Figure 1.
Alignments and Phylogenetic analysis of McinOBP1.
(A) Alignment of McinOBP1(KF900276) from hymenopteran insects as well as other insects. Microplitis mediator (MmedOBP8, AEF14409)\ Rhynchophorus palmarum (RpalOBP2, AAD31883)\ Tribolium castaneum (TcasOBP5, EFA05677)\ Aedes aegypti (AaegOBP56a, XP_001658489)\ Nasonia vitripennis (NvitGOBP lush, XP_001603472)\ Aedes albopictus (AalbOBP10, AGI04311)\ Nasonia vitripennis (NvitOBP3a, CCD17772)\ Nasonia vitripennis (NvitOBP3b, CCD17860)\ Sclerodermus guani (SguaOBP2, ABE68831)\ Microplitis mediator (MmedOBP10, AEO27860)\ Dendroctonus ponderosae (DponOBP, ENN77432)\ Aedes albopictus (AalbOBP14, GI04314)\ Aedes albopictus (AalbOBP2, AFC60563)\ Aedes albopictus (AalbOBP13, AGI04313)\ Delia antiqua (DantOBP8, BAI82448)\ Argyresthia conjugella (AconOBP5, AFD34173)\ Locusta migratoria (LmigOBP1e, ACR39385)\ Laodelphax striatella (LstrOBP4, AEQ19910)\ Nasonia vitripennis (NvitOBP72, CCD17841). Six conserved cysteine residues are highlighted with black background (B) Phylogenetic tree of McinOBP1 amino acid sequences in others insects.
Figure 2.
Expression pattern analysis of McinOBP1.
Transcript level of M. cingulum odorant binding protein 1 (McinOBP1) in different tissues of adult male and females measured by qPCR. cDNAs were amplified with specific primers from antennae, heads (without antennae),thoraxes, abdomens, legs, and abdomen.
Figure 3.
SDS-PAGE analysis of expressed recombine McinOBP1.
Lane-1: Non-induced pET32a (+)/McinOBP1 transformed BL21 (DE3) cells; 2: IPTG induced E. coli pET32a (+)/McinOBP1 transformed BL21(DE3) cells; 3. Supernatant after ultrasonic treated pET32a (+)/McinOBP1 cells; 4: Inclusion body of pET32a (+)/McinOBP1 cells; 5: Ni–NTA columns purified proteins; 6. Purified protein cleaved His-tag by rProtease; 7: Molecular weight marker.
Figure 4.
Binding of 1-NPN and various ligands to McinOBP1.
(A) Binding curves and relative Scatchard plot (insert) of N-phenyl-1-naphthylamine (1-NPN) and McinOBP1 protein. Two μM solutions of protein was titrated with 1 μM solution of 1-NPN in methanol to final concentrations of 2–20 μM. (B) Competitive binding affinities of McinOBP1-wt to different aldehyde ligands. (C) Binding affinities of various terpenoids to McinOBP1-wt and (D) Affinities of McinOBP1-wt to aliphatic alcohol and others.
Table 1.
IC50 values and calculated dissociation constants, KD (μM) for the complexes between McinOBP1-wt and different ligands at pH = 7.4.
Figure 5.
Affinities of selected odorants: trans-2-nonenal, cis-3-hexen-1-ol, and β-caryophelle to M. cingulum odorant-binding protein 1 (McinOBP1). The fluorescence intensities are plotted against increasing ligand concentrations.
Figure 6.
3D structure model of the M. cingulum odorant binding protein 1.
(A) Predicted 3D model of McinOBP1 was built on the reference of structure of OBP1 from the Mosquito Aedes aegypti. Six α-helixes, N-terminal (Nt) and C-terminal (Ct) are marked. (B) Sequence alignment of McinOBP1 and AaegOBP1. In the alignment of the two proteins, conserved cysteines are highlighted in green and identical residues are highlighted in light blue.
Figure 7.
Docking of M. cingulum odorant binding protein 1 with ligands.
Molecular docking of McinOBP1-wt with (A) trans-2-nonenal; (B) cis-3-hexen-1-ol and (C) β-caryophelle. The predicted key amino acids residues are shown in red and respective ligands shown in green.
Table 2.
The interaction energies (kcal/mol) between the key residues of McinOBP1-wt and ligands.
Figure 8.
Binding of 1-NPN and ligands to McinOBP1 mutants.
(A) Affinities of 1-NPN to the McinOBP1-wt and three mutants. Dissociation constants (KD) were 6.60 μM for McinOBP1-wt, 14.62 μM for McinOBP1-m1, 13.69 μM for McinOBP1-m2 and 6.53 μM for McinOBP1-m3 are shown in inset. (B) Dissociation constants of complexes between McinOBP1-wt and its three mutants (McinOBP1-m1to 3) and the three selected odorants. These three mutants showed lower binding affinity to selected three odorants with respect to the wild type, indicating that the respective amino acids are involved in binding these ligands.
Table 3.
Affinities of McinOBP1-wt and three mutants to trans-2-nonenal, cis-3-hexen-1-ol and trans- caryophelle.