Repellent activity
Our team tested the repellent activity of terpenoids (1R)-(+)-α-Pinene and β-Caryophyllene against T. castaneum, which are widely present in the EOs with repellent effect and have high content. The experimental results are shown in Table 1. The repellent activity of the two terpenoids gradually increased with increasing sample concentration and β-Caryophyllene showed the strongest repellent activity (Class Ⅴ) at the dose of 78.63 nL/cm2. At the range of 78.63–0.63 nL/cm2, β-Caryophyllene has better repellent activity than (1R)-(+)-α-Pinene. At the five testing concentrations, the PR values of β-Caryophyllene was higher than the positive control, DEET. The repellent activity of (1R)-(+)-α-Pinene was slightly higher than that of DEET and the class are very close. The RP values of these two compounds did not change significantly at 2h and 4h respectively. In brief, the results indicated that (1R)-(+)-α-Pinene and β-Caryophyllene had obvious repellent effect against T. castaneum.
Table 1
Percentage repellency (PR) of T. castaneum exposed to different concentrations of (1R)-(+)-α-Pinene and β-Caryophyllene for 2 h and 4 h.
Sample | concentration | PR(%) (Mean ± SE) | Class | PR(%) (Mean ± SE) | Class |
(nL/cm2) | 2h | | 4h | |
(1R)-(+)-α-Pinene | 78.63 | 78 ± 13.04 | Ⅳ | 78 ± 14.04 | Ⅳ |
15.73 | 68 ± 22.81 | Ⅳ | 60 ± 24.50 | Ⅲ |
3.15 | 44 ± 24.08 | Ⅲ | 44 ± 15.17 | Ⅲ |
0.63 | 34 ± 11.40 | Ⅱ | 36 ± 16.73 | Ⅱ |
0.13 | 28 ± 8.37 | Ⅱ | 26 ± 8.94 | Ⅱ |
β-Caryophyllene | 78.63 | 82 ± 16.43 | Ⅴ | 82 ± 13.04 | Ⅴ |
15.73 | 78 ± 8.37 | Ⅳ | 78 ± 17.89 | Ⅳ |
3.15 | 66 ± 11.40 | Ⅳ | 64 ± 13.42 | Ⅳ |
0.63 | 58 ± 21.68 | Ⅲ | 58 ± 13.04 | Ⅲ |
0.13 | 26 ± 11.40 | Ⅱ | 32 ± 14.83 | Ⅱ |
DEET* | 78.63 | 70 ± 2.56 | Ⅳ | 67 ± 2.57 | Ⅳ |
15.73 | 56 ± 4.34 | Ⅲ | 58 ± 2.46 | Ⅲ |
3.15 | 48 ± 3.58 | Ⅲ | 45 ± 5.82 | Ⅲ |
0.63 | 35 ± 6.87 | Ⅱ | 30 ± 3.67 | Ⅱ |
0.13 | 22 ± 2.48 | Ⅱ | 18 ± 3.43 | Ⅰ |
* DEET as positive control |
We are the first to study the repellent activity of (1R)-(+)-α-Pinene and β-Caryophyllene against T. castaneum. The research indicated that α-Pinene and β-Caryophyllene have repellent effect on a variety of insects. Jacob D et al.found that α-pinene had repellent activity (Class IV) against Sitophilus zeamais at the testing concentrations of 2,3,4 ppm and moderate repellent activity (Class III) at the concentrations of 1 ppm (Langsi et al. 2020). Carmenza E et al. found that β-Caryophyllene repelled Hypothenemus hampei by olfactometer and behavioral tests (Góngora et al.2020). This may be due to the presence of OBPs in these insects that can bind to α-Pinene and β-Caryophyllene to exert repellent effect, and the specific mechanism needs further verification.
Analysis Of Homologous Modeling And Molecular Simulation Docking Results
Table 2 displayed TcOBP-9B and TcOBP-10B were the target proteins with higher template protein identity, while the expression of TcOBP-9B in antennae was significantly higher than that of TcOBP-10B (Sun et al. 2012; Li et al. 2020).
Table 2
Part homology modeling results of TcOBPs for template protein identity greater than 30%.
TcOBPs | Template | GMQE | Identity |
TcOBP-5B | 3k1e | 0.59 | 37.07 |
2erb | 0.59 | 36.21 |
3ogn | 0.59 | 35.34 |
TcOBP-5C | 2erb | 0.60 | 36.61 |
3ogn | 0.60 | 36.61 |
2l2c | 0.56 | 36.61 |
TcOBP-5D | 6hhe | 0.53 | 34.55 |
6jpm | 0.62 | 33.33 |
3ogn | 0.54 | 30.91 |
TcOBP-5F | 2erb | 0.54 | 39.73 |
TcOBP-9A | 6jpm | 0.52 | 38.66 |
TcOBP-9B | 6jpm | 0.74 | 45.38 |
3r72 | 0.57 | 31.93 |
TcOBP-4A | 6qq4 | 0.60 | 31.09 |
TcOBP-4C | 4z39 | 0.52 | 30.28 |
TcOBP-7B | 1c3z | 0.64 | 37.96 |
1c3y | 0.64 | 37.96 |
TcOBP-9C | 3r1v | 0.58 | 30.00 |
3r1o | 0.58 | 30.00 |
TcOBP-10B | 1c3z | 0.68 | 61.68 |
1c3y | 0.50 | 31.68 |
Molecular simulation docking could more intuitively observed the interaction between protein and ligands. The possible binding modes and binding sites of ligands and protein could be predicted by the molecular simulation docking software Auto-Dock Tools 1.5.6. Through the molecular simulation docking of all 50 TcOBPs with 4 terpenoids, 11 TcOBPs interacted with anti-insect active components, including 6 antennal binding proteins (ABPIIs), namely TcOBP-5B, 5C, 5D, 5F, 9A and 9B, two classic OBPs, including TcOBP-4A and 4C, and three C-OBPs were TcOBP-7B, 9C and 10B, respectively. Through homologous modeling, it is found that the TcOBP-9B protein possessed a total of 119 amino acid residues, 6 α-helices, and its three-dimensional structure was shown in Fig. 2. Among them, the 4th to 22nd residues constitute the α-1 helix; the 26th to 34th residues constitute the α-2 helix; the 41st to 54th residues constitute the α-3 helix; residues 64th to 73th constitute the α-4 helix; residues 76th to 89th constitute the α-5 helix; and residues 98th to 111st constitute the α-6 helix. A hydrophobic cavity is formed between the α-helices.
The small molecule conformation with the lowest binding energy was selected for subsequent analysis of molecular simulation docking results (Hamdi et al. 2015). The molecular simulation docking results of the interaction between TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene were shown in Fig. 3. As we know, protein-ligand binding is usually affected by many factors, such as protein-binding cavity size, shape, ligand binding sites, protein key residues and pH, etc. Related studies have shown that hydrophobic forces play an important role in the interaction between insect OBPs and odorants, and the three-dimensional hydrophobic domain formed by six α-helixes in insect OBPs may be a key position in the interaction with external odorants (Qin er al. 2021; Li et al. 2015). In addition, some residues in the binding cavity also play a role in protein binding ligands. It can be seen from the figure that (1R)-(+)-α-Pinene mainly acts on the two α-helices of α-5 and α-6, and β-Caryophyllene mainly acts on several other α-helices except α-2, and mainly through hydrophobic forces (red eyelash line in Fig. 3).
(1R)-(+)-α-Pinene, β-caryophyllene and TcOBP-9B were selected for follow-up research, because TcOBP-9B was highly expressed in antennae, and in molecular simulation docking, ligand pinene and caryophyllene showed good interaction with TCOBP-9B. The above reasons were the basis for us to screen ligands and target proteins for further study.
Analysis Of Target Protein Expression And Purification Results
This study theoretically evaluated the hydrophobicity, signal peptide, transmembrane domain, etc. of TcOBP-9B target protein sequence. According to the evaluation results, the plasmid construction scheme was designed. The plasmid was constructed by total gene synthesis and subcloned into the pCOLDII expression vector, further transformed into E. coli competent cells, cultured, induced expression, collected bacteria, and purified the protein. The results of expression and purification assays of TcOBP-9B were shown in Fig. 4. After the fusion protein was purified, it was analyzed by SDS-PAGE electrophoresis, and an obvious band appeared at the position near the theoretical molecular weight, as shown in Fig. 4. Therefore, it can be judged that the fusion protein was successfully purified. Finally, 3.2 mg of recombinant TcOBP-9B with the His-tag was obtained through validation. The protein concentration was determined to be 0.64 mg/ml using a non-interfering protein quantification kit Cat. No.: C503071.
Analysis Of Fluorescent Probe Suitability Test Results
At room temperature, the suitability of 1-NPN as a fluorescent probe of TcOBP-9B was tested. The experimental results showed that the maximum fluorescence value of TcOBP-9B combined with 1-NPN gradually increased and tended to be saturated with the increase of 1-NPN concentration. The binding curve of the fluorescence values of TcOBP-9B and 1-NPN was analyzed by the Scatchard equation, which showed a linear correlation and the linear equation of y=-0.3936x + 0.8296 (R²=0.9763). The binding curve and equation analysis result of TcOBP-9B-1-NPN were shown in Fig. 5. According to the above experimental results, 1-NPN and TcOBP-9B were bound in a 1:1 binding manner. Thus, 1-NPN could be used as a fluorescent probe for TcOBP-9B for subsequent fluorescence experiments.
Analysis Of Fluorescence Quenching Results
Relevant studies have shown that aromatic amino acid residues in protein molecular structure, such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr), were the main sources of protein fluorescence (Levine 1977). When the TcOBP-9B interacted with (1R)-(+)-α-Pinene and β-Caryophyllene compounds, the fluorescence intensity of the protein would change. Therefore, the interaction between the protein and the ligand could be studied by monitoring the fluorescence quenching of the protein fluorescent group. As shown in Fig. 6, in the presence of (1R)-(+)-α-Pinene and β-Caryophyllene, the TcOBP-9B exhibited fluorescence quenching at three different temperatures with a concentration-dependent trend, indicating there were the interaction between the ligand and TcOBP-9B. These results confirmed the conclusion of molecular docking in part 3.1 and proved the stability and compatibility of docking.
Analysis Of Fluorescence Quenching Mechanism
The types of fluorescence quenching included dynamic and static quenching. When the temperature increased, if it was dynamic quenching, the collision between excited fluorescent molecules and the quencher would become more intense, and the value of fluorescence quenching rate constant (Kq) value would also increase; while static quenching was the opposite, the complexes would be formed between the protein and quencher during the static quenching, and the relevant quenching parameters would decrease with the increase of temperature (Wang et al. 2017; Lakowicz 1991). Therefore, the fluorescence quenching mechanism could be studied by the variation of relevant parameters such as Kq and fluorescence lifetime. The Stern-Volmer equation was used to analyze the fluorescence quenching mechanism (Lakowicz 1973; Wang et al. 2019):
F 0 /F = 1 + Kqτ0[Q] = 1 + Ksv[Q]
Among them, Ksv and Kq were the constants of Stern-Volmer equation and fluorescent quenching rate, separately; τ0 (τ0 = 6.0×10− 9 s) was the average fluorescence lifetime of TcOBP-9B without quencher; Ksv=Kqτ0; [Q] referred to the concentration of (1R)-(+)-α-Pinene and β-Caryophyllene; F0 and F were the fluorescence intensities of TcOBP-9B before and after adding the quenchers. F0/F and [Q] were linearly related, the slope of the linear regression line was Ksv, and Kq could be obtained by further calculation. Relevant studies have shown that when Kq < 2×1010 L/(mol·s), it was a dynamic quenching process, on the contrary, if Kq > 2×1010 L/(mol·s), it was a static quenching model (Kazemi et al. 2015).
The fluorescence quenching mechanism of TcOBP-9B-(1R)-(+)-α-Pinene, TcOBP-9B-β-Caryophyllene were shown in Fig. 7 and Table 3. Under three temperature conditions, the quenching curves were well related linearly, and the Kq values were much greater than 2×1010 L/(mol·s), and the Kq values decreased with increasing temperature. Therefore, the fluorescence quenching mechanisms of TcOBP-9B-(1R)-(+)-α-Pinene, TcOBP-9B-β-Caryophyllene were both static quenching types.
Table 3
Fluorescence quenching constants for the interaction of TcOBP-9B with (1R)-(+)-α-pinene and β-caryophyllene.
| T (K) | Ksv (mol/L) | Kq L/(mol·s) | R2 |
TcOBP-9B+(1R)-(+)-α-Pinene | 289 | 1.7424 × 105 | 0.2904 × 1014 | 0.982 |
298 | 1.1278 × 105 | 0.1880 × 1014 | 0.9812 |
308 | 0.6582 × 105 | 0.1097 × 1014 | 0.9843 |
TcOBP-9B + β-Caryophyllene | 289 | 1.6111 × 105 | 0.2685 × 1014 | 0.9815 |
298 | 1.0319 × 105 | 0.1720 × 1014 | 0.9938 |
308 | 0.5903 × 105 | 0.0984 × 1014 | 0.9947 |
Uv Analysis Of Quenching Mechanism
Amino acid residues containing benzene rings in the TcOBP-9B have UV absorption at a wavelength of 278 nm. The static quenching type is due to the formation of a complex between the protein and the quencher, which would change the ultraviolet absorption spectrum of the fluorescent molecules in the static quenching state, while the dynamic quenching process only affected its fluorescence excitation state without changing the UV absorption of fluorescent molecules.
As shown in the Fig. 7, with the increase of the concentration of (1R)-(+)-α-Pinene, β-Caryophyllene, the UV absorption values of TcOBP-9B at 278 nm gradually increased, which further verified that the fluorescence quenching mechanisms of the interaction between TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene were static quenching.
Analysis Of Protein-ligand Binding Constants And Number Of Binding Sites
The strength of the protein-ligand interaction could be measured by calculating binding constants and number of binding sites of the interaction between TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene. The binding constant (Ka) and the number of binding sites (n) could be calculated by using the modified logarithmic regression equation (Lissi and Abuin 2011):
lg[(F0-F)/F] = lgKa+nlg/[Q]
According to the above equation, when lg[(F0-F)/F] was a regression line against lg[Q], the binding constant Ka is the negative logarithm of the intercept of the line and the number of binding sites n is the slope of the line.
As shown in Fig. 8, the interaction between TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene conformed to the revised double logarithmic equation, and the linear relationship was good. In addition, as shown in Table 4, the Ka values TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene were all greater than 104 under different temperature conditions, indicating that the ligands had strong binding force to the protein, which was beneficial to the stability and action time of the drug (Chen et al. 2011).
Table 4
Binding constant and number of binding sites for the interaction of TcOBP-9B with (1R)-(+)-α-Pinene and β-Caryophyllene.
| T (K) | Ka(mol/L) | n | R2 |
TcOBP-9B+(1R)-(+)-α-Pinene | 289 | 2.3153 × 106 | 1.2479 | 0.9925 |
298 | 2.6044 × 105 | 1.0851 | 0.9876 |
308 | 1.7450 × 104 | 0.8705 | 0.9927 |
TcOBP-9B + β-Caryophyllene | 289 | 7.7660 × 106 | 1.3574 | 0.9957 |
298 | 5.6689 × 105 | 1.1582 | 0.9955 |
308 | 1.7306 × 104 | 0.8823 | 0.9982 |
The above experimental results have shown that the binding forces of complexes formed between the TcOBP-9B and the (1R)-(+)-α-Pinene, β-Caryophyllene was strong, which made the complex formed between the external odor information molecule and the OBP in the olfactory receptors of the insect T. castaneum more stable. It was convenient for the external hydrophobic odorant molecules to pass through the water-soluble sensory lymph fluid by binding with insect OBP, and finally reached the ORs and activated the corresponding ORs to complete the olfactory conduction process. The above data showed that the stability of the complexes formed by insect OBPs and ligands decreased when the temperature increased. In addition, the number of binding sites (n) between the TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene were approximately 1, indicating that the relationship between the TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene was close to 1:1 binding ratio.
Thermodynamic Parameter Analysis Of Protein Binding Force Types
Since the binding force was temperature-dependent, the thermodynamic parameters were applied to analyze the interaction between protein and ligands (Davis 1964). The main types of interaction between protein and ligands include hydrogen bonds, electrostatic forces, hydrophobic forces, van der Waals forces, etc. The thermodynamic parameters in the Van't-Hoff equation were used to calculate and analyze the types of interaction force between TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene (Pace et al. 1996).
lnKa=-ΔH/RT + ΔS/R
ΔG = ΔH-TΔS
Among them, T is the absolute temperature, R is the gas constant, ΔS is the entropy change, ΔH is the enthalpy change, and Ka is the binding constant. The lnKa of this equation was plotted against 1/T as a regression line, then ΔS, ΔH and Gibbs free energy change (ΔG) could be calculated via the slope and intercept of the equation. The results of the Van't-Hoff diagram and thermodynamic parameter data of the interaction between TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene were shown in Fig. 9, Table 5.
Table 5
Thermodynamic parameters for interaction of TcOBP-9B with (1R)-(+)-α-Pinene and β-Caryophyllene.
| T (K) | ΔG (KJ/mol) | ΔH (KJ/mol) | ΔS (J/mol·K) |
TcOBP-9B+(1R)-(+)-α-Pinene | 289 | -12.53 | -14.4 | -6.48 |
298 | -12.47 |
308 | -12.4 |
TcOBP-9B + β-Caryophyllene | 289 | -12.29 | -13.61 | -4.54 |
298 | -12.26 |
308 | -12.21 |
As shown in the above results, the thermodynamic parameters of the interaction between TcOBP-9B and β-Caryophyllene, (1R)-(+)-α-Pinene were ΔG < 0, ΔS < 0, suggesting that the interaction was a spontaneous molecular interaction process with reduced entropy and Gibbs free energy, which may be related to the existence of van der Waals forces and hydrogen bonds (Dufour and Dangles 2005; Ross 1981; Tayyab et al. 2018). According to the thermodynamic rules reported in related studies, the types of force in the interaction between TcOBP-9B and (1R)-(+)-α-Pinene, β-Caryophyllene could be inferred. Ross et al. had proved that the negative value of ΔH and ΔS indicated the existence of Van der Waals forces, electrostatic and hydrophobic interactions (Ross 1981).
Analysis Of Competitive Binding Site
The results of the fluorescence probe suitability test showed that with the increase of the concentration of probe 1-NPN, the maximum fluorescence value of TcOBP-9B bound to the probe gradually increased and tended to be saturated. After linearization by Scatchard equation, it was found that the binding of probe 1-NPN to TcOBP-9B was linearly correlated, indicating that 1-NPN and TcOBP-9B were combined in a ratio of 1:1. Therefore, the competitive displacement experiment of the (1R)-(+)-α-Pinene, β-Caryophyllene could preliminarily confirm whether the odorant chemical information can compete for the binding site of the fluorescent probe and the TcOBP-9B.
As shown in the Fig. 10, with the increase of the dosage of (1R)-(+)-α-Pinene and β-Caryophyllene, the fluorescence intensity of 1-NPN-TcOBP-9B system gradually decreased. Therefore, (1R)-(+)-α-Pinene and β-Caryophyllene can competitively bind to the binding site of 1-NPN and TcOBP-9B, thereby insecticidal effect, and combined with molecular simulation docking results, the competitively binding site maybe the α-helices.
Previous studies have found that OBPs in a variety of insects can bind to these two terpenoids. HparOBP14, DhelOBP21 and SmosOBP11 bind to α-Pinene, and β-Caryophyllene has a strong affinity for AlucOBP22, CvesOB1and CvesOBP4. By converting their results, the values of Ka were calculated between 3.0460×104-4.1152×105 mol/L (Liu et al. 2019; Li et al. 2017; Li et al.2015; Qu et al. 2021; Cheng et al. 2020). By comparing the Ka values of TcOBP-9B with the two terpenoids at room temperature, it was found that the two terpenoids had higher affinity for TcOBP-9B, and the affinity of β-caryophyllene to TcOBP9B was stronger than other proteins. Therefore, we speculate that EOs with high content of these two terpenoids can cause the behavioral response of T. castaneum by binding to TcOBP-9B.
Analysis Of Circular Dichroism Data
Circular dichroism spectroscopy was an experimental method with simple operation and high sensitivity, which was often used in the study of protein structure (Chen and Yang 1971; Greenfield 2006). The circular dichroism spectrum of TcOBP-9B with α-helical structures showed two obvious negative peaks at 208 and 222 nm, and related studies have shown that the peak at 208 nm represents the π-π* transition of the α-helix, and the peak at 222 nm indicates π-π* transitions of α-helix and random coil (Hu and Ying 2015). The circular dichroism signal of TcOBP-9B decreased with the addition of (1R)-(+)-α-Pinene and β-Caryophyllene, but the type and position of the peaks did not change significantly. Such results suggested that when (1R)-(+)-α-Pinene and β-Caryophyllene interacted with TcOBP-9B, its α-helical secondary structures were altered. The change of α-helix structure content in protein could be calculated by the following equation (Suo et al. 2018; Greenfield and Fasman 1969; Shahabadi et al.2014).
$$MRE=\frac{Observed CD \left(mdeg\right)}{{C}_{p}nl\times 10}$$
$$\alpha -helix \left(\%\right)=\frac{{-MRE}_{208}-4000}{33000-4000}\times 100$$
In the above equation, Observed CD (mdeg) refers to the ellipticity; n refers to the number of amino acid residues in the protein; Cp refers to the mole fraction of the protein; l refers to the optical path of the circular dichroic sample cell; MRE (Mean Residue Ellipticity) refers to the average residue ellipticity of the protein. 33000 refers to the MRE value with only α-helical structure at 208nm in the circular dichroic chromatogram; MRE208 refers to the MER value at 208 nm in the circular dichroic chromatogram; 4000 refers to the random coil at 208 nm in the circular dichroic chromatogram the MRE value. The circular dichroism of the interaction between TcOBP-9B and (1R)-(+)-α-Pinene and β-Caryophyllene were shown in Fig. 11.
The experimental results were calculated and analyzed. After adding (1R)-(+)-α-Pinene and β-Caryophyllene to the TcOBP-9B, the content of the α-helix structure in TcOBP-9B protein decreased from 60.8% and 65.4–59.1% and 64.3%, respectively. The experimental results showed that (1R)-(+)-α-Pinene and β-Caryophyllene were bound to the inside of the α-helix structure of TcOBP-9B. This result was the same as that of molecular docking presented in Fig. 3 and Fig. 4.
The study on the mechanism of odor molecules induce behavioral responses of T.castaneum was mainly to use RNA-seq to first screen out the related TcOBP, and then Silence the target gene by RNA interference and test the behavior of T.castaneum, by comparing with the previous behavioral test results to confirm that the target protein involved in the action of odorant molecules (Gao et al. 2020; Gao et al. 2021b), but this method does not directly prove that odorant molecules do bind to the target protein. Fluorescence competitive binding assay is the most commonly used method to detect the binding characteristics between OBPs and odor molecules, but this method may ignore odor molecules with weak binding to OBPs. Therefore, it is necessary to combine with other methods to verify and supplement the experimental results. Yi et al. also used circular dichroism to detect the interaction of SspOBP7 with multiple host plant volatiles; Liu et al. detected the binding properties of DcitOBP2 and DcitOBP7 to odorant molecules in Diaphorina citri by microscale thermophoresis technology (MST) (Yi et al. 2018; Liu et al. 2020). Further research on the binding properties between TcOBPs and odor molecules requires more sensitive and accurate experimental methods.