From the ¶ Architecture et Fonction des Macromolécules
Biologiques, Unité Mixte de Recherche 6098, CNRS and
Universités Aix-Marseille I and II, 31 Chemin Joseph Aiguier,
13402 Marseille Cedex 20, France, Istituto di
Biochimica Veterinaria, Facoltà di Medicina Veterinaria,
Università di Parma, Via del Taglio 8, 43100 Parma, Italy, and
Institut National de Recherche Agronomique, Unité
de Phytopharmacie et des Médiateurs Chimiques, Route de
Saint-Cyr, F-78026 Versailles Cedex, France
Received for publication, November 15, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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Bovine odorant-binding protein (bOBP) is a
dimeric lipocalin present in large amounts in the respiratory and
olfactory nasal mucosa. The structure of bOBP refined at 2.0-Å
resolution revealed an elongated volume of electron density inside each
buried cavity, indicating the presence of one (or several) naturally
occurring copurified ligand(s) (Tegoni et al. (1996)
Nat. Struct. Biol. 3, 863-867; Bianchet et al.
(1996) Nat. Struct. Biol. 3, 934-939). In the present
work, by combining mass spectrometry, x-ray crystallography (1.8-Å
resolution), and fluorescence, it has been unambiguously established
that natural bOBP contains the racemic form of 1-octen-3-ol. This
volatile substance is a typical component of bovine breath and in
general of odorous body emanations of humans and animals. The compound
1-octen-3-ol is also an extremely potent olfactory attractant for many
insect species, including some parasite vectors like
Anopheles (Plasmodium) or Glossina
(Trypanosoma). For the first time, a function can be
assigned to an OBP, with a possible role of bOBP in the ecological
relationships between bovine and insect species.
Several isoforms of odorant-binding protein
(OBP)1 have been purified and
characterized from the nasal mucosa of many mammalian species (1-5).
These 19-kDa soluble proteins are produced in large amounts in
seromucous glands of the respiratory and olfactory epithelium and are
secreted in the mucus that layers the surface of nasal mucosa (5). OBPs
belong to the lipocalin family, which is composed of structurally
related soluble proteins that bind different types of small hydrophobic
molecules (6, 7). These proteins are usually monomeric and are formed
of a nine-strand The recent characterization of the binding properties of porcine OBP
(pOBP) in solution and in the crystal (13) has established that a high
degree of hydrophobicity coupled to a molecular mass between 160 and 200 daltons is the main requirement for a ligand to match the
OBP Purification and Extraction of the Ligand--
Single bovine
nasal mucosa samples were collected during fall and winter from
freshly slaughtered 16-18-month-old males of different geographical
origins (Northern Italy and France). OBP was purified according to the
procedure previously reported by Bignetti et al. (20).
Briefly, purification consists of an ammonium sulfate fractionation of
the soluble proteins in the extract of nasal mucosa, followed by two
rounds of fast protein liquid anion exchange chromatography
(Baker bond and Mono-Q) and a final acidic step. The purity of the
protein was checked by SDS-polyacrylamide gel electrophoresis, and its
functionality was determined in binding tests with 1-aminoanthracene
(AMA) as fluorescent probe (21-23). The molar absorption coefficient
of 47,000 (M GC/MS Identification and Chiral Determination--
Analyses were
conducted on a Varian Saturn II trap spectrometer coupled to a Varian
3400 gas chromatograph. Mass spectra were recorded in electronic impact
mode, with a mass range of 40-300 atomic mass units. A 30-m,
0.32-mm internal dimension, 0.5-µm df RTX-5MS column (Restek,
Bellefonte, PA) was used for analysis, and the program temperature was
from 50 °C (1 min) to 280 °C at 10 °C/min with helium as
carrier gas at 10 p.s.i. pressure. Samples were injected in a
septum programmable injector heated at 250 °C. Chemical
ionization was conducted on a Nermag R10-10C quadrupole mass
spectrometer, with ammonia as reactant gas at 10
To determine the R or S configuration of the
natural product, derivatization of standard and natural compound were
conducted as follows. A solution of pyridine (50 mg/ml) in dry
ether (15 µl) and a solution of S-lactyl chloride reagent
(26) (25 mg/ml) in dry methylene chloride (30 µl) was added to a
solution of standard commercially available 1-octen-3-ol (racemic or
R-( Crystallization, Data Collection, and Refinement of Bovine
OBP--
OBP crystals were obtained by micro-dialysis of a 10 mg/ml
protein solution against 28-32% ethanol, in 50 mM
citrate, pH 5.4 at 4 °C. The crystals belong to the space group
P21, with cell dimensions a = 55.9 Å,
b = 65.5 Å, c = 42.7 Å, and
The refinement of bovine OBP containing its natural ligand or AMA made
use of the previously determined bOBP structure as a starting model (8;
1OBP). The atomic structure of 1-octen-3-ol and of AMA were built with
the program TURBO-FRODO (29), and topology and force field data were
defined in the suitable files of CNS (30) by the automated
procedure XDICT (G.L. Kleywegt). The models were then refined using
CNS version 1.0 (30). Cycles of refinement were alternated with
manual refitting into sigmaA-weighted electron density maps (31) with
the graphic program TURBO-FRODO (29). The final model of native bOBP
has Rwork and Rfree
values of 20.3 and 22.7%, respectively (see Table I), and the final model of the bOBP·AMA complex has Rwork
and Rfree values of 21.0 and 23.7%,
respectively (Table I). The
(Fobs Fluorescence Binding Assay--
The fluorescence binding assay
of AMA to bOBP and the competition between AMA and 1-octen 3-ol were
realized according to the method published by Paolini et al.
(22), with minor modifications. To avoid interference with the protein
absorption band, the excitation wavelength was chosen at 380 nm, where
an AMA absorption band is observed, instead of 295 nm (22) or 255 nm
(23). The stock solutions of AMA (1-10 mM) in ethanol were
preserved in the dark at 4 °C and used within 4 days. The influence
of the concentration of ethanol on the chasing process of AMA was
tested and found to be negligible up to 1% v/v, a result in contrast
with the behavior of rat OBP reported in Briand et al. (23).
In the case of the direct binding, the titration curves were prepared
incubating different samples of bOBP (0.76 µM dissolved
in 20 mM Tris-HCl, pH 7.8, 0.5% ethanol (v/v)) with
various amounts of AMA ranging from 0.019 to 10 µM for
24 h at 4 °C. In the case of the competition curves, the bOBP
samples were incubated with a fixed amount of 2 µM AMA
and increasing concentrations of 1-octen-3-ol (0.39-50 µM). Fluorescence emission spectra between 450 and 550 nm
were recorded at a fixed excitation wavelength of 380 nm using a
PerkinElmer LS 50 luminescence spectrometer, and the formation of the
bOBP·AMA complex was detected by the increase of the fluorescence
emission intensity at 480 nm. The concentration of the complex was
evaluated on the basis of a calibration curve obtained by incubating
increasing concentrations of AMA (0.076-5 µM) with a
saturating amount of bOBP (10 µM). The binding and
competition curves were analyzed using the nonlinear fitting facility
of Sigma Plot 5.0 (Cambridge Soft Corp., Cambridge, MA).
Identification of the Ligand--
The bOBP organic extract
analyzed by gas chromatography yields a prominent peak (Fig.
1A). Comparison of control and
native or denatured bOBP showed a single peak difference in mass
spectra. After comparison with the Environmental Protection
Agency/National Institutes of Health library, the electronic
impact mass spectrum of this compound (ions at
m/z 41, 43, 55, 57 (100% base pic), 72, 81, 99, 110) was found to correspond to a C8 monoethylenic alcohol
(Fig. 1C). This was confirmed with CI/NH3 data exhibiting ions at m/z 128 and 146 ((M + NH4)+),
leading to a C8H16O formula. Ions at
m/z 57 and 72 (McLafferty rearrangement) in the
electronic impact mass spectrum and comparison of the retention
times of standards indicated the presence of a secondary alcohol and
led us to propose the structure of 1-octen-3-ol (Fig.
1C).
Tentative separation of the racemic standard on a chiral gas
chromatography column failed. The derivatization of 1-octen-3-ol yielded two peaks in the same gas chromatography conditions (50/50 ratio and electronic impact mass spectra with characteristic
ions at m/z 115 and 127 atomic mass
units). The derivatization of the natural compound under identical
conditions also yielded two peaks with identical mass, retention time,
and intensities (Fig. 1B).
Overall Three-dimensional Structure--
As described previously,
bOBP is a dimer consisting of 2 × 159 residues at neutral or
basic pH and monomerizes at pH values below 4.5 (20). The 2.0-Å
resolution structure of bOBP has been reported elsewhere (8). Briefly,
each monomer is composed of a lipocalin-type nine-strand Internal Cavities and the Buried Ligands--
Inside the
The structure of the bOBP·AMA complex reveals two very well ordered
AMA molecules in the buried cavities (Fig. 2A). This
indicates clearly that these cavities are the general binding site of
bOBP, as in pOBP (6, 13), and discredits the existence of a
putative third binding pocket previously proposed (8). The orientation of AMA in the binding pocket is comparable with that of 1-octen-3-ol, with the long axis perpendicular to the axis of the
The walls of these cavities are mostly composed of hydrophobic
residues. The water-accessible surface area of bOBP alone or with its
ligand was calculated for all the residues. The ligands cover 150 Å2 of each cavity surface. Residues Phe-36, Phe-89,
Asn-103, Tyr-83, and Phe-54 display the larger loss of surface
accessibility upon complexation (10-12 Å2) (Table
II). The residues involved in the
interaction with the ligand are identical in the two cavities and have
similar variation of accessibility. Furthermore, all the residues of
both cavities are superimposable, including Phe-89, which is found with
two different conformations in each cavity (Fig. 2E).
Fluorescence Binding Assay with AMA and Competition between AMA and
1-Octen 3-ol--
The fluorescence properties of AMA when surrounded
by a hydrophobic environment have already been used to probe binding in pOBP (20) and porcine salivary OBP (34). We have determined the
binding affinity of AMA for ligand-depleted bOBP by titrating at 480 nm
the increase in fluorescence signal of AMA upon complexation to the
protein (Fig. 3A). The hyperbolic titration curve, with a
maximum saturation level of 1.61 molecules of AMA per bOBP molecule, indicates a clear stoichiometry of two AMA molecules per bOBP dimer. The titration of non-extracted
bOBP yields a stoichiometry of 1.85 AMA molecules per bOBP dimer (data
not shown), indicating some degradation of bOBP during ligand
extraction. The Kd of AMA for bOBP is 1.0 µM, a value within the range found for most odors toward
OBPs (7, 12-16). Because x-ray crystallography experiments indicated
that AMA binds in the internal cavities of bOBP described in
the previous paragraph, competition of AMA by odors should titrate the
same binding site of bOBP, which is the functionally relevant one.
We have determined the binding affinity of 1-octen-3-ol (racemic) by
chasing saturating amounts of AMA bound to the internal cavities of
bOBP. The decrease of AMA fluorescence at 480 nm was recorded as a
function of 1-octen-3-ol concentration (Fig. 3B). The
competition curve has a hyperbolic decay with an apparent Kd value of 9.6 µM. The almost
complete displacement of AMA indicates a stoichiometry of two
1-octen-3-ol molecules per bOBP dimer. Taking into account the
K Identification of lipocalin-bound compounds based on mass
spectroscopy coupled to x-ray structure determination has already been
successful in the past, for example in the case of the major urinary
protein, where four pheromonal components were identified as copurified
ligands (11). This procedure failed, however, with aphrodisin, another
pheromonal lipocalin (35). In the present study, by using the same type
of procedure, we have identified a unique compound, the racemic
1-octen-3-ol, as the naturally occurring copurified ligand of bOBP.
Furthermore, both R and S isomers were found to
match nicely the electron density maps of the bOBP natural ligand at
1.8-Å resolution. The contacts between the ligand and amino acid
residues of the Previous studies in solution (1-5) and the recent structural
characterization of porcine OBP binding properties (13) have shown that
a high degree of hydrophobicity coupled to a molecular mass
between 160 and 200 daltons is the main requirement for a ligand to fit
the Moreover, among all potential ligands of OBPs, two groups can be
distinguished: exogenous compounds inhaled from the environment and
endogenous compounds produced by the animal. The endogenous compounds
can be produced in the nasal mucosa itself as consequence of
physiological epithelium turnover, inflammation, and injuries resulting
from the action of physical (temperature and humidity), chemical
(pollutants and oxidants passing through respiratory airways), and
biological (parasites, bacteria, and viruses) aggression. They can also
be produced and released by organs (lung, stomach, bloodstream, etc.)
and flow through the nasal cavity as breath components. In this
context, it is particularly striking to observe that 1-octen-3-ol has
an endogenous origin in bovine species and in ruminants in general
(37). It is produced in large quantities from fatty acids by
lipoxigenases (38) probably associated to the ruminal microflora and
then released in the environment as a component of the breath of the
animal (37).
It has also been demonstrated that 1-octen-3-ol has a relevant role in
the olfactory chemoreception-driven behavior of many worldwide insect
species, including parasite vehicles like Anopheles and
Glossina (38, 39). These insects express in their antennae two receptors specific for the breath components 1-octen-3-ol and
carbon dioxide, respectively. Their simultaneous stimulation drives
host-seeking behavior toward bovines through their breath components
(37). The identification of 1-octen-3-ol as the bOBP naturally observed
ligand suggests that bOBP might be used by bovines to remove parts of
1-octen-3-ol from the breath flowing through the nasal cavities and to
make them less appealing for several insect species. This would result
in a general decrease of the number of insect bites and furthermore
might partially protect the animal from parasitosis and infectious
diseases carried by these insect vectors.
This hypothesis on the possible role of OBP in the ecological
relationships between bovine and insect species must be limited, at
present, to the case of bovine species, because only in this case has
the presence and the role of the breath component 1-octen-3-ol in the
ecological relationships with insects been extensively documented.
Besides, this function should not be taken as an alternative to the
other roles previously proposed, such as that of a binder of exogenous
compounds. All the possible putative roles of OBPs in mammalian
physiology, toward either endogenous or exogenous compounds, are
compatible with their above-mentioned capacity to bind several classes
of small molecules, with their stability, and with their abundant
production and presence in nasal mucosa.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-barrel and a C-terminal
-helix (6, 7). Bovine
OBP is a dimer in which the C-terminal domain (residues 125-159) is
swapped between the two monomers, a peculiar feature that seems to be specific to the bovine species (8, 9). An elongated volume of electron
density was observed in each of the two internal buried cavities. In
the 2.0-Å resolution electron density we assigned this electron
density to an unknown molecule of 8-10 non-hydrogen atoms (8),
whereas Bianchet et al. (9) tentatively assigned it to a
terpenoid compound, citronnellyl acetate. Several structures of
lipocalins have revealed the presence of specific or nonspecific copurified ligand in their binding sites, such as retinol in
retinol-binding protein (10) and pheromones in the major urinary
protein (11). The structure of porcine OBP, instead, revealed an empty
cavity (12).
-barrel cavities, independently of its odorous properties, chemical
class, and molecular structure. Some of the ligands of pOBP have a very
low olfactory threshold (13), whereas others, for instance the toxic
7-11 carbon alkyl aldehydes (14), cannot be considered real odorous
compounds (15). The Kd values, obtained or estimated
from direct and competitive equilibrium binding experiments, are in the
micromolar range for most of the OBP ligands (1, 13-18). The peculiar
binding properties of OBPs with respect to other lipocalins (low ligand
specificity and low affinities) and the very high quantities of protein
found in nasal epithelium have suggested that OBPs might
function as odorant carriers and/or scavengers for olfactory receptors
or that they might be involved in endogenous detoxification processes
(19). In this latter hypothesis it has been proposed that OBPs might deliver, to the appropriate degradative pathways, some toxic compounds produced during nasal epithelium turnover and inflammatory processes in
charge of the nasal mucosa (19). These two hypotheses are not mutually
exclusive, and at present, no direct experimental evidence supports or
excludes any of them. On the contrary, the present data suggest that
the two functions might be carried out in parallel. In the present
work, the unambiguous identification of the copurified natural ligand
of bovine OBP as the insect attractant 1-octen-3-ol makes it possible
to hypothesize a role for bOBP in the ecological relationships between
bovine and several insect species.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
cm
1/bOBP dimer) at 280 nm was determined
according to the Edelhoc method (24) as described by Pace
et al. (25). The natural ligand was extracted from samples
of native and denatured OBP with different organic solvents (diethyl
ether, methylene chloride, and hexane). The protein (a 7.0-mg/ml OBP
solution in 20 mM Tris-HCl, pH 7.8) was previously
denatured by overnight incubation at room temperature in the presence
of 7.5 M urea. The ligand extraction was performed by
vortexing mixtures of OBP-organic solvent in glass tubes (1:1
volumetric ratio), and the organic phases were analyzed by GC/MS.
Aliquots of Tris buffer and Tris buffer containing 7.5 M
urea were treated with the same extraction procedure, and the organic
phases were analyzed by GC/MS as blanks.
4
pressure in the source housing. Spectra were recorded with a mass range
of 70-300 atomic mass units. The mass spectrometer was coupled
to a Varian 3400 gas chromatograph equipped with a Ross injector heated
at 240 °C and a similar column used with the same program temperature.
)) (50 ng) in hexane (30 µl) or natural extract in
hexane (40 µl) placed in a micro-conic flask. Closed flasks were kept
at room temperature for 30 min. The solution was then diluted with
hexane (50 µl) and washed successively with water (50 µl), aqueous
5% sodium bicarbonate (2 × 50 µl), and water (50 µl). The
samples were directly analyzed by GC/MS. Four samples of bOBP coming
from animals of different geographical origin yielded indistinguishable results.
= 98.8°, and contain one homodimer in the asymmetric unit. Data were
collected on a Mar-research 345 image plate placed on a Rigaku RU2000
rotating anode. Data collection was performed at room temperature,
because cryocooling always yielded data sets that could not be used in refinement. Indexation and integration were performed with DENZO (27);
data scaling was performed with SCALA (28); and data reduction was
performed by TRUNCATE (28) (see Table I). The bOBP·AMA complex
was obtained by soaking the crystals in a synthetic crystallization
solution containing 2 mM AMA overnight at 4 °C. Data
collection, scaling, and reduction were performed as with native bOBP
(see Table I).
Fcalc)exp(ia calc) maps did not show any uninterpretable features. The final models have a
good geometry according to the PROCHECK criteria (32), with 91.1% of
residues located in the most favorable area, 8.9% in additional
allowed regions, and no residues in forbidden zones. The coordinates
have been deposited in the Protein Data Bank with accession numbers
1G85 (native) and 1HN2 (AMA).
Data collection and final refinement statistics
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Gas chromatography-coupled mass spectroscopy
of the bOBP extracts. A, chromatogram of the bOBP
extract displaying a prominent peak (arrow). B,
derivatization of the compound identified in A to identify
the presence and quantity of each enantiomer (see "Material and
Methods"). C, mass spectra of the main peak identified by
gas chromatography in A (bottom) and of the
authentic 1-octen-3-ol (top).
-barrel
comprising residues 15-121 (strands 1-8) and residues 145-149
(strand 9) from the other monomer (Fig.
2A). From residue 123 onwards,
the topology diverges from the consensus lipocalin fold. The
-barrel
is connected by an extended stretch of residues 123-126 to the
-helix protruding out of the
-barrel and crossing the dimer
interface (Fig. 2A). As a consequence, the
-helix of one
monomer is placed close to where the
-helix of the other monomer
would be if bOBP had a classical lipocalin fold, in a special
arrangement called domain swapping (33). In the present structure, the
two bOBP polypeptidic chains are visible from residues 1-159 and
3-157 for molecules A and B, respectively.
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Fig. 2.
The internal ligand binding site of bovine
OBP. A, view of bovine OBP C trace with the two
buried cavities, containing the fluorescent probe AMA and
(B) the natural ligand 1-octen-3-ol. C,
2Fo - Fc
A electron density
maps of the racemic 1-octen-3-ol natural ligand in the cavity A of bOBP
and (D) in the cavity B. E, stereo view of the
superimposed binding cavities A and B with the racemic 1-octen-3-ol
natural ligand bound; note the alternative conformations of the ligand
and of Phe-89.
-barrel of each monomer, a large buried cavity of about 407 Å3 is observed (Fig. 2A), at a location similar
to that observed in other closed lipocalins, such as the major urinary
protein (11). Both cavities contain an elongated patch of electron
density map, which has been attributed to an 8-10 non-hydrogen atom
linear compound in the structure at 2.0-Å resolution (8). Because of the significant improvements in resolution (1.8 Å), data treatment, map calculation, and refinement procedures, interpretable features came
out in the difference maps within the buried cavities. Having determined the nature of the ligand by GC/MS, we could fit a molecule of 1-octen-3-ol in each difference electron density map. After CNS refinement, significant patches of electron density still remained in the difference Fourier maps. The two enantiomers of 1-octen-3-ol were then fitted in the most appropriate conformations, and their occupancies were refined in CNS. The resulting
structure contains both enantiomers in a ratio close to 1, a result in
accord with the GC/MS data (Fig. 2, C and D). In
cavity A, the two isomers have very similar orientations and are
quasi-superimposed (Fig. 2C). The aliphatic chains of
the two isomers are extended and remain close to each other.
The hydroxyl groups point in the same direction, toward an area of the
cavity where no hydrogen bond donor is available, however. The
positions of the two 1-octen-3-ol enantiomers in the two cavities are
slightly different (Fig. 2, C and D), because of
a rotation around the
atom. This movement leaves an empty space in
the cavity, between the aliphatic chain of the ligands and the O
atom of Thr-38, which is filled by a water molecule hydrogen-bonded to
the O
atom (Fig. 2E).
-barrel (Fig. 2,
A and B).
Interactions of the natural ligand, the racemic 1-octen-3-ol, with the
residues of bOBP internal cavities
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Fig. 3.
Fluorescence titration of the bOBP binding
site. A, fluorescence titration curve of bOBP with AMA.
Before the experiment, the natural ligand was removed from the protein
by urea denaturation and organic solvent extraction (see "Materials
and Methods" for details). B, competition curve between
1-octen-3-ol and AMA on bOBP. The concentration of 1-octen-3-ol is
reported versus the residual concentration of AMA bound to
bOBP (see "Materials and Methods" for details).
(Eq. 1)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-barrel cavities have been characterized at the
atomic level. The x-ray structure of the fluorescent probe AMA has
revealed that it binds in the same cavity as 1-octen-3-ol. Although
several reports of OBP fluorescence titration with AMA are available in
the literature, we provide here the first direct evidence that AMA
binds in the lipocalin internal pocket. This gives more weight to
fluorescence experiments based on ligand displacement, such as
those described here. All these results converge to unambiguously
identify 1-octen-3-ol (R,S) as the natural
copurified ligand of bOBP.
-barrel cavities of OBP, irrespective of the chemical class,
substituents, and molecular structure. These compounds can be odorous
chemical messengers that strongly contribute to mediate the
relationship between an individual animal and its environment, in food
search, mating, etc. (36), or toxic compounds such as natural and
synthetic pollutants (19).
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FOOTNOTES |
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* This work was supported in part by a BIOTECH contract from the European Union (BIO4-98-0420).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G85 and 1HN2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence may be addressed. Tel.: 39-0521-90-27-67; Fax: 39-0521-90-27-70; E-mail: vetbioc@unipr.it.
** To whom correspondence may be addressed. Tel.: 33-491-164-512; Fax: 33-491-164-536; E-mail: tegoni@afmb.cnrs-mrs.fr.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M010368200
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ABBREVIATIONS |
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The abbreviations used are: OBP, odorant-binding protein; pOBP, porcine OBP; bOBP, bovine OBP; AMA, 1-aminoanthracene; GC/MS, gas chromatography/mass spectrometry.
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