Table 1.
Gene-specific primers used for expression analyses of GmolGOBPs and for site-directed mutagenesis of GmolGOBP2 key amino acid residues.
Fig 1.
SDS-PAGE analysis of rGmolGOBP1 (A) and rGmolGOBP2 (B) in Escherichia coli.
M. Protein molecular weight marker; 1. Non-induced pET28a(+)/GmolGOBP1transformed BL21 (DE3) cells; 2. Induced pET28a(+)/GmolGOBP1transformed BL21 (DE3) cells; 3. Supernatant of disrupted pET28a(+)/GmolGOBP1 IPTG induced cells; 4. Inclusion body of disrupted pET28a(+)/GmolGOBP1 IPTG induced cells; 5. Purified rGmolGOBP1; 6. Non-induced pET28a (+)/GmolGOBP2 transformed BL21 (DE3) cells; 7. Induced pET28a(+)/GmolGOBP2 transformed BL21 (DE3) cells; 8. Supernatant of disrupted pET28a(+)/GmolGOBP2 IPTG induced cells; 9. Inclusion body of induced pET28a(+)/GmolGOBP2 IPTG induced cells; 10. Purified rGmolGOBP2.
Fig 2.
Immunolocalization of GOBP1 and 2 in the antennae of G.molesta.
Whole mount preparations were probed with antisera specific for GmolGOBP1 (A. female, B. male) and GmolGOBP2 (C. female, D. male). Immunoreactivity was visualized by 488-AffiniPure secondary antibody. A high number of GmolGOBP1 and 2-expressing cells are visible and can be located in sensilla basiconica. E (female), F (male) and G (female), H (male) represent control experiments with GmolGOBP1 and 2 pre-immune serums producing no labeling, respectively.
Fig 3.
Binding curves of 1-NPN and relative Scatchard plots for rGmolGOBP1 and 2.
A. rGmolGOBP1; B. rGmolGOBP2. A 2 μM solution of each protein in 20 mM Tris-HCl buffer (pH 7.4) was titrated with a 1 mM 1-NPN solution in spectrophotometric-grade methanol to final concentrations of 2 μM to 22 μM, and the emission spectra were recorded between 370 and 550 nm. The dissociation constants of rGmolGOBP1 and 2 were 11.44 and 4.73 μM, respectively.
Table 2.
Binding affinities of ligands to GmolGOBP1 and 2 evaluated via competitive binding assays by using the fluorescent probe, 1-NPN.
Fig 4.
Fluorescence competitive binding curves of rGmolGOBP1 to sex pheromone components and host plant volatiles.
A. rGmolGOBP1 to sex pheromone components; B, C, D, F. rGmolGOBP1 to aldehydes, alcohols, esters and alkanes; E. rGmolGOBP1 to terpenoids and benzonitrile. Mixtures of proteins and 1-NPN, at a 2 μM concentration, were titrated with 1 mM of each sex pheromone component and host-plant volatile to final concentrations of 0 μM to 22 μM and 0 μM to 64 μM, respectively. Relative fluorescence intensities are shown as a percent of the initial fluorescence without competitors.
Fig 5.
Fluorescence competitive binding curves of rGmolGOBP2 to sex pheromone components and host plant volatiles.
A. rGmolGOBP2 to sex pheromone components; B, C, D, F. rGmolGOBP2 to aldehydes, alcohols, esters and alkanes; E. rGmolGOBP2 to terpenoids and benzonitrile. Mixtures of proteins and 1-NPN, at a 2 μM concentration, were titrated with 1 mM of each sex pheromone component and host-plant volatile to final concentrations of 0 μM to 22 μM and 0 μM to 100 μM, respectively. Relative fluorescence intensities are shown as a percent of the initial fluorescence without competitors.
Fig 6.
Effects of pH on the binding of rGmolGOBP2 with dodecanol.
The rGmolGOBP2 and 1-NPN were both at 2 μM. The mixture solution was titrated with 1 mM of dodecanol to final concentrations of 14 μM. The Ki value was calculated as previously mentioned. The solutions were excited at 337 nm, and emission spectra were monitored between 370 and 550 nm. All data represent a mean of three independent measurements, and the bars denote mean±SEM. Values that not share the same letter have significant difference (S–N–K, P<0.05).
Fig 7.
Modeled 3D structure and molecular docking experiments of GmolGOBP2.
(A) Sequence alignment of GmolGOBP2 and BmorGOBP2. α-helices are displayed as squiggles. Strictly identical residues are framed and highlighted with a red background. (B) Overall structure of the GmolGOBP2 and the docking result. Three disulfide bridges are colored in yellow. N-terminal, C-terminal and α-helices are labeled. The top three potential key residues, Thr9, Val111 and Val114, are labeled in black font. Dodecanol is shown as a stick model with the hydroxyl oxygen in red.
Table 3.
The interaction energies between dodecanol and potential binding sites in the binding pocket of GmolGOBP2.
Fig 8.
SDS-PAGE analysis of rGmolGOBP2 wild-type (WT) and mutants T9A, V111A and V114A.
Fig 9.
Comparison of the binding properties of GmolGOBP2 wild-type (A) and mutants T9A (B), V111A (C), V114A (D).
The recombinant protein and 1-NPN were diluted to a fixed concentration of 2 μM. The mixture solution was titrated with 1 mM of each ligand to final concentrations of 0 μM to 14 μM. Relative fluorescence intensities are shown as a percent of the initial fluorescence without competitors. All data are an average of three independent measurements. Structures of ligands decanol, dodecanol, tetradecanol and hexadecanol (E) were plotted by ChemSketch.
Table 4.
Binding affinities of dodecanol and its analogs to rGmolGOBP2 wild-type and mutants.