Physicochemical Evidence on Sublethal Neonicotinoid Imidacloprid

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Physicochemical evidences on the sublethal neonicotinoid imidacloprid interacting with an odorant-binding protein from the tea geometrid moth, Ectropis obliqua Hongliang LI, Lei Zhao, Xiaobin Fu, Xinmi Song, Fan Wu, Mingzhu Tang, Hongchun Cui, and Jizhong Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00597 • Publication Date (Web): 02 Apr 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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Journal of Agricultural and Food Chemistry

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Physicochemical evidences on the sublethal neonicotinoid

2

imidacloprid interacting with an odorant-binding protein from

3

the tea geometrid moth, Ectropis obliqua

4 Hongliang Li, *, †, Lei Zhao †, Xiaobin Fu †, Xinmi Song †, Fan Wu †, Mingzhu Tang †,

5

，‡

Hongchun Cui ‡, Jizhong Yu *

6 7

†

8

Sciences, China Jiliang University, Hangzhou 310018, China;

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‡

Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life

Tea Research Institute, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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ACS Paragon Plus Environment


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ABSTRACT: Nowadays the excessive usage of neonicotinoid insecticides always

28

results in the residues in Chinese tea fields. It is not clear whether the insecticide

29

residue at the sublethal level influence on the physiological processes of tea pests.

30

Here, we provided the interacting evidences between neonicotinoid imidacloprid and

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a general odorant-binding protein, EoblGOBP2, from the tea geometrid moth,

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Ectropis obliqua. The interacting process was demonstrated through multiple

33

fluorescence spectra, UV absorption spectra, circular dichroism (CD) spectra and

34

molecular docking, etc. The binding mode was determined to be static (from 300 to

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310 K) and dynamic quenching (from 290 to 300 K). The binding distance was

36

calculated to be 6.9 nm based on the FRET theory. According to the thermodynamic

37

analysis, the process was mainly driven by enthalpy (∆H97 % purity) and E-2-hexenal (>98 % purity) were both

113

purchased from TCI co. Japan, and dissolved in spectroscopic pure grade methanol

114

(Tedia, USA) to prepare 1.0 ×10

115

dark. Milli-Q water (18.2 MΩ, Millipore, US) was used, and all the other solvents and

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chemicals used in this study were of analytical reagent grade.

−3

mol/L stock solutions, and stored at 4 °C in the

117 118

Insects and Extract of Total Moth Antennae RNA

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The caterpillars of tea geometrid Ectropis obliqua Prout were collected from tea

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plantation in HangZhou Daqinggu, and then kept with fresh tea leaves until eclosion

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in the laboratory at the College of Life Sciences, China Jiliang University at 16 h

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light : 8 h dark. The antennae of female or male moths were excised, and immediately

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frozen in liquid nitrogen, and stored at –70 °C until use.

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According to the kits manual of TRIzol® Reagent (Invitrogen, USA), the total

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RNA was extracted from the antennae of 100 male and female moths, respectively.

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The UV A260/280 values for all total antennal RNA were ranging from 1.90 to 2.0.

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Using 2 µg antennal total RNA, the first-strand cDNA was synthesized by PrimeScript

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Reverse Transcriptase System (Takara, Japan) according to the kits manual.

129 130 131

Preparation of Recombinant EoblGOBP2 Protein The recombinant pET32-EobGOBP2 plasmid, constructed and obtained in the 24

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previous study,

was transformed into BL21(DE3) E. coli competent cell, then the

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positive clone was inoculated into BL medium with ampicillin. The recombinant

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EoblGOBP2 protein was induced in the presence of 1.0 ×10

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thiogalactoside (IPTG) with at 37 °C with 200 rpm for 4 hours. After ultrasonication, 6


−3

mol/L isopropyl-β-d-

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from broken cells, the recombinant EoblGOBP2 protein was purified by the Ni2+-NTA

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column with gradient concentration imidazole washing. When the primary purified

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protein was dialyzed by PBS buffer (pH 7.4) until imidazole free, the final purified

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protein including N-terminal tag was digested by thrombin, and then obtained by

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using general gel filtration method. After detected by SDS-PAGE, the protein was

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diluted into 1.0×10

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until further analysis.

−6

mol/L stock solutions in PBS (pH 7.4), and stored at −20 °C

143 144

Absolute Quantification of EoblGOBP2 in Moth Antennae

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For the assessment of EoblGOBP2 amounts in E. oblique, the absolute

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quantification of EoblGOBP2 in total mRNA of E. oblique was calculated based on

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the standard curve of pMD-18T/EoblGOBP2 plasmid. The primers of qRT-PCR were

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designed as sense primer is: 5’-CAGAGGCTCGTGAGGATG-3’, and antisense

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primer is: 5’- CCTGACCATTAGGGAAGC-3’, and synthesized by Corporation

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Sangon Bietech (Shanghai, CN).

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Real time PCR was performed in Bio-Rad iQ5 (Bio-Rad, USA). The 20 µL

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reaction system consisted of 10 µL of 2 ×SYBR Premix EX Taq, 0.4 µL each primer

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(10 µM), 2 µL cDNA sample (total 12 tissues), 7.2 µL ddH2O. The PCR program was

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at 95℃ for 30 sec, 40 cycles of 95℃ for 10 sec, and 59℃ for 30 sec. The negative

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controls were set, and the interaction was replaced with ddH2O instead of cDNA

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samples. Three experimental technical replications were performed for each sample.

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Fluorescence Spectra Measurements

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The fluorescence spectral data of EoblGOBP2 with imidacloprid were recorded

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using a RF-5301 PC spectrofluorimeter (Shimadzu, Japan) with a 1.0 cm quartz cell at

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different temperatures (290, 300, and 310 K). An electronic thermostat water bath 7



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(9012, PolyScience, USA) was used to precisely control the temperature of the

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reaction. For the fluorescence quenching spectra, the stock solution of EoblGOBP2

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(1.0 × 10 −6 mol/L) was titrated with the working solution of imidacloprid (1.0 × 10 −3

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mol/L). Three individual experiments included:

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(1). Fluorescence quenching spectra. The excitation and emission slit widths were set

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at 5.0 nm. The optimum excitation wavelength was 282 nm, determined by the

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scanning of fluorescence excitation spectra of protein alone (Fig. S1), and then the

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fluorescence quenching / emission spectra were recorded ranging from 290 to 550 nm.

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(2). Synchronous fluorescence spectra. The spectra were recorded when ∆λ (the

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difference between excitation and emission wavelength) was set as 15 nm and 60 nm,

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respectively.

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(3). Competitive fluorescence spectra. E-2-hexenal (1.0 × 10

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into the unique EoblGOBP2 stock solution (1.0 × 10 −6 mol/L) and the complex of the

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equal concentration of EoblGOBP2 with imidacloprid (1.0 × 10

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respectively. The spectra scanning parameters are the same to the fluorescence

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quenching spectra, and then the both dissociation constants of E-2-hexenal with both

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systems were calculated and compared.

−3

mol/L) was titrated

−6

mol/L),

179 180

UV Measurements

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The UV absorption spectra of imidacloprid and EoblGOBP2 were measured

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using a Shimadzu UV-1800 UV spectrophotometer (Shimadzu, Japan) in the

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wavelength range of 190~400 nm with a 1.0 cm quartz cell at room temperature.

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Based on the results of fluorescence quenching, the UV absorption spectra of the

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recombinant EoblGOBP2 protein alone, imidacloprid alone, and the corresponding

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ratio between imidacloprid and EoblGOBP2 were recorded and obtained. All the UV

8


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measurements were carried out in PBS buffer (pH 7.4) at room temperature.

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Circular Dichroism (CD)

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The CD spectra were measured using a CD spectrometer (Jasco-815, Japan) with

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a 1.0 cm path length quartz cuvette (200 µL of total volume). In the presence of

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imidacloprid, the CD spectra of recombinant EoblGOBP2 (1.0 × 10

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recorded in the range of 190~270 nm at room temperature. The molar ratios of

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imidacloprid to EoblGOBP2 were varied as 0:1, 3:1, and 5:1. All the observed CD

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spectra were baseline-subtracted for PBS buffer (pH 7.4), and the results were taken

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as CD ellipticity. The contents of the secondary conformation forms of EoblGOBP2,

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e.g., α-helix, β-sheet, β-turn, and random coil, were analyzed from the CD

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spectroscopic data using the Raussens’s method. 29

−6

mol⋅L−1) were

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Molecular Docking The binding docking analysis of EoblGOBP2 with imidacloprid was performed 30

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by the Molegro Virtual Docker (MVD 4.1, free trial) software.

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dimensional (3D) conformer structure of imidacloprid was downloaded from the

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chemical compound database of PubChem.

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EoblGOBP2 was obtained from the Swiss-Model Workspace

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crystal template in Protein Data Bank (PDB). The potential binding sites of

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EoblGOBP2 were determined using the developed grid-based cavity algorithm. The

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binding pose of EoblGOBP2-imidacloprid complex was obtained according to the

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searching algorithm of MolDock Optimizer and the energetic evaluation of MolDock

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Score. By default, the torsions in the imidacloprid ligand are set flexible during the

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docking simulation. The optimized binding pose and hydrogen bonds were then

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analyzed by Ligplot+ software 33 and displayed by Pymol software.34

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The three-

The predicted 3D crystal structure of

9


32

based on the 3D


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RESULTS AND DISCUSSION

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Protein Purification and Fluorescence Quenching Assay

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After the recombinant EoblGOBP2 protein induced and purified, as seen in the

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SDS-PAGE gel in Fig. 1(A), from the expressional bacteria including the pET32-

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EoblGOBP2 plasmid, lane 2 and 3 were the uninduced and induced full lysis proteins

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of bacteria, respectively. As the predicted molecular weight of the recombinant

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EoblGOBP2 protein are about 17 kDa, and lane 4 and 5 showed the purified target

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recombinant EoblGOBP2 protein with and without N-terminal tags, respectively. It

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showed that the recombinant protein was successfully obtained.

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Since the EoblGOBP2 protein contains a tryptophan residue (Trp37) which

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consists of a fluorescence group of indole ring, the EoblGOBP2 protein can emit the

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fluorescence when excited by appropriative ultraviolet light. 35 After diluted into 1.0 ×

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10

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EoblGOBP2 in the absence and presence of an increasing amount of imidacloprid are

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shown in Fig. 1(B). The molecular structure of imidacloprid is shown in the inset of

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Fig. 1(B). Upon excitation at 282 nm, EoblGOBP2 exhibited a strong fluorescence

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emission signal and the maximum emission spectrum was observed at 340 nm. The

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fluorescence intensity of EoblGOBP2 gradually decreased upon increasing the

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imidacloprid concentration, without changing the maximum emission wavelength and

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the overall shape of the peaks. This latter result is a clear indication for interactions

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between imidacloprid and EoblGOBP2. Therefore, the fluorescence quenching effect

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can most likely be attributed to the generation of non-fluorescent complexes.

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−6

mol·L −1 stock solutions in PBS (pH 7.4), the fluorescence emission spectra of

[Fig.1 insert here]

236 237 238

Mechanism of Binding Interaction The fluorescence quenching of biological macromolecules by small-molecule 10


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fluorescent probes can be explained through dynamic quenching and static quenching

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phenomena. The fluorescence quenching mechanism can be commonly analyzed by

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using the following Stern-Volmer36 and Lineweaver-Burk equation:37

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243 244

F0 = 1 + K qτ o [Q ] = 1 + K sv [Q ] F

(1)

1 1 KD = + F0 − F F0 F0 [ Q ]

(2)

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Here F0 and F are the fluorescence intensity in the absence and presence of a

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quenching species at a given concentration [Q]. Kq is the quenching rate constant of

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the biomolecule, τ0 is the average lifetime of the molecule with a value of 10 −8 s and

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without a quenching species, Ksv is the Stern-Volmer dynamic quenching constant and

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KD is the dissociation constant.38, 39 Both the Stern-Volmer and Lineweaver-Burk plots

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can be found in Fig. 2(A) and (B), respectively; Ksv, Kq and KD are listed in Table 1.

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When the quenching process follows a dynamic process, the value of Ksv, i.e. the

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slope of the plot, increases with increasing temperature, on the contrary, being a static

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process if Ksv decrease with increasing temperature. 40 As shown Fig. 2-A and Table 1,

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interestingly, the binding of imidacloprid with EoblGOBP2 seems to be a complex

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process. For example, the value of Ksv at 300 K was higher than those at both 290 K

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and 300 K, while that at 290 K was closer to that at 300 K than 310 K (Fig.2-A). It

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indicates that the interaction between imidacloprid and EoblGOBP2 is a probably

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dynamic process from 290 to 300 K, while clearly static ranging from 300 to 310 K

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(Table 1). The latter is most notably, due to the fact that the static quenching is

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generated by a new complex, and the stability of the complex decreases with

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increasing temperature. 41

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[Fig.2 insert here] 11



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In addition, UV spectra of EoblGOBP2 in the absence and presence of

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imidacloprid were recorded at room temperature to further elucidate the quenching

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mechanism. As showed in Fig. 3, when imidacloprid was added to EoblGOBP2 ([c] =

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1.0 × 10

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curve of EoblGOBP2 with imidacloprid (the both [c] = 1.0 × 10

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maximum peak of UV spectra of EoblGOBP2 evidently increased. This also indicates

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that the binding interaction between imidacloprid and EoblGOBP2 is static quenching.

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Because the static quenching usually results from the ground state of the fluorescent

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molecule, while the overall shape of the spectrum does not change as it is the case in

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dynamic quenching. 42

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−6

mol·L

−1

) at equal concentration, compared with the theoretical merging −6

mol·L

−1

), the

[Fig. 3 insert here]

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All the above data obtained demonstrate that fluorescence quenching between

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EoblGOBP2 and imidacloprid follows a stable static quenching mode at or above

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room temperature. Nevertheless, as other OBP type, ASP2 specifically expressed in

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the antennae of honeybee, shows a binding mode that is static upon binding with

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imidacloprid in the range of 290 to 300 K.

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natural floral volatile β-ionone proves to follow a dynamic mode in the range of 300

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to 310 K. 44 Therefore, the detailed binding mode between OBP and the ligand might

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result from the difference of molecular polarity.

43

However, the binding of ASP2 to the

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To sum up, as a water-soluble systemic insecticide, imidacloprid can be easily

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absorbed and transported via a binding protein like EoblGOBP2, which is widely

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distributed in the hemolymph of the insect. This binding in the range of 300 to 310 K

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is similar to the static binding interaction between serum albumin and numerous drug

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molecules or other bioactive components,

45

requiring highly stable temperatures

12


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when transported in vivo. This result is also accordance with the characteristics that

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the pesticide effect of imidacloprid was greater at the higher temperatures in the range

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of 30 to 39 ℃ (303 to 312 K). 46

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Determination of Interaction Force

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It has already been shown that a variety of interactions exist between proteins

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and organic molecules, e.g. electrostatic forces, hydrophobic interactions, hydrogen

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bonds, and van der Waals interactions.

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EoblGOBP2 in this study, the interaction forces can be calculated using the

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thermodynamic equations as follows:

47

For the interaction of imidacloprid with

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∆ G = − RT ln K = ∆ H − T ∆ S

300

∆H =

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∆S = ( ∆H − ∆G ) T

(2)

RT1T2 ln( K 0 , 2 K 0 ,1 ) T2 − T1

(3) (4)

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where ∆H, ∆S, and ∆G are enthalpy change, entropy change, and the free energy

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change, respectively. The values of ∆G, ∆H, and ∆S for imidacloprid and EoblGOBP2

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are calculated. As listed in Table 1, the interactions between EoblGOBP2 and

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imidacloprid could be divided into two cases, one is the temperature range of 290 to

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300 K and another is from 300 to 310 K. According to the characteristics of the

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theoretical parameters, ∆H0 indicate a typical hydrophobic

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interaction; ∆H0 indicate hydrophobic and electrostatic interactions.

310

suggested that these two cases were spontaneous (due to ∆G

Physicochemical Evidence on Sublethal Neonicotinoid Imidacloprid

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