chemosensory protein from the moth mamestra brassicae

Chemosensory protein from the moth Mamestra brassicaeExpression and secondary structure from 1H and 15N NMR

Valerie Campanacci1, Amor Mosbah1, Olivier Bornet2, Rainer Wechselberger3, Emmanuelle Jacquin-Joly4,Christian Cambillau1, Herve Darbon1 and Mariella Tegoni1

1AFMB, UMR 6098-CNRS and Universites d’Aix-Marseille I and II, Marseille, France; 2IBSM, IFR1, Marseille, France; 3Bijvoet Center

for Biomolecular Research, University of Utrecht, the Netherlands; 4INRA, Unite de Phytopharmacie et des Mediateurs Chimiques,

Versailles, France

A group of ubiquitous small proteins (average 13 kDa) has

been isolated from several sensory organs of a wide range of

insect species. They are believed to be involved in chemical

communication and perception (olfaction or taste) and have

therefore been called chemo-sensory proteins (CSPs). Several

CSPs have been identified in the antennae and proboscis of

the moth Mamestra brassicae. We have expressed one of the

antennal proteins (CSPMbraA6) in large quantities as a

soluble recombinant protein in Escherichia coli periplasm.

This 112-residue protein is a highly soluble monomer of

13 072 Da with a pI of 5.5. NMR data (1H and 15N) indicate

that CSPMbraA6 is well folded and contains seven a helices

(59 amino acids) and two short extended structures (12

amino acids) from positions 5 to 10 and from 107 to 112.

Thirty-seven amino acids are involved in b turns and coiled

segments and four amino acids are not assigned in the NMR

spectra (the N-terminus and the residue 52 in the loop

48–53), probably due to their mobility. This is the first

report on the expression and structural characterization of a

recombinant CSP.

Keywords: biochemical characterization; chemosensory

protein; Lepidoptera; NMR; periplasmic expression.

Chemical communication in insects is a process ofprimordial importance for reproduction and survival, asmating and searching for food depend on it. In fact, othersenses such as vision and hearing are poorly developed inmany insect species and this is particularly true of moths.Studies on olfaction started with the identification ofchemicals that are perceived by the animals and that elicitmating or food searching behavior. The response ofindividual animals exposed to different pheromones wascharacterized by behavioral assays and was found to bespecies- and sex-specific in the case of pheromones, andnonspecific in the case of odors. The organs devoted toolfactory perception were identified as sensilla trichodeaand basiconica, the former being tuned to the perception ofpheromones [1]. In Mamestra brassicae, long and shortsensilla trichodea were found to react with (Z)-11-hexa-decenyl-1-acetate and (Z)-11-hexadecenal, respectively [2].Typically, lepidopteran pheromones are volatile hydro-phobic compounds of 12–20 carbon atoms. Because of thehydrophobic character of pheromones and odors, a generalscheme was proposed in which the transport of the volatile

molecule from the interface air/sensilla to the olfactoryreceptor required a carrier, which might also operate adiscrimination among different volatile molecules [3,4].

Two classes of small proteins (150 amino-acids long),very soluble and very abundant, were detected in sensillae ofLepidoptera. They contained six conserved cysteines thatformed three disulfide bridges and were distinguished on thebasis of N-terminal sequence, sexual and topologicaldistribution. General odorant binding proteins (GOBPs)are expressed in sensilla basiconica and sensilla trichodeaand are distributed equally in the two sexes; pheromonebinding proteins (PBPs) are present mainly in males and insensilla trichodea [5], but recently they were also found tobe expressed at high levels in the antennae of the females ofsome species [6]. GOBPs can show up to 95% sequenceidentity between species and are supposed to carry one typeof odorant molecule, signaling the presence of food forexample. Nevertheless, recently they have been proposed tobe involved in the discrimination between pheromone andantagonist [7]. PBPs show a low degree of sequence identity(30–40%) and have been postulated to ferry specificpheromones. The direct receptor–PBP contact or phero-mone release on the receptor may produce a chemical signalthat is converted into an electric signal by means ofa G-protein [8] inducing the production of cAMP [9] orinositol 1,4,5-trisphosphate which activates ionic channels.

A major progress in the molecular characterization ofthe olfactory process has been the cloning and theexpression of PBPs and GOBPs, as they are otherwisevery scarce in nature. Nevertheless, the intrinsic difficulty ofobtaining pure, homogeneous PBPs makes reports onfunctional and structural characterization of this class ofprotein rare. Very recently, the three-dimensional structureof a Bombyx mori PBP, a structural paradigm for all PBPs

Correspondence to M. Tegoni and H. Darbon, AFMB, UMR

6098-CNRS and Universites d’Aix-Marseille I and II, 31 chemin

J. Aiguier, F-13402 Marseille Cedex 20, France.

Fax: 1 33 491 16 45 35, Tel.: 1 33 491 16 45 12,

E-mail: [email protected], [email protected]

(Received 14 May 2001, revised 4 July 2001, accepted 9 July 2001)

Note: V. Campanacci and A. Mosbah contributed equally to this work.

Abbreviations: CSP, chemosensory protein; PBP, pheromone binding

protein; GOBP, general odorant binding protein; Mbra, Mamestra

brassicae; IPTG, isopropyl thio-b-D-galactoside; HSQC, heteronuclear

single quantum coherence; LTP, lipid transfer proteins

Eur. J. Biochem. 268, 4731–4739 (2001) q FEBS 2001

and GOBPs, has been solved in complex with bombykol[10]. This crystallographic structure revealed a fold con-taining six a helices delimiting a buried cavity filled withthe alkyl alcohol, bombykol.

A third class of small proteins (average 13 kDa) has beenidentified in antennae more recently (OS-D from Drosophilamelanogaster ) and several sensorial organs (chemosensoryproteins; CSPs) from a wide range of species of the insectorder [11–20]. They were not classified as PBPs as theycontain only four conserved cysteines and share no sequencehomology with PBPs. Because of their localization inantennae, tarsi and labrum [18,21], these proteins have beenproposed to be involved in the CO2 detection [12], in thechemical signal transmission in regenerating legs [14] or inchemo-perception, either olfaction or taste. In this respect,they may also play a role in transport of hydrophobic chemi-cals (volatile or not) from air or water to olfactory or tastereceptors. However, the physiological role of CSPs has stillto be identified, although their wider distribution makesthem of great functional interest.

In the moth Mamestra brassicae (Mbra), several CSPshave been identified in the proboscis [21] and in theantennae [22]. MbraCSPs have been shown to bind severalcomponents of the pheromonal blend, and, therefore aresupposed to have a function analogous to that of PBPs [21].Indeed, few reports are available on CSPs as compared withGOBPs and PBPs; in particular, neither expression norbiochemical and structural characterization of CSPs hasbeen reported to date. In order to improve our knowledge onthese important insect proteins, we have expressedCSPMbraA6 originating from the antennae of M. brassicaeas a recombinant protein in the Escherichia coli periplasm.We have already published the crystallization and a pre-liminary crystallographic study of this same protein [23]. Inthe present paper, we describe the biochemical character-istics and the secondary structure of CSPMbraA6, which weshow to be a mainly helical protein like the PBPs, asdetermined by 1H- and 15N-NMR.

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

Subcloning of CSPMbraA6 in pET22b(1)

A detailed description of the molecular cloning ofCSPMbraA6 will be described elsewhere [11]. We beganthe subcloning procedure from a plasmid pCRII (Invitrogen)in which the CSPMbraA6 gene was inserted between twoEco RI restriction sites. At position 15 of the cloned gene acytosine base was lacking.

The primers (forward primer: 50-CGGAGGACAAGTACACCGATAAGTACGAT-30; reverse primer: 50-CCGGGCGGCCGCTTATTCTTCTGGGAT-30) were designed tointroduce a blunt end 50 of the gene, a cytosine at position 15of the gene (in bold in the sequence) and the Not I site (initalic) 30 of the sequence.

The primers (15 pmol each) were mixed with 20 ng DNAtemplate, 2mL each 10 mM dNTPs (PerkinElmer), 1mL Pfupolymerase (2.5 U:mL21, Stratagene), 5mL 10� Pfu Buffer(Stratagene) and water to a final volume of 50mL. Thereaction cycles were carried out as follows: 94 8C for 5 min,followed by 25 cycles of 1 min at 94 8C, 1 min at 45 8C and1 min at 72 8C, and finally 8 min at 72 8C. The PCR productwas analyzed on an agarose gel (Tris/borate/EDTA),

purified using QIAquick gel extraction kit (QIAGEN), andcloned into the Msc I and Not I sites of the pET22b(1)expression vector (Novagen). The ligation reaction was usedto transform XL1-Blue cells. Transformants were selectedon Luria–Bertani agar plates containing 50mg:mL21

ampicillin. Plasmids extracted from randomly selectedtransformant clones were screened for the presence of theinsert by PCR, using the forward and reverse primersdescribed above. DNA of positive candidates wastransformed into BL21(DE3) cells. The production of therecombinant CSPMbraA6 was checked by analyzing thecrude cell extracts from BL21(DE3) transformant clonesinduced by isopropyl thio-b-D-galactoside (IPTG) on SDS/PAGE. Positive candidates were characterized further byrestriction map analysis. Automated DNA sequencing(ESGS, Evry, France) was used to confirm the presence ofthe CSPMbraA6 gene and to check for PCR fidelity. Theresulting construct is referred to as pET22b(1)/CSPMbraA6.

Expression in E. coli

Cell culture conditions. Expression was carried out in E. coliBL21(DE3) expression hosts. Cultures of BL21(DE3)transformed by the recombinant pET22b(1)/CSPMbraA6plasmid were grown in Luria–Bertani medium supple-mented with carbenicillin (50mg:mL21). After optimizationof the expression conditions (see Results), the cultureswere grown at 37 8C without induction. For production ofthe 15N-labeled sample, cultures were grown at 37 8C in M9minimal medium supplemented with 0.4 g:L21 of 15NH4Cl(Martek Biosciences, USA) and carbenicillin (50mg:mL21).When the D600 reached 0.6, expression of the target proteinwas induced with 50mM IPTG, and the temperature wasdecreased to 28 8C. Cells were harvested after 16 h ofinduction.

Preparation of periplasmic fraction. Induced cultures(0.5–4 L) were harvested by centrifugation after 20–24 h(D600 . 4.0). The periplasmic proteins were released byosmotic shock as described in the pET system manual. Allsteps from the preparation of the periplasmic fraction to theend of the purification were carried out at 4 8C.

Purification procedure of the periplasmic CSPMbraA6

Anion exchange on ResourceQ column. Large scalepreparations of periplasmic proteins, obtained from inducedor noninduced cultures of BL21(DE3) [pET22b(1)/CSPMbraA6] were dialyzed against 50 mM Tris/HCl,50 mM NaCl, pH 8.0, concentrated on an Amicon stirredcell with a 3-kDa cut-off membrane, and purified by anionexchange on a ResourceQ column (Pharmacia, Akta FPLC)pre-equilibrated with the same buffer. Elution wasperformed with a linear gradient to 50 mM Tris/HCl, 1 M

NaCl, pH 8.0. Fractions (1 mL each) were collected andanalyzed by SDS/PAGE. Fractions containing theCSPMbraA6 were pooled and concentrated by Amiconstirred cell.

Gel filtration. The FPLC gel filtration column (Superdex200 Column, Pharmacia) was pre-equilibrated with 10 mM

4732 V. Campanacci et al. (Eur. J. Biochem. 268) q FEBS 2001

Tris/HCl, 50 mM NaCl, pH 8.0. Elution was carried out at0.25 mL:min21. Fractions (0.5 mL each) were analyzed bySDS/PAGE. Fractions containing the CSPMbraA6 werepooled, dialyzed against 10 mM Tris, 25 mM NaCl, pH 8.0and submitted to a second ResourceQ, equilibrated in thesame buffer. To eliminate the Tris buffer before performingthe NMR experiments the pure protein was dialysed against10 mM sodium phosphate buffer, 25 mM NaCl, pH 6.9.

Characterization of the recombinant CSPMbraA6 andsecondary structure prediction

N-Terminal sequencing, MS, mass determination in solutionand secondary structure prediction. The N-terminalsequence of CSPMbraA6 was obtained with an automatedprotein sequencer, using a standard Edman degradationprotocol. Mass analysis of recombinant CSPMbraA6 wasobtained by MALDI-TOF using a Voyager-DE RP(PerSeptive Biosystems). Samples (0.7mL containing15 pmol) were mixed with an equal volume of sinapinicacid matrix solution and spotted on the target, then dried atroom temperature for 10 min. The mass standard was apo-myoglobin (molecular mass 16 951.6 Da). Hydrodynamicsize determination was achieved by gel filtration on aSuperdex 200 FPLC column (Pharmacia). The elution wascarried out with 10 mM Tris, 150 mM NaCl, pH 8.0 at0.5 mL:min21. BSA (67 kDa), ovalbumin (43 kDa), chymo-trypsinogen (25 kDa) and ribonuclease (13.7 kDa) wereused as standards. The hydropathy profile was obtained byPROTSCALE (http://www.expasy.ch/cgi-bin/protscale.pl)using the method of Kyte and Doolittle [24] with a windowsize of nine amino acids.

Electrophoresis. Proteins were analyzed by SDS [25] andnative PAGE [26] on 15% acrylamide gels. Proteins werevisualized by staining with Coomassie blue. Molecular massmarkers used in SDS/PAGE were: lysozyme (14.4 kDa),trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa),ovalbumin (45 kDa), albumin (66.2 kDa), phosphorylase b(97.4 kDa). IEF was carried out with Novex system(Invitrogen) using a pH 3–7 precast gel and following theinstructions of the manufacturer.

NMR spectroscopy

Homonuclear NMR experiments were performed with2.0 mM of unlabeled protein sample at 296 K, in 90% H2Oand 10% D2O, containing 10 mM sodium phosphate buffer,25 mM NaCl, at pH 6.9. NMR spectra were recorded on a500-MHz DRX Bruker spectrometer equipped with a 5-mmtriple-resonance HCN probe with self-shielded triple axisgradients. DQF-COSY [27,28], clean-TOCSY [29] andNOESY [30] spectra were acquired in a phase-sensitivemode using States-TPPI [31]. NOESY spectra were acquiredwith mixing times of 80 and 100 ms. Clean-TOCSY spectrawere recorded with mixing times 80 ms using the WALTZ-16 mixing sequence [32] and a spin-lock field strength of8 kHz. Spectra were recorded with 2000 complex points inthe directly acquired dimension and 512 (for COSY andClean-TOCSY) or 1000 (for NOESY) complex points inthe indirectly detected dimension, with 64 transients pert1 increment over a spectral width of 6000 Hz in both

dimensions. Water suppression was achieved using pre-saturation during the relaxation delay (1.3 s), and during themixing time in the case of NOESY experiments or through aWATERGATE 3-9-19 pulse train [33,34] using a gradient atthe magic angle obtained by applying simultaneously x-, y-,and z-gradients prior to detection. Data were processedusing the program XWINNMR (Bruker). Time domain datawere multiplied by a sine function and zero filled to give afinal matrix size of 2000� 2000 (real) points after Fouriertransformation. A fifth-order polynomial baseline correctionin both dimensions was applied. Proton chemical shifts werecalibrated relative to H2O at 296 K at 4.79 p.p.m.

Heteronuclear NMR experiments were performed on a0.5-mM uniformly 15N-labeled protein sample at 296 K. The3D NOESY-(1H,15N)-FHSQC [35,36] and 3D TOCSY-(1H,15N)-HSQC [37] and 2D (1H,15N)-HSQC [38] wererecorded on the labeled sample under the same physico-chemical conditions as for the unlabeled sample. Thesespectra were recorded on a Varian Unity INOVA 750-MHzspectrometer. The NOE mixing time was 100 ms for the3D NOESY-(1H,15N)-HSQC. A total of 1536 acquisitionpoints, 128 complex points in t1 and 64 complex points int2 for the 3D NOESY-(1H,15N)-HSQC and 1024 acquisitionpoints, 128 complex points in t1 and 64 complex pointsin t2 for the 3D TOCSY-(1H,15N)-HSQC were recordedand processed. Spectra were processed using NMRPIPE

software [39] to a final size of 2000� 256� 128 matrix,and assigned using XEASY software [40]. The 3-9-19 com-posite WATERGATE scheme was applied for water suppres-sion in the ‘fast HSQC’ part of the pulse sequence.

NMR spectral analysis

The NMR spectral analyses were performed using theXEASY software, running on a Silicon Graphic R10000workstation. The identification of amino-acid spin systemsand the sequential assignment were performed by using thestandard strategy described by Wuthrich [41]. The com-parative analysis of COSY, TOCSY and the 3D TOCSY-(1H,15N)-HSQC spectra recorded in water gave the spinsystem signature of the protein. The spin systems wereconnected sequentially using the NOESY and 3D (1H,15N)-NOESY-HSQC spectra.

3JHN-Ha coupling constant measurements3JHN-Ha coupling constant measurements were estimated bythe INFIT program [42]. For a given residue, separatedNOESY cross-peaks with the backbone amide proton in thev2 dimension were used. Several cross-sections throughthese cross-peaks that exhibited a good signal-to-noise ratiowere selected; these were summed and only those datapoints of the peak region that were above the noise levelwere retained. The left and the right ends of the peak regionwere then brought to zero intensity by a linear baselinecorrection. After extending the baseline-corrected peakregion with zeros on both sides, which is equivalent to over-sampling in the time domain, an inverse Fouriertransformation was performed. The value of the 3JHN-Ha

coupling constant was obtained from the first localminimum.

q FEBS 2001 NMR characterization of a moth chemosensory protein (Eur. J. Biochem. 268) 4733

R E S U L T S

Subcloning of CSPMbraA6 and periplasmic expression

The correct reading frame was obtained by inserting by PCRa C in position 15 of CSPMbraA6 cDNA. After PCRamplification, gel purification, digestion and precipitation,the CSPMbraA6 cDNA was subcloned into the pET22b(1)vector. This vector allows the expression of the recombinantproteins fused to the pelB signal peptide, which targets themto the periplasmic space, where the oxidative environment isfavorable for the formation of disulfide bonds. This systemhas been used successfully for the expression of thepheromone binding protein of B. mori [43], carrying threedisulfide bridges [44,45]. By this expression system, wecould achieve a yield of 10–15 mg pure CSPMbraA6 perlitre culture; optimal conditions of cell growth were 37 8Cfor 24 h, without induction. Indeed, the addition of IPTGincreased further the amount of the recombinant protein, butin inclusion bodies (data not shown).

Two bands at < 13 and < 15 kDa (Fig. 1A, lane 1) arepresent in the SDS/PAGE of whole BL21(DE3) cell lysatesupon transformation with pET22b(1)/CSPMbraA6. Theband at 15 kDa present in the cytoplasmic fraction (Fig. 1A,lane 2) and absent from the periplasmic fraction (Fig. 1A,lane 3) is probably CSPMbraA6 with its signal peptide(13 072 Da for CSPMbraA6 1 2228 Da for the pelB signalpeptide). The band at < 13 kDa (Fig. 1A, lane 3), presentonly in the periplasmic fraction is CSPMbraA6 after cleav-age of the signal peptide. A single band of pure CSPMbraA6is visible in 15% SDS (Fig. 1B) and native PAGE (Fig. 1C)after three steps of purification (see Experimentalprocedures)

Biochemical characterization and secondary structureprediction of periplasmic CSPMbraA6

CSPMbraA6 migrated as a unique band between pH 5.4 and5.9 (Fig. 1D) in an IEF gel between pH 3 and 7. This value isconsistent with the theoretical value of 5.54, calculated onthe basis of the amino acids composition.

The N-terminus of CSPMbraA6 verified by proteinsequence analysis is EDKYTDKYDNI. This is in agreementwith the sequence deposited in GenBank (AF211178) andindicates that the signal peptide has been processed correctly.The molecular mass of 13 067.2 Da obtained by MS is also inagreement with the molecular mass of 13 072.8 Da calculatedon the basis of the sequence. By gel filtration on a Superdex

200 column we have determined the hydrodynamic size ofCSPMbraA6 in solution under native conditions. Compari-son with the protein standards gave a hydrodynamic size of13.2 kDa, which is close to the monomer molecular mass.The protein thus appears to be monomeric and folded into acompact and approximately spherical structure. The primarysequence of CSPMbraA6 was analyzed by the method ofKyte and Doolittle [24]: the hydropathy profile (data notshown) corresponds to a hydrophilic protein with a tentativehydrophobic portion between residues 22 and 25.

NMR spectral analysis and sequential assignment of therecombinant CSPMbraA6

The sequence-specific assignment was performed accordingto the standard method of Wuthrich. The spin systems wereidentified on the basis of both COSY and TOCSY recordedin water at 296 K. The almost complete assignment ofintraresidue HN–Ha cross-peaks was obtained with theexception of the N-terminal residues (E1 to K3) and theresidue E52 for which no NOE were found, probablybecause of their mobility. Starting from fingerprint cross-peaks of the COSY, each spin system was characterized byits connectivities in the COSY and further checked in the2D TOCSY and 3D TOCSY-(1H,15N)-heteronuclear singlequantum coherence (HSQC) spectra. Few methylene groupspresent only one resonance, either because of their strictdegeneracy or because they resonate in overcrowded regions. All of the chemical shifts are listed in Table 1 and anexample of the assigned HSQC spectrum is shown in Fig. 2.The sequential connectivities were obtained from NOESYand 3D NOESY/HSQC spectra, recorded in water at 296 K,with a mixing time of 80 or 100 ms. The sequential- andmedium-range NOE observed in the NOESY and3D NOESY/HSQC spectra of CSP2 are summarized inFig. 3. Characteristic extended strand NOE patterns (strongconsecutive Ha(i)-HN(i 1 1), Hb(i)-HN(i 1 1) and weakto medium HN(i)-HN(i 1 1) are observed from T5 to D10and from I107 to E112, which reveal the presence ofextended regions. The presence of strong consecutiveHN(i)-HN(i 1 1) NOE together with Ha(i)-HN(i 1 3) andHa(i)-HN(i 1 4) connectivities reveals the presence ofseven helical regions: 12–18, 21–31, 39–45, 61–65, 68–76, 78–86, and 91–102. Some Ha(i)-HN(i 1 2) NOE aredetected at the ends of the helices, as is generally the case.Secondary structure prediction by PSIPRED (Fig. 3) shows avery good correlation with the NMR-determined secondarystructures, but with no evidence of extended structures at the

Fig. 1. Expression of CSPMbraA6 in

BL21(DE3). (A) SDS/PAGE (15% acrylamide)

of crude cell extracts (lane 1) and of cytoplasmic

(lane 2) and periplasmic (lane 3) fractions after

osmotic shock. SDS (B) and native (C) PAGE,

and IEF (D) on recombinant CSPMbraA6 after

purification.


C- or N-termini. The location of the secondary structures issubstantiated by the chemical shift index shown in Fig. 4, inwhich the experimental chemical shifts of alpha protons arecompared with their standard values [41]. A negative or a

positive sign indicates the involvement of the residue in ahelix or in an extended region, respectively [46]. The sevenhelices detected by analysis of the NOE intensities are alsopredicted by the chemical shift index. However, the helix

Fig. 2. Two-dimensional 15N-1H HSQC spectrum of uniformly 15N-labeled sample.

Fig. 3. Amino-acid sequence of CSPMbraA6 and a survey of the sequential assignment. NOEs involving NH and CaH are represented as bars;

the intensity of NOE cross-peaks is indicated by the heights of the bars. The location of secondary structures are identified at the top: extended regions

are indicated by arrows and helical regions by cylinders.


from residue 92 to 102 contains two residues having aslightly positive value (0.03 p.p.m.) of chemical shift index.This positive value may result from the proximity of thestrong aromatic cluster detected around the W94 and formedby Y67, Y98, W94 and W81.

Moreover, we have measured only 96 3JHN-Ha couplingconstants on the NOESY spectrum, using the INFIT software.The 3JHN-Ha coupling constants of residues 51, 66 and 89cannot be measured because of signal overlapping. All ofthe residues in helical regions show 3JHN-Ha couplingconstants lower than 7 Hz (Table 1).

D I S C U S S I O N

In this work we report for the first time the heterologousexpression of an insect chemosensory protein. This expres-sion makes it possible to characterize the CSPMbraA6 withbiophysical methods. CSPs belong to the family of the OS-Dlike protein, first identified in the antennae of the fruit flyD. melanogaster [47]. To date, several other proteins havebeen detected in the chemosensory organs, such as antennae,tarsi, proboscis and labrum, of other insects orders (seeReferences in Fig. 5). Two classes of CSPs with differentsequences have been reported in the literature [20,48,49].CSPs are not sequentially related to the GOBPs and PBPsdescribed mainly in insects and they have only four

cysteines instead of six. In the case of Schistocerca gregariaCSP, it has been demonstrated that the four cysteines areinvolved in two disulfide bridges (C29–C38 and C57–C60)[18].

It has been shown that the CSPs of Eurycantha calcaratado not undergo post-translational modification [20], whichmakes the use of a prokaryotic expression system such asE. coli suitable for the expression of this class of proteins.The CSPMbraA6 was overproduced successfully in E. coliusing the pET22b(1) vector, without additional aminoacids at its N-terminus. The identity of the recombinantCSPMbraA6 to the native protein has been shown byN-terminal sequencing and molecular mass determination;the structural integrity has been shown by hydrodynamicmeasurements and NMR study, and by the crystallization ofCSPMbraA6 [23]. By gel filtration we have estimated amolecular mass of 13.2 kDa, which corresponds to the massestimated for a monomer. The CSPs of S. gregaria [18] andCarausius morosus [48] have also been found to be mono-meric. In this respect also CSPs differ from the insect PBPs,which were found to be dimeric under native conditions[50–53], although this result had been a serious matter ofdiscord [54].

The NMR study indicates a largely a helical structure forCSPMbraA6 (59 residues out of 108 observed). However,significant amounts of extended structures have also been

Fig. 4. Chemical shift index analysis of

CSPMbraA6. Dd (p.p.m.) is plotted against the

sequence position. Extended regions are indicated

by arrows and helical regions are represented by

cylinders.

Fig. 5. Sequence alignment of CSPs from different species. 1 [11], Mbra, Mamestra brassicae; 2 [12], Ccac, Cactoblastis cactorum; 3 [13], Msex,

Manduca sexta; 4 [14], Pamer, Periplaneta americana; 5 [15], 6 [16], 7 [17], Dmel, Drosophila melanogaster; 8 [18], Sgreg, Schistocerca gregaria; 9

[19], Lmig, Locusta migratoria; 10 [20], Ecal, Eurycantha calcarata.


observed (12 residues) mainly at the N- and C-termini,although they were not predicted by PSIPRED. Interestingly,a region is well conserved in CSPs (Fig. 5), containing thepattern A(LI)xxxCxKCx(DE)(KN)Q, and including a puta-tive disulfide bridge between C55 and C58.

CSPMbraA6 has an a helical secondary structure that isobserved in several other transport proteins, PBPs andGOBPs (see above), and also in nonspecific lipid transferproteins (LTPs) and the B1 and B2 proteins, secreted by thetubular accessory glands of the adult male mealworm beetleTenebrio molitor. LTPs are small proteins (< 100 aminoacids) that facilitate the transfer of lipids through mem-branes. Their structure, solved by NMR [55–57] and X-raycrystallography [58], reveals four helices delineating acavity extending through the entire molecule. B1 and B2proteins share common features with CSPs: their molecularmass is around 13 kDa, their pI is acidic around 4.2, they arehighly abundant and soluble and they have four conservedcysteines. They have been proposed to be lipid carriers ableto keep hydrophobic compounds in solution in the aqueousseminal fluid [59]. The NMR structure of a homologousprotein of T. molitor, THP12, a nonbundle structure of six a

helices, has shown binding of fatty acids (nonanoic acid andoctanoic acid), the T. molitor pheromone, 4-methylnonanoland ergosterol [60]. However, the sequences, disulfidebridge pattern and the secondary structure of LTP andTHP12 differ strongly from those of CSPMbraA6, suggest-ing a new fold for CSPMbraA6. It has been hypothesizedthat the class of proteins including CSPMbrcA6 could beinvolved in CO2 detection [12] or in the chemical signaltransmission in regenerating legs [14]. The structuralanalogies with various transport proteins of lipidiccompounds clearly indicate a lipid transport function forCSPMbraA6, which could be for pheromones or otherlipidic compounds.

A C K N O W L E D G E M E N T S

The authors thank J. Bonicel and D. Moinier for performing mass

spectrometry and N-terminal sequencing, respectively. We also thank

C. Lewandowski and A. Lartigue for their technical assistance.

The750 MHz spectra were recorded at the SON NMR Large Scale

Facility in Utrecht, which is supported by the Large Scale Facility

program of the European Union. This study was supported in part by the

PACA region (no. 9811/2177) and by the EU BIOTECH Structural

Biology project (BIO4-98–0420 OPTIM).

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S U P P L E M E N T A R Y M A T E R I A L

The following material is available from http://blackwell-science.com/ejb/

Table S1. Resonance assignments. Proton chemicalshifts.


chemosensory protein from the moth mamestra brassicae

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