Three-dimensional folding of CquiOBP1 (acc. No. 3OGN) of <i>Culex quinquefasciatus</i> and model of HarmOBP10.

<p>The model of HarmOBP10 was based on the structure of CquiOBP1. The two proteins share only 19% of their residues, but the overall folding appears rather similar, apart from two extra loops in HarmOBP10. The amino acid residues lining the binding pockets in the two proteins, however, appear rather different. HarmOBP10 contains a relatively large number of aromatic groups, Phe10, Tyr50, Tyr68, Phe114 and Phe126, as well as other hydrophobic residues, such as Ile14, Val45 and Ile64. Of these only Phe126 is also present in CquiOBP1,while Tyr50 is replaced in the mosquito protein by a Phe. Such differences can be related to the different structures of the ligands in the two proteins, a cyclic terpenoid molecule for CquiOBP1 and linear ligands for our OBP10. Amino- and carboxy termini are indicated as N and C, respectively. Models have been generated using the programme Swiss-Model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030040#pone.0030040-Guex1" target="_blank">[42]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030040#pone.0030040-Arnold1" target="_blank">[44]</a>. Models were displayed using the SwissPdb Viewer programme “Deep-View” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030040#pone.0030040-Guex1" target="_blank">[42]</a> (<a href="http://www.expasy.org/spdbv/" target="_blank">http://www.expasy.org/spdbv/</a>).</p

SDS-PAGE (upper panels) and Western blot analysis (lower panels) of crude extracts from reproductive organs.

<p>Reproductive organs include male accessory glands and testes, and female accessory glands and ovaries, dissected from virgin individuals of different ages. Numbers indicate days after emergence. Only male extracts of both species present an intense cross-reacting band at the expected molecular weight of OBP10. Molecular weight markers (<b>M</b>) are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030040#pone-0030040-g002" target="_blank">Figure 2</a>.</p

Values of [IC]₅₀ and calculated dissociation constants (µM) for the complexes between HarmOBP7-wt and various ligands.

<p>Values of [IC]₅₀ larger than 16 have been extrapolated from the competitive binding curves.</p

Amino acid sequences for the new OBPs identified in <i>H. armigera</i> and <i>H. assulta</i>.

<p>Differences between orthologous proteins in the two sibling species are limited to only one or few amino acid substitutions. The similarity tree on the right illustrates graphically the sequence similarity relationships between the 18 OBPs of <i>H. armigera</i> so far available. In particular, the newly identified OBP11 (a C-minus OBP with only four cysteines) and OBP12 (a C-plus OBP with 14 cysteines) fall outside the main branches of the tree. Accession numbers are as follows. HarmOBP10: JN571544; HarmOBP11: JN571545; HarmOBP12: JN571543; HassOBP1: JN571537; HassOBP2: JN571536; HassOBP4: JN571541; HassOBP5:JN571540; HassOBP6: JN571535; HassOBP8: JN571542; HassOBP10: JN571539; HassOBP12: JN571538.</p

Immunostaining of OBP10 on the eggs of <i>H. assulta.</i>

<p>Eggs that had been laid on a piece of cotton were directly stained with the antiserum against OBP10, following the same protocol as for Western blot analysis. The upper panels show fertilized eggs at different magnifications. The lower panels show parallel experiments performed with unfertilized eggs, collected from females that had never been in contact with males. The heavy staining is specifically associated with fertilized eggs on the tip opposite to the micropyle. Calibration bars: 100 µm.</p

Bacterial expression and purification of HarmOBP10.

<p>The protein was obtained in high yields (about 30 mg/L of culture) as insoluble inclusion bodies and had to be denatured and renatured in order to be solubilised. Purification was accomplished by two chromatographic steps on anion-exchange resin DE-52 (Whatman). The figure reports the SDS-PAGE analysis relative to crude bacterial extracts before (<b>Pre</b>) and after (<b>Ind</b>) induction with IPTG, the crude solubilised protein (<b>SN</b>) and fractions relative to the last step of purification. Molecular weight markers (<b>M</b>) are: Bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa) and a-lactalbumin (14 kDa).</p

GC/MS analysis of dichloromethane extracts of reproductive organs of males and females <i>H. assulta.</i>

<p>The selected fractions (upper trace) from a Superose-12 fractionation of male reproductive organs. SDS-PAGE and Western blot analysis revealed the presence of OBP10 mainly in fraction 15. The same fraction also showed to contain 1-dodecene at higher concentration than the others. Peaks in the GC/MS profile were identified as (1) (<i>E</i>)-2-heptenal, (2) 1-dodecene, (3) dodecene (position of double bond not identified), (4) nonanal, (5) decanal. The identity of 1-dodecene has been confirmed with an authentic sample analysed in the same conditions.</p

SDS-PAGE and Western blot of extracts from parts of the body of adults <i>H. armigera</i> and <i>H. assulta</i>.

<p>Upper panels: SDS-PAGE; lower panels: Western blot. (A); antennae, (P): proboscis, (T): tarsi, (W): wings of males (m) and females (f). The expression of OBP7 is limited to antennae with no significant differences between sexes or species. A weak staining in the extract of tarsi might indicate low levels of expression of OBP7 in such organ or cross-reactivity with other OBPs. Molecular weight markers (<b>M</b>) are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055132#pone-0055132-g001" target="_blank">Figure 1</a>.</p

Binding affinities of ligands to recombinant HarmOBP10.

<p>The maximum concentration used for the binding assays and the percent of fluorescence measured at that concentration are reported, together with the concentration of ligand halving the initial fluorescence value (IC₅₀) and the calculated dissociation constants (K_D).</p

Affinities of <i>Harm</i>OBP7-wt to series of linear saturated primary alcohols, aldehydes and ethyl esters.

<p>(<b>A–C</b>) Displacement curves obtained with compounds of the three series. Solutions of 2 µM proteins and 2 µM 1-NPN were titrated with 1 mM solution of each ligand in methanol to final concentrations of 0.5 to 6 µM. (<b>D</b>) Dissociation constants as function of chain length. Best affinities are observed with linear compounds of 13–14 carbon atoms.</p