by growing E. coli BL21(DE3) transformed by the recombinant pET22b(+)/Mbra1-M6 plasmid at 37 °C in LB medium supplemented with 50 μg/ml carbenicillin. WhenA600 reached the value of 0.8, the cultures were induced with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside, and the temperature was decreased to 28 °C.Purification ProcedureThe purification of recombinant MbraPBP1 and Mbra1-M6 mutant was initiated by centrifugation harvesting of 0.5–4 liters of induced cultures after 20–24 h of induction. The periplasmic proteins were released by osmotic shock as described in the pET system manual. Periplasmic proteins, obtained from induced cultures of BL21(DE3) (pET22b(+)/MbraPBP1) were submitted to a 100% ammonium sulfate precipitation, dialyzed overnight against 50 mm Tris-HCl, 50 mm NaCl, pH 8.0, and purified by anion exchange on a ResourceQ column (Amersham Pharmacia Biotech, Äkta FPLC) pre-equilibrated in the same buffer. Elution was carried out with a linear gradient of 0–1 m NaCl in 50 mm Tris, pH 8.0. Fractions (1 ml each) were collected and analyzed by SDS-PAGE. Fractions containing the MbraPBP1 were pooled and concentrated by a 10-kDa Microsep (Filtron), and loaded onto a Superdex 200 gel filtration column (Amersham Pharmacia Biotech) pre-equilibrated with 50 mm Tris, 150 mm NaCl, pH 8.0; the elution was carried out at 0.25 ml/min. Purification of Mbra1-M6 followed the same procedure, with the exception of the ammonium sulfate precipitation step. The periplasmic fraction was directly dialyzed overnight against 50 mm Tris-HCl, 25 mm NaCl, pH 8.0, before purification by anion exchange on a ResourceQ column followed by gel filtration (see above). A second anion exchange on a ResourceQ column was performed with a linear gradient of 0–1 m NaCl in 10 mm Tris, pH 8.0. Fractions (0.5 ml each) were analyzed by SDS-PAGE. Both proteins were shown to be >95% pure in SDS and native PAGE. The integrity of the hexamutant was checked by mass spectrometry and N-terminal sequencing.Purification of the recombinant ApolPBP1 and BmorPBP proteins was performed by adjusting the periplasmic fraction to 10 mmTris, pH 8.0, and loading it onto a 20-ml DEAE HR16/10 column (Tyopearl 650S, TOSOH). Proteins were eluted with a linear gradient of 0–300 mm NaCl in 10 mm Tris, pH 8.0. The collected fractions (1 ml) were analyzed by SDS-PAGE. Fractions containing PBP were pooled, concentrated in a Centriprep-10 (Amicon), and separated on a Superdex 75 gel filtration column (Amersham Pharmacia Biotech) pre-equilibrated with 10 mm Tris, 150 mm NaCl, pH 8.0. PBP containing fractions (1 ml each) were determined by SDS-PAGE, pooled, and desalted by dialysis against 20 mmTris, pH 8.0. Purified ApolPBP1 and BmorPBP protein was shown to be >95% pure in SDS and native PAGE.Mass Spectrometry and Circular Dichroism AnalysisMass analysis of recombinant PBPs was performed with a Voyager-DE RP spectrometer (PerSeptive Biosystems). Samples (0.7 μl containing 15 pmoles) were mixed with an equal volume of sinapinic acid matrix solution and spotted on the target, then dried at room temperature for 10 min. The mass standard was apomyoglobin.The CD spectra were measured on a CD6 spectropolarimeter (Jobin Yvon). Spectra were recorded in 10 mm Na phosphate, pH 7.0, at 20 °C between 178 and 260 nm, with a 30-s averaging.Fluorescence AssaysIn the case of MbraPBP1, BmorPBP, and Mbra1-M6, the fluorescence spectra were recorded on a Jobin-Yvon FL 3–21 spectrofluorimeter at 20 °C using a front face fluorescence accessory. Slit widths of 1 and 10 nm were used for excitation and emission, respectively. The spectra were processed with DataMax software. In the case of ApolPBP1, the spectra were recorded in a right angle configuration on a PerkinElmer LS 50B spectrofluorimeter using a 1-cm light path fluorimeter quartz cuvette. Slit width of 5 nm were used for both excitation and emission.Intrinsic Fluorescence of PBPsThe interaction of AMA and bombykol with PBPs was monitored by following the quenching of the intrinsic protein fluorescence (excitation at 280 nm and emission 290–420 nm, slits as described above). Spectra were recorded with 1 μm protein in 20 mm Tris buffer, pH 8.0, 0.3% methanol and under the same conditions in the presence of different concentrations of AMA (0–10 μm).Fluorescence Emission Spectra Using AMAThe effects of solvents, such as methanol, ethanol, and dimethyl sulfoxide on AMA binding have previously been tested with rat OBP (27Briand L. Nespoulous C. Perez V. Remy J.J. Huet J.C. Pernollet J.C. Eur. J. Biochem. 2000; 267: 3079-3089Crossref PubMed Scopus (59) Google Scholar). In that study, methanol was demonstrated to be less effective than ethanol in displacing the bound AMA from the binding site. In our study, the displacement of AMA by ethanol and methanol has been determined by successively adding aliquots of solvent to the AMA·MbraPBP1 complex solution. As with rat OBP, ethanol was found to compete significantly with AMA bound to MbraPBP1, and methanol was found to have a lesser effect (data not shown). For this reason, methanol was used for dissolving AMA and its competitors in the fluorescence titrations.MbraPBP1, Mbra1-M6, and BmorPBPAll the fluorescence experiments were carried out in 20 mm Tris, pH 8.0, at 20 °C (excitation 298 nm, emission 400–575 nm). The binding affinity for AMA was titrated by adding to the protein sample (1 μm) aliquots of a stock solution of AMA (10 mm) solubilized in 100% methanol. The fluorescence of AMA was recorded after stabilization of the signal (5–6 min). In competition experiments, PBP (1 μm) was incubated in the presence of AMA (5 μm) for 1 h at room temperature, the fluorescence signal of AMA was then monitored and the decrease in fluorescence intensity upon addition of different test compounds was recorded. The competition experiments were carried out at constant methanol concentration (0.3%) and with a test compound concentration between 0 and 10 μm.ApolPBP1Binding and competition experiments were carried out in 20 mm Tris, pH 8.0, at 20 °C (excitation 256 nm, emission 440–600 nm). All values reported were obtained from three independent measurements. The curves for binding of AMA to ApolPBP1 were obtained by titration of 2 μm protein with increasing concentrations of chromophore dissolved in methanol. In competition assays, we monitored the fluorescence signal of AMA (2 μm) equilibrated with ApolPBP1 (2 μm) upon addition of increasing amounts of competitor. Fluorescence intensities at the maximum of emission (487 nm) were determined for different concentrations of competitor and were corrected before further data analysis by the extent of AMA fluorescence decrease due to the methanol present in the cuvette.Data AnalysisThe affinity of different compounds for MbraPBP1 and ApolPBP1 was estimated by plotting the decrease of intensity of AMA fluorescence at the emission maximum, calculated as (I −Imin)/(I0 −Imin) against the competitor concentration;I0 is the maximum of fluorescence intensity of the complex AMA·MbraPBP1 and AMA·ApolPBP1, at 492 and 487 nm, respectively, I is the fluorescence intensity after addition of an aliquot of competitor, and Imin the fluorescence intensity at saturating concentration of the competitor. The IC50 values were estimated on the direct plot by non-linear regression with equation corresponding to a single binding site using Prism 3.02 (GraphPad software, Inc).Kdiss values were calculated according toKdiss = [IC50]/(1 + [AMA]/KAMA), in which [AMA] = free AMA concentration and KAMA = dissociation constant for MbraPBP1/AMA and ApolPBP1/AMA, respectively.RESULTSDesign of the Mutant of MbraPBP1Residues were chosen using two criteria: first, they are localized in the binding pocket defined in the three-dimensional structure ofB. mori PBP (17Sandler B.H. Nikonova L. Leal W.S. Clardy J. Chem. Biol. 2000; 7: 143-151Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar) and second, they are not conserved among the PBPs. Six residues of MbraPBP1 were thus replaced by their counterparts in B. mori PBP (Fig.1). Three primers were designed to introduce these mutations by PCR. The final DNA sequence, subcloned in pET22b(+), was verified by automated DNA sequencing to check the introduction of mutations and PCR fidelity.Subcloning, Expression, and PurificationAfter PCR amplification, gel purification, digestion, and precipitation, the MbraPBP1, hexamutant Mbra1-M6, BmorPBP, and ApolPBP1 cDNAs were subcloned into the pET22b(+) vector. This vector leads to expression of a pelB leader/PBP fusion protein in E. coli, which targets the recombinant proteins from the cytoplasm to the periplasm of the bacteria. Upon translocation, the pelB signal peptide is cut off and the PBP is released into the oxidative environment of the periplasm, favorable to the formation of disulfide bonds. This system has been successfully used for the expression of the pheromone-binding protein ofBombyx mori (26Wojtasek H. Leal W.S. J. Biol. Chem. 1999; 274: 30950-30956Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), which contains 3 disulfide bridges (28Leal W.S. Nikonova L. Peng G. FEBS Lett. 1999; 464: 85-90Crossref PubMed Scopus (196) Google Scholar), and in the case of a chemosensory protein from M. brassicae. 2V. Campanacci, A. Mosbah, O. Bornet, R. Wechselberger, E. Jacquin-Joly, H. Darbon, C. Cambillau, and M. Tegoni, submitted for publication.PBP1 and Hexamutant of M. brassicaeUsing this system, we routinely obtained 2–3 mg of pure recombinant MbraPBP1 and Mbra1-M6 per liter of culture. Two predominant bands at around 17 and 20 kDa (Fig.2 A, lane 1) are present in the SDS-PAGE of whole cell lysates of BL21(DE3) transformed with pET22b(+)/MbraPBP1. The band at 20 kDa is absent in the periplasmic fraction (Fig. 2 A, lane 2), and present in the cytoplasmic fraction (data not shown) suggesting that it corresponded to the MbraPBP1 with its signal peptide (16,168 Da for MbraPBP1 + 2,228 Da for the pelB signal peptide), whereas the band at around 17 kDa, present only in the periplasmic fraction, probably corresponded to the recombinant MbraPBP1 after cleavage of the signal peptide. Pure MbraPBP1 and Mbra1-M6 were obtained after two or three steps of purification (see “Experimental Procedures”) as shown on 15% SDS-PAGE (Fig. 2 A, lanes 3 and 4). N terminus sequencing and mass spectrometry confirmed the identity of the recombinant MbraPBP1 and the mutant Mbra1-M6.Figure 2Expression of moth PBPs in E. coliBL21(DE3). Protein fractions from PBP expressing bacteria were analyzed on 15% SDS gels and visualized by Coomassie Blue staining. A, MbraPBP1; B, ApolPBP1. Lane 1, whole cell proteins; lane 2, periplasmic fraction;lane 3, purified protein. The same low molecular weight markers were used in A and B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PBP1 of A. polyphemus and PBP of B. moriIn the case of ApolPBP1, the expression was slightly better than MbraPBP1 and up to 5 mg of pure PBP per liter of culture could be purified. Two bands at about 16 and 18 kDa (Fig. 2 B, lane 1), likely corresponding to recombinant ApolPBP1 (15,783 Da) and the pelB/ApolPBP1 fusion protein (15,783 Da + 2,228 Da) can be observed in the SDS-PAGE of whole cell lysates of BL21(DE3) cells transformed with the pET22b(+)/ApolPBP1 construct. In contrast, only the 16-kDa band can be seen in the SDS-PAGE of the periplasmic fraction (Fig.2 B, lane 2). This size is identical to that of the purified ApolPBP1 (Fig. 2 B, lane 3). The identity of the isolated recombinant ApolPBP1 protein was confirmed by mass spectrometry and Western blot analysis using an antiserum directed against the wild type protein. Results similar to those described in Wojtasek and Leal (26Wojtasek H. Leal W.S. J. Biol. Chem. 1999; 274: 30950-30956Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) were obtained for BmorPBP.Circular DichroismAnalytical methods such as CD have been used to define structural features of insect PBPs. The CD spectra of the four PBPs were recorded and showed that they are all well folded and have similar secondary structures. The two minima in the spectra around 208 and 222 nm are typical of a fold with a majority of α-helical secondary structure, in agreement with the crystal structure of B. mori PBP (17Sandler B.H. Nikonova L. Leal W.S. Clardy J. Chem. Biol. 2000; 7: 143-151Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The α-helical content obtained by CD (55–70%) is in agreement with the Psi-Pred secondary structure predictions (30Jones D.T. J. Mol. Biol. 1999; 292: 195-202Crossref PubMed Scopus (4386) Google Scholar), which indicates 61–66% helical content.Fluorescence Binding Assays Fluorescence Emission of AMA Upon Binding to PBPs—When excited at 256 or 298 nm, AMA in aqueous buffer shows a weak fluorescence emission with a maximum at 563 nm. In a hydrophobic environment, such as the binding pocket of MbraPBP1 or ApolPBP1, there is a blue shift of the fluorescence emission maximum and a large increase of intensity (Fig.3 A), resulting from the modification in the AMA environment. The fluorescence emission spectra show a maximum at 492 and 487 nm for MbraPBP1 and ApolPBP1, respectively (Fig. 3,B and C, inset). The concentration dependence of AMA binding to MbraPBP1 and ApolPBP1 can be described by a hyperbolic curve as expected for a one-site binding model (Fig. 3, B and C), andKdiss values of 4.5 and 0.95 μm, respectively, were calculated. The affinity of ApolPBP1 for AMA is thus greater than that of MbraPBP1.Figure 3AMA fluorescence. A, AMA fluorescence emission spectra. MbraPBP1 (- -), free AMA (···), MbraPBP1 + AMA (—). Excitation wavelength was 298 nm; MbraPBP1 was 1 μm, and AMA was 6 μm. B, titration of MbraPBP1 with AMA. Relative fluorescence intensity is plotted as a function of AMA concentration. Conditions were as follows: 1 μm MbraPBP1 in 20 mm Tris, pH 8.0, at 20 °C. AMA fluorescence was monitored at 490 nm with excitation at 298 nm. Data represent the mean of three independent measurements. Standard deviations are indicated by error bars. The curve corresponds to the theoretical binding curve for n = 1 andKdiss = 4.5 μm. Inset, fluorescence emission spectra of AMA in the presence of MbraPBP1. Recombinant MbraPBP1 (1 μm in 20 mm Tris, pH 8.0) was titrated with increasing amounts of AMA (0–9 μm). Excitation wavelength was 298 nm. Fluorescence emission spectra were recorded at 20 °C between 440 and 600 nm. The emission maximum was at 492 nm. C , binding of AMA to recombinant ApolPBP1. The protein (2 μm in 20 mm Tris, pH 8.0) was titrated with increasing amounts of AMA (0–7 μm). The relative fluorescence intensities at the emission maxima (487 nm) are plotted as a function of the AMA concentration. Data represent the mean of three independent measurements. Standard deviations are indicated by error bars. A dissociation constant of 0.95 μm was determined from the best fit to the data. Inset ,fluorescence emission spectra of AMA in the presence of ApolPBP1. Recombinant ApolPBP1 (2 μm in 20 mm Tris, pH 8.0) was titrated with increasing amounts of AMA (0–7 μm). Excitation wavelength was 256 nm. Fluorescence emission spectra were recorded at 20 °C between 440 and 600 nm. The emission maximum was at 487 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the case of BmorPBP, no increase in fluorescence emission and no saturation are observed with increasing AMA concentration. Interestingly, the Mbra1-M6 mutant behaves like BmorPBP. In these cases, the fluorescence increase observed is not significantly greater than the experimental error, furthermore, the lack of saturation suggests either some nonspecific interaction between AMA and the proteins, not related to binding, or the formation of superstructures" @default.
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• W2037953375 date "2001-01-01" @default.
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• W2037953375 title "Revisiting the Specificity of Mamestra brassicaeand Antheraea polyphemus Pheromone-binding Proteins with a Fluorescence Binding Assay" @default.
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