Combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis reveal the cognitive characteristics of honey bee chemosensory protein to plant semiochemical

Accepted Manuscript Combinatorial multispectral, thermodynamics, docking and sitedirected mutagenesis reveal the cognitive characteristics of honey bee chemosensory protein to plant semiochemical

Jing Tan, Xinmi Song, Xiaobin Fu, Fan Wu, Fuliang Hu, Hongliang Li PII: DOI: Reference:

S1386-1425(18)30371-8 doi:10.1016/j.saa.2018.04.074 SAA 16059

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

23 July 2017 18 April 2018 29 April 2018

Please cite this article as: Jing Tan, Xinmi Song, Xiaobin Fu, Fan Wu, Fuliang Hu, Hongliang Li , Combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis reveal the cognitive characteristics of honey bee chemosensory protein to plant semiochemical. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi:10.1016/j.saa.2018.04.074

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ACCEPTED MANUSCRIPT Combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis reveal the cognitive characteristics of honey bee chemosensory protein to plant semiochemical

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

Sciences, China Jiliang University, Hangzhou 310018, China

College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

* Corresponding author. Tel.:

+86 571 86835774; Fax: +86 571 86914449.

[email protected] (H. Li).

These authors contributed equally to this work.§

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†

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E-mail address:

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b

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a

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Jing Tan a, †, Xinmi Song a, †, Xiaobin Fu a, Fan Wu a,§, Fuliang Hu b, Hongliang Li a, *

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The present address is “Institute of Apicultural Research, Ministry of Agriculture, Chinese Academy of

Agricultural Science, Beijing 10093, China”

1

ACCEPTED MANUSCRIPT

ABSTRACT In the chemoreceptive system of insects, there are always some soluble binding proteins, such as some antennal-specific chemosensory proteins (CSPs), which are abundantly distributed in the chemosensory sensillar lymph. The antennal-specific CSPs usually have strong capability to bind

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diverse semiochemicals, while the detailed interaction between CSPs and the semiochemicals

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remain unclear. Here, by means of the combinatorial multispectral, thermodynamics, docking and

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site-directed mutagenesis, we detailedly interpreted a binding interaction between a plant semiochemical β-ionone and antennal-specific CSP1 from the worker honey bee. Thermodynamic

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parameters (ΔH < 0, ΔS > 0) indicate that the interaction is mainly driven by hydrophobic forces

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and electrostatic interactions. Docking prediction results showed that there are two key amino acids, Phe44 and Gln63, may be involved in the interacting process of CSP1 to β-ionone. In order

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to confirm the two key amino acids, site-directed mutagenesis were performed and the binding

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constant (KA) for two CSP1 mutant proteins was reduced by 60.82% and 46.80% compared to wild-type CSP1. The thermodynamic analysis of mutant proteins furtherly verified that Phe44

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maintained an electrostatic interaction and Gln63 contributes hydrophobic and electrostatic forces.

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Our investigation initially elucidates the physicochemical mechanism of the interaction between antennal-special CSPs in insects including bees to plant semiochemicals, as well as the development of twice thermodynamic analysis (wild type and mutant proteins) combined with multispectral and site-directed mutagenesis methods.

Keywords: Honey bee; Chemosensory protein; Multispectral analysis; Thermodynamics; Molecular docking; Site-directed mutagenesis. 2

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1. Introduction Insects’ complex behavior relies on their sensitive chemoreceptive ability to sense many kinds of semiochemicals in the natural environments (Burchell 1991). Especially with respect to subsistence and reproduction, insects’ chemoreception plays an important role in the recognition

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of diverse odors such as sex pheromones, plant volatiles, and the semiochemicals from enemies or

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oviposition sites (Schröder and Hilker 2008). In the sensillar lymph of chemosensory organs such

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as antennae, mouth organs and other chemosensory structures, there are generally two kinds of soluble chemoreceptive proteins: chemosensory proteins (CSPs) and odorant-binding proteins

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(OBPs) (Pelosi et al. 2006). These proteins are highly concentrated in the lymph of chemosensilla,

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and regarded as carriers of pheromones and odorants from the external environment to the membrane of chemosensing neurons (Pelosi et al. 2017).

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In general, OBPs exhibit a higher binding specificity or affinity for common environmental

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volatiles, while CSPs show relatively conservation and were related to sense semiochemicals involved in chemical communication (Pelosi et al. 2017), for instance, the recognition of diverse

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host plant volatiles (Gu et al. 2012; Yi et al. 2014), cuticular hydrocarbon or lipids (Gonzalez et al.

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2009; Ozaki et al. 2005), some pheromone components (Briand et al. 2002; Sun et al. 2015) and so on. All the evidences above show that CSPs, especially and abundantly expressed in the lymph of chemosensilla, participate in the semiochemical signal sense and transduction involved in the chemoreception (Sanchez-Gracia et al. 2009). However, although the physiological functions of CSPs in vitro have been widely reported, the detailed physicochemical mechanism of CSPs in sensing semiochemicals still remains unclear. As an Asian hive bee, Apis cerana is widely applied as a agricultural pollinator in vegetable 3

ACCEPTED MANUSCRIPT seed production (Verma and Partap 1993). The pollination efficiency of bee is closely related to its susceptibility to plant volatiles, that is, to the chemoreceptive sensitivity to semiochemicals (Tumlinson et al. 1999). In the previous study, CSP1, an antennal-specific CSP was identified from the significant chemoreceptive organ, antennae of A. cerana worker bee (Li et al. 2016b). By

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means of the fluorescence competitive binding assay, CSP1 was identified to exhibit a strong

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affinity for some semiochemicals including floral odors, pheromones and so on (Li et al. 2016b).

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Nowadays the fluorescence competitive binding assay has been widely used to investigate the ligands binding functions of insects CSPs (Iovinella et al. 2013; Sun et al. 2015; Zhang and Lei

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2015). Nevertheless, the competitive binding assay only obtained the binding constants of CSPs

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with candidate competitive ligands due to the presence of fluorescence probe (Pelosi et al. 2017; Pelosi et al. 2006), which results in other detailed binding parameters and characteristics of the

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proteins and ligands being ignored.

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Therefore, for the further description and elucidation of how CSP1 directly interacts with the candidate ligand, here, the twice multispectral and thermodynamics methods of wild type and

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mutant proteins were developed to analyze the binding interaction. The key amino acid sites of

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CSP1 binding to the semiochemical were predicted by molecular docking based on the homology crystal structure (Campanacci et al. 2003), and then confirmed by the site-directed mutagenesis. To sum up, our study not only reveals the significant physicochemical characteristics of antennal-special CSPs in insects including bees to plant semiochemicals, but also facilitate further understanding of the dynamic interacting details involved in the honey bee pollination process of based on its chemoreceptive system. 2. Materials and methods 4

ACCEPTED MANUSCRIPT 2.1. Materials and apparatus β-ionone (purity>96%, ACROS, Belgium) was dissolved into 10 mmol·L-1 with methanol (TEDIA, USA) as the solvent. A RF-5301PC spectrofluorophotometer (Shimadzu, Japan) was used to conduct all fluorescent measurements, equipped with a 1 cm-width quartz cell. A UV-1800

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UV spectrofluorophotometer (Shimadzu, Japan) was used to record the UV spectra. A Jasco-815

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CD spectrometer (Jasco, Japan) was used to measure the CD spectra with a 1 cm-width quartz cell.

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A sensitive electronic thermostat water-bath (9012, PolyScience, USA) was used to control all the interaction temperatures in the thermodynamic experiments. A BS224S electronic analytical

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balance (Sartorius, Germany) was used to weigh all the chemical materials. All other chemical

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reagents were spectral purity.

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2.2 Expression and purification of the recombinant CSP1 protein

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For the expression of the recombinant CSP1 protein, the prokaryotic expression plasmid pET32-CSP1 constructed in the previous study(Li et al. 2016b) was transformed into the BL21 competent

cells.

After

induced

and

expressed

with

1

mmol∙L-1

isopropyl

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(DE3)

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β-D-1-thiogalactopyranoside (IPTG) at 30 ℃ for 6 h, the recombinant CSP1 protein was purified by ProteinIsoTM Ni2+-NTA Resin (TransGen, China). Based on the quantification of Bradford method, the final concentration of the purified recombinant CSP1 proteins were diluted into 1 μmol∙L-1 with phosphate buffered saline (PBS; pH=7.4).

2.3. Multispectral analysis (1). Fluorescence quenching assay and thermodynamic analysis of CSP1 wide type. When 5

ACCEPTED MANUSCRIPT the excitation wavelength was set 281 nm, the fluorescence emission spectra were recorded from 290 to 450 nm, and the width of excitation and emission slit was set at 5.0 nm. β-ionone (10 mmol∙L-1) was titrated into the stock solution of CSP1 with concentration of 1 μmol∙L-1. Fluorescence spectra data of CSP1 with β-ionone were recorded with a 1.0 cm quartz cell at 284 K

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and 294 K, respectively.

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(2). Synchronous fluorescence (SF) assay. Synchronous fluorescence spectra of CSP1 were

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measured with increasing concentrations of β-ionone, by setting ∆λ (the difference between the wavelength of monochromatic excitation and the emission light) = 15 nm and ∆λ = 60 nm for

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tyrosine and tryptophan residues, respectively. Using Shimadzu UV-1800 spectrophotometer with

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a 1.0 cm quartz cell, the UV spectra of CSP1 and mixture (CSP1 and β-ionone) were recorded from 230 to 320 nm at room temperature, respectively. The molar ratio of β-ionone to CSP1 was

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1:1 in the mixture.

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(3). Circular dichroism (CD) spectra assay. The CD spectra of CSP1 and that with β-ionone were recorded at the wavelength from 200 to 260 nm at room temperature under constant nitrogen

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flush. The molar ratio of β-ionone to CSP1 was 0:1, 1:1, and 10:1, respectively. All CD spectra

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data were baseline-subtracted through PBS buffer (pH = 7.4). The CD spectra results were then taken as CD ellipticity, and the secondary conformation forms of CSP1 were calculated using by the online SELCON3 program (Sreerama and Woody 2000) according to the CD spectroscopic data.

2.4. Molecular docking analysis The characteristic mode in the binding interactions of the organic compound and proteins 6

ACCEPTED MANUSCRIPT could be visually analyzed by molecular docking analysis (Hu et al. 2012). The predicted 3D crystal structures of CSP1 were generated by homology modeling on SWISS-MODEL Workspace (Schwede et al. 2003), based on the crystal structure of CSPMbraA6 from the moth Mamestra brassicae (PDB entry code, 1n8v) (Campanacci et al. 2003) as the template (Two sequence

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similarities reach 37.23%). The 3D structure of β-ionone was downloaded from pubchem (register

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number: 638014) database. By using a Molegro Virtual Docker (MVD) 4.2 program (trial version),

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the 3D structure of β-ionone was docked into the predicted binding cavity of CSP1 3D structure. MolDock Optimizer and MolDock Score were used as the searching algorithm and scoring

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algorithm, respectively (René and Christensen 2006). According to the minimum value of

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MolDock Score, the optimal pose of CSP1 to β-ionone was determined. The putative two key amino acids binding sites of CSP1 and β-ionone were predicted based on the energy contribution

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of the best docking pose.

2.5. Site-directed mutagenesis

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For the verification of two key amino acid sites of CSP1 binding to β-ionone, the

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recombinant plasmids including two CSP1 mutants are required to be prepared through the site-directed mutagenesis assay, respectively. The forward and reverse primers including the mutation sites of the two key amino acid CSP1 plasmids (CSP1-F44G and CSP1-Q63G) were first designed (Table S1), then two CSP1 mutants, CSP1-F44G and CSP1-Q63G, were mutated by using Fast Mutagenesis System (TransGen, China) referred to the kit instructions. After the recombinant plasmids including two CSP1 mutants were transformed into the BL21 (DE3) competent cells, the corresponding recombinant CSP1 mutant proteins were induced and purified 7

ACCEPTED MANUSCRIPT according to the method of section 2.2 above.

2.6 Thermodynamic analysis of CSP1 mutant proteins In order to study and verify the thermodynamic contribution of key amino acids in CSP1 to

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ionone, the fluorescence quenching spectra of CSP1-F44G and CSP1-Q63G and β-ionone were

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re-recorded at 284 K and 294 K based on the method described in section 2.3-(1) above. The

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thermodynamic parameters were also calculated and the docking analysis of two CSP1 mutants to

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β-ionone was also performed.

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3. Results and discussion 3.1. Expression and purification of CSP1

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Recombinant pET32-CSP1 plasmid was prepared as previously described (Li et al. 2016b)

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and re-transformed into BL21 (DE3) strain E. coli., Recombinant protein was extracted after bacteria induced and collected. As seen by SDS-PAGE in Fig. 1, lane 1 and lane 2 showed

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extracted bacterial proteins from the pET32-CSP1 plasmid without and with induction of IPTG,

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respectively. It was evident that IPTG induction (lane 2) resulted in expression of the recombinant protein, compared with bacteria not induced with IPTG (lane 1). Lane 3 shows that the purified recombinant CSP1 protein has a molecular weight of approximately 28 kDa. The final concentrations of the recombinant CSP1 protein were adjusted to 1 μmol∙L-1 to be used in the following assays. 3.2. Multispectral analysis (1) Fluorescence quenching assay and thermodynamic analysis of CSP1 wild type 8

ACCEPTED MANUSCRIPT The CSP1 recombined protein was prepared (Fig. 1) and to be used in the following assays. Fluorescent quenching spectra are used to study the binding interactions between chemical molecules (e.g. organic drugs) and binding proteins (e.g. BSA) (Shahabadi et al. 2015). In the previous study, we have used the competitive fluorescence probe to find strong binding of

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β-ionone to CSP1 (Li et al. 2016b). Here, when β-ionone was directly titrated into purified

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recombinant CSP1 without the competitive fluorescence probe, as seen in Fig. 2, it was found that

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the maximum fluorescence intensity of CSP1 was significantly decreased as the β-ionone concentration increased in the range of 334 to 350 nm. It was evident that there were direct

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binding interactions between CSP1 and β-ionone due to the generation of a CSP1-β-ionone

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complex. In addition, the binding process exhibited a slight red shift when β-ionone added, indicating that the hydrophobicity of the binding site of interaction was reduced and the peptide

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chain was stretched.

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(2) Synchronous fluorescence (SF) assay

Synchronous fluorescence (SF) spectra have been used to analyze the fluorescence

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contribution of the two fluorescent amino acids (tyrosine and tryptophan) to proteins through

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scanning excitation and recording emission spectra simultaneously (Juqun and Rong 2007). The values of ∆λ = 15 and 60 nm separately correspond to the fluorescent contributions of tyrosine and tryptophan (Burstein et al. 1973). As seen in Fig. 3, the fluorescence intensity of tryptophan was higher than that of tyrosine, indicating that the fluorescence of CSP1 was mainly caused by the tryptophan residue. The occurrence of a blue shift at the maximum emission wavelength of the fluorescence spectra demonstrated that the polarity of tryptophan and tyrosine residues decrease in the 9

ACCEPTED MANUSCRIPT interacting microenvironments (Goldouzian et al. 2011). Therefore, when β-ionone was added, in the SF spectra, the blue shift at the maximum emission wavelength occurred in tyrosine more than that of tryptophan (Fig. 3). This implies that the polarity of tyrosine in the microenvironments declined more severely than that of tryptophan residues, and tyrosine seems closer to binding

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cavity than tryptophan residues.

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(3) Circular dichroism (CD) spectra assay

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CD spectra have been used to study the secondary conformational changes of proteins when binding with small molecules (Greenfield 2007). Here, when β-ionone was added into the

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recombinant CSP1 protein, as shown in Fig. 4, two negative peaks could be observed at 208 nm

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and 222 nm (line a), indicating the presence of the α-helix. However, with the increase in the concentration of β-ionone, the intensity of the negative peaks decreased (lines b and c). This

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indicates that the conformational structure of CSP1 changed with an increase in the percentage of

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α-helices of CSP1. However, the opposite result was reported with OBP2 binding to β-ionone, where the negative ellipticity of OBP2 significantly increased and the percentage of α-helices

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decreased (Li et al. 2013), which indicate that the both proteins exhibit distinct conformational

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changes when interacting with the same floral odor.

3.3. Fluorescence quenching mechanism Dynamic and static quenching are the two types of fluorescent quenching modes (Ghalandari et al. 2014). They can be distinguished by a temperature changing assay followed by measuring UV absorption spectra (Hu et al. 2005). Typically, dynamic quenching is caused by loss of excitation energy of molecules at the excited state, while static quenching is caused by formation 10

ACCEPTED MANUSCRIPT of a stable complex between the quencher and fluorescent substance at the ground state (Xu and Wang 2006). Dynamic quenching was analyzed by Stern-Volmer equation (Lakowicz 2013):

F0  1  K q o [Q ]  1  K sv [Q ] F

(1)

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where F0 and F are the fluorescence emission intensities in the absence and presence of a

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quencher at [Q] concentration, respectively. Kq τ0 and Ksv represent the quenching rate constant of

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the molecule, the average lifetime (τ0 = 10-8 s (Lakowicz and Weber 1973a)) of the molecule without the quencher present, and the Stern-Volmer dynamic quenching constant (Lakowicz and

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Weber 1973b). The fluorescence intensity of CSP1 binding β-ionone at 284 K and 294 K was

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measured. As shown in Fig. 5 and Table 1, the Ksv at 294 K was higher than that at 284 K, which suggests that the interaction of CSP1 with β-ionone is likely dynamic. This is because dynamic

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temperature rises (Ware 1962).

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quenching usually results from the diffusion of molecules and the diffusion rate increases as the

As the dynamic binding interaction between proteins and small molecules does not result in

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the formation of novel complex, the ground state of the mixed system will also remain stable. That

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is, the UV absorption spectra of the protein will also remain unchanged after the ligand is added (Karolin et al. 1994). Therefore, as shown in Fig. 6, the since UV spectra shape showed almost no change after β-ionone was added into CSP1, it is likely that the interaction between β-ionone and CSP1 is a dynamic process. This is similar to what was found in the binding interaction between β-ionone and OBP2, which was also determined to be a dynamic quenching process (Li et al. 2013), but contrasts with the static quenching that was found between OBP2 and a neonicotinoid insecticide (Li et al. 2015). Therefore, these findings indicate that several bee olfactory related 11

ACCEPTED MANUSCRIPT proteins exhibit similar interacting mechanisms when binding to the similar floral odor molecule.

3.4. Thermodynamic analysis of CSP1 wide type The forces governing binding interactions between macromolecules and organic

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micro-molecules includes hydrophobic interaction, electrostatic force, hydrogen bonds, and Van

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der Waals interactions (Hu et al. 2005), which can be deduced by the thermodynamic equations as

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follows (Hu et al. 2006):

G   RT ln K  H  TS RT1T2 ln( K 0, 2 K 0,1 )

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H 

T2  T1

(3) (4)

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S  ( H  G) T

(2)

where ΔG, ΔH and ΔS are the free energy change, enthalpy change, and entropy change. K

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and R represent the binding constant at the corresponding temperature T and the universal gas

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constant, respectively. Even when the temperature changes slightly, the enthalpy change is always regarded as a constant. As a result, the values of ΔG, ΔH and ΔS in the reaction between CSP1 and

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β-ionone were calculated (Table 1). Theoretically, When ΔH < 0 and ΔS < 0, the main acting

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forces are van der Waals and hydrogen bonds, when ΔH < 0 and ΔS > 0, the main acting forces are hydrophobic and electrostatic forces, and when ΔH > 0 and ΔS > 0, the main acting force is hydrophobic (Ross and Subramanian 1981). When ΔG < 0, it implies that the binding interactions of CSP1 and β-ionone occur spontaneously. In Table 1, ΔH < 0 and ΔS > 0, which suggests that the acting forces between β-ionone and CSP1 are mainly be driven through hydrophobic and electrostatic forces. It is not exactly the same that the force of the binding of OBP2 and floral odor is driven only by the hydrophobic interaction force (Li et al. 2013). This indicates that the 12

ACCEPTED MANUSCRIPT electrostatic force of CSP1 to bind the floral odor has distinct characteristics, which are different from the interactions of OBP2.

3.5. Molecular docking analysis

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Three 3D crystal structures of CSP1 were predicted by SWISS-MODEL Workspace based on

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that of CSPMbraA6 from the M. brassicae (PDB entry code, 1n8v) (Campanacci et al. 2003). The

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sequence alignment between CSP1 and CSPMbraA6 (1n8v) is shown in Fig. 7C. According to the MolDock Score in MVD software, the best docking pose of CSP1 with β-ionone was predicted

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and obtained. As shown in Fig. 7A, the binding cavity of CSP1 is closer to the N-terminal flexible

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region, surrounding the β-ionone along with two α-helix structures. β-ionone is located in the binding cavity composed of 12 hydrophobic residues, including five hydrophobic amino acid

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residues (Phe43, Phe44, Ile48, Ala51, and Leu67), five polar neutral residues (Tyr4, Tyr8, Cys56,

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Cys59, and Gln63), one acidic residue (Asp40), and one alkaline residue (His47) (Fig. 7B). There are no hydrogen bonds between β-ionone and CSP1 found in the binding site, which is in

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agreement with the results of thermodynamic analysis above.

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On the other hand, as found in the SF analysis above, the binding site might be closer to the tyrosine than tryptophan residues, due to the occurrence of blue shift at maximum emission wavelength. In accordance with this finding, there are two tyrosine residues (Tyr4 and Tyr8) predicted in the binding cavity, while no tryptophan residues are found in the binding site (Fig. 7B). Similarly, some tryptophan residues are also involved in the ligand binding process, for example, it has been found that Tyr26 side chain might be rotated toward the protein surface in CSPMbraA6 from the M. brassicae (Lartigue et al. 2002). Anyway, these results indicate that the 13

ACCEPTED MANUSCRIPT docking analysis coincides with the experimental results of thermodynamic analysis and SF spectra in this study.

3.6. Site-directed mutagenesis and comparison of association constant

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Based on the docking analysis, there are two key residues (Phe44 and Gln63) predicted to

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contribute most of the energy of CSP1 binding to the ligand (Table 2). In order to confirm whether

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each of the amino acids play an important role in the binding process, site-directed mutagenesis was performed. Based on the wild-type pET32-CSP1 plasmid, two CSP1 recombinant mutated

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proteins (CSP1-F44G and CSP1-Q63G) were produced, and both purified mutant CSP1 proteins

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and the corresponding wild-type protein are shown in Fig. 8. To assess the key amino acids functioning in the binding process, the fluorescence quenching abilities of the mutant CSP1-F44G

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and CSP1-Q63G proteins with β-ionone were measured. The results can be expressed by the

F0  F  lg K A  n lg[ Q ] F

(5)

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lg

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double logarithmic equation (Liu et al. 2015):

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where F0 is the fluorescent intensity in the absence of a quencher and F is the fluorescent intensity in the presence of a quencher at [Q] concentration, KA is the apparent association constant, and n is the number of binding sites per protein. The corresponding parameters, n and KA, were obtained as a plot of lg [(F0-F)/F] versus lg [Q] (Fig. 9, Table 3). From Table 3, the number of binding sites n is approximately equal to 1 and the apparent association constants (KA) of CSP1-F44G and CSP1-Q63G with β-ionone respectively were 1.565×104 L∙mol-1 and 2.125×104 L∙mol-1, which declined 60.82% and 46.80% compared with the KA of wild-type CSP1 protein 14

ACCEPTED MANUSCRIPT with β-ionone, which was 3.994×104 L∙mol-1. The site-mutagenesis of key site amino acid always resulted in the decline of the binding affinity in chemoreceptive proteins, such as in Thr9 of GmolGOBP2 in Grapholita molesta (Li et al. 2016a). Thus, the decrease in the binding constants implies that Phe44 and Gln63 in CSP1 are significant amino acid residues to maintain the

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hydrophobic and electrostatic forces in the binding process of CSP1 to β-ionone.

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3.7. Thermodynamic analysis of CSP1 mutant proteins and docking analysis

Although it is predicted that Phe44 and Gln63 in CSP1 play an important role in the decline

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of the binding affinity of the CSP1 mutant to β-ionone, it is necessary to clarify whether the

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detailed interaction of the two residues has also changed. Thus, the thermodynamic analysis of CSP1 mutant proteins binding to β-ionone was preformed, and exactly produced evidently change

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after site-mutant. As seen in Table 1, since ΔH >0 and ΔS >0, the only acting forces between

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CSP1-F44G and β-ionone remains as hydrophobic interactions, while the original electrostatic force in CSP1 wild-type is absent. This indicates that Phe44 may be an important residue that

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produces the electrostatic force in the binding process of CSP1 to β-ionone. On the other hand, it −1

, for

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was known that the ΔH value for van der Waals force ranges from −10 to −4 kJ·mol

hydrogen bonding from −40 to −2 kJ·mol −1, and for chemical bonding below −60 kJ·mol −1 (Fu et al. 2013a). Since ΔH <0 and ΔS <0, the acting force between CSP1-Q63G and β-ionone is greatly changed from hydrophobic and electrostatic forces to hydrogen bonds and Van der Waals interactions. Additionally, it was found that the binding properties of CSP1-Q63G and β-ionone were changed to static binding. Taken together, this evidence suggests that Gln63 may be the significant residue in CSP1 to be involved in the hydrophobic and electrostatic forces in the 15

ACCEPTED MANUSCRIPT binding process with β-ionone. In the interactions between protein and ligands, mutations of some amino acid residues in protein can change the weak interactions, which include hydrophobic interaction, electrostatic force, hydrogen bonds, and Van der Waals interactions (Williams and Westwell 1998). To further

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understand the CSP1 mutant proteins binding with β-ionone, CSP1-F44G and CSP1-Q63G

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interacting with β-ionone were evaluated by docking analysis again (detailed energy of individual

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residue contributions are listed in Table S2, S3). As seen in Fig. S1, CSP1-F44G and β-ionone do not show hydrogen bond interaction, while hydrogen bonds are found between CSP1-Q63G and

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β-ionone, which is consistent with the 2nd round thermodynamic results of previous mutants and

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β-ionone (Table 1).

OBPs are widely considered to be the significant odor recognition proteins in insects and the

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detailed interactions of these proteins with specific molecules have been investigated by using

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site-directed mutagenesis (Rusconi et al. 2012). This approach has identified several predicted key binding site residues that form hydrogen bonds, such as Lys74 in AlinOBP5 in the alfalfa plant

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bug Adelphocoris lineolatus (Fu et al. 2013b), Tyr111 in HoblOBP1 in Holotrichia oblita (Zhuang

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et al. 2014), and Thr15 in LstiGOBP1 in Loxostege sticticalis (Yi et al. 2015). However, in this study we found hydrophobic and electrostatic forces participating in the interaction between a CSP and the plant volatile while hydrogen bonds did not. This suggests that there might be some differences in the mechanisms of semiochemicals discrimination between the two kinds of olfactory soluble proteins involved in the chemoreceptive system in insects. 4. Conclusions This study demonstrates the detailed binding interactions between CSP1 of A. cerana and a 16

ACCEPTED MANUSCRIPT general plant semiochemical, β-ionone. The intrinsic fluorescence of CSP1 was found to be quenched by β-ionone. Based on the thermodynamic analysis, the binding process was found to be of the dynamic quenching mode and mainly driven by hydrophobic interaction and electrostatic forces. Docking analysis and site-directed mutagenesis assays revealed that two key amino acids,

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Phe44 and Gln63, are mainly involved in the binding process. The 2nd round of thermodynamic

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analysis of CSP1 mutants determined that Phe44 maintains the electrostatic interactions while

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Gln63 contributes the hydrophobic force. The combinatorial multispectral approaches we have developed in this study not only provides a further understanding of the chemosensory details of

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honey bees to cognize semiochemicals, but also helps to thoroughly elucidate the functional

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mechanism of the insect chemoreceptive system.

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Acknowledgements

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31772544, 31372254).

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This work was supported by the National Natural Science Foundation of China (No.

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References Briand, L, Swasdipan, N, Nespoulous, C, Bezirard, V, Blon, F, Huet, JC, Ebert, P and Penollet, JC (2002) Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier. Eur. J. Biochem. 269: 4586-96. Burchell, B (1991) Turning on and turning off the sense of smell. Nature 350: 16-7. Burstein, E, Vedenkina, N and Ivkova, M (1973) Fluorescence and the location of tryptophan residues in protein molecules. Photoch. photobio. 18: 263-279. Campanacci, V, Lartigue, A, Hallberg, BM, Jones, TA, Giudici-Orticoni, MT, Tegoni, M and Cambillau,

PT

C (2003) Moth chemosensory protein exhibits drastic conformational changes and cooperativity on ligand binding. Proc. Natl. Acad. Sci. U S A 100: 5069-74.

RI

Fu, Q, Wang, Y, Zhang, J, Zhang, H, Bai, C, Li, J, Wang, W, Wang, H, Ye, Q and Li, Z (2013a) Soil microbial effects on the stereoselective mineralization, extractable residue, bound residue, and

SC

metabolism of a novel chiral cis neonicotinoid, paichongding. J Agric Food Chem 61: 7689-95.

Fu, Q, Zhang, J, Xu, X, Wang, H, Wang, W, Ye, Q and Li, Z (2013b) Diastereoselective metabolism of

NU

a novel cis-nitromethylene neonicotinoid paichongding in aerobic soils. Environ Sci Technol 47: 10389-96.

Ghalandari, B, Divsalar, A, Saboury, AA, Haertlé, T, Parivar, K, Bazl, R, Eslami-Moghadam, M and Amanlou, M (2014) Spectroscopic and theoretical investigation of oxali–palladium

MA

interactions with β-lactoglobulin. Spectrochim. Acta A Mol. Biomol. Spectrosc. 118: 1038-1046.

Goldouzian, Z, Goldouzian, F, Momen-Heravi, M and Chamani, J (2011) The investigation of the

D

interaction between human serum transferring with colchicine in the presence of Pb Ions: synchronous fluorescence Measurements. J. Chem. Heal. Risks 1: 13-15.

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Gonzalez, D, Zhao, Q, McMahan, C, Velasquez, D, Haskins, WE, Sponsel, V, Cassill, A and Renthal, R (2009) The major antennal chemosensory protein of red imported fire ant workers. Insect Mol. Biol. 18: 395-404.

Greenfield, NJ (2007) Using circular dichroism spectra to estimate protein secondary structure. Nat.

CE

Protocols 1: 2876-2890.

Gu, SH, Wang, SY, Zhang, XY, Ji, P, Liu, JT, Wang, GR, Wu, KM, Guo, YY, Zhou, JJ and Zhang, YJ (2012) Functional characterizations of chemosensory proteins of the alfalfa plant bug

AC

Adelphocoris lineolatus indicate their involvement in host recognition. PLoS One 7: e42871. Hu, WJ, Yan, L, Park, D, Jeong, HO, Chung, HY, Yang, JM, Ye, ZM and Qian, GY (2012) Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose. Int. J. Biol. Macromol. 50: 694-700. Hu, YJ, Liu, Y, Zhang, LX, Zhao, RM and Qu, SS (2005) Studies of interaction between colchicine and bovine serum albumin by fluorescence quenching method. J. Mol. Struct. 750: 174-178. Hu, YJ, Yi, L, Sun, TQ, Bai, AM, Lü, JQ and Pi, ZB (2006) Binding of anti-inflammatory drug cromolyn sodium to bovine serum albumin. Int. J. Biol. Macromol. 39: 280-285. Iovinella, I, Bozza, F, Caputo, B, Della Torre, A and Pelosi, P (2013) Ligand-binding study of Anopheles gambiae chemosensory proteins. Chem. Senses 38: 409-19. Juqun, X and Rong, G (2007) Interactions between flavonoids and hemoglobin in lecithin liposomes. Int. J. Biol. Macromol. 40: 305-311. 18

ACCEPTED MANUSCRIPT Karolin, J, Johansson, LBA, Strandberg, L and Ny, T (1994) Fluorescence and absorption spectroscopic properties of dipyrrometheneboron difluoride (BODIPY) derivatives in liquids, lipid Membranes, and proteins. J. Am. Chem. Soc. 116: 7801-7806. Lakowicz, JR (2013) Principles of fluorescence spectroscopy. Springer Science & Business Media. Lakowicz, JR and Weber, G, . (1973a) Quenching of fluorescence by oxygen. Probe for structural fluctuations in macromolecules. Biochemistry 12: 4161-4170. Lakowicz, JR and Weber, G, . (1973b) Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale. Biochemistry 12: 4171-4179. Lartigue, A, Campanacci, V, Roussel, A, Larsson, AM, Jones, TA, Tegoni, M and Cambillau, C (2002)

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X-ray structure and ligand binding study of a moth chemosensory protein. J. Biol. Chem. 277: 32094-8.

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Li, GW, Chen, XL, Li, BL, Zhang, GH, Li, YP and Wu, JX (2016a) Binding properties of general odorant binding proteins from the oriental fruit moth, Grapholita molesta (Busck)

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(Lepidoptera: Tortricidae). PLoS One 11: e0155096.

Li, HL, Ni, CX, Tan, J, Zhang, LY and Hu, FL (2016b) Chemosensory proteins of the eastern honeybee, Apis cerana: identification, tissue distribution and olfactory related functional characterization.

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Comp. Biochem. Phys. B 194-195: 11-9.

Li, HL, Wu, F, Zhao, L, Tan, J, Jiang, HT and Hu, FL (2015) Neonicotinoid insecticide interact with honeybee odorant-binding protein: Implication for olfactory dysfunction. Int. J. Biol.

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Macromol. 81: 624-630.

Li, HL, Zhang, LY, Ni, CX, Shang, HW, Zhuang, SL and Li, J, K. (2013) Molecular recognition of floral volatile with two olfactory related proteins in the Eeastern honeybee (Apis cerana). Int. J. Biol. Macromol. 56: 114-121.

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Liu, J, Yue, Y, Wang, J, Yan, X, Liu, R, Sun, Y and Li, X (2015) Study of interaction between human serum albumin and three phenanthridine derivatives: fluorescence spectroscopy and

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computational approach. Spectrochim. Acta A Mol. Biomol. Spectrosc. 145: 473-81. Ozaki, M, Wada-Katsumata, A, Fujikawa, K, Iwasaki, M, Yokohari, F, Satoji, Y, Nisimura, T and Yamaoka, R (2005) Ant nestmate and non-nestmate discrimination by a chemosensory sensillum. Science 309: 311-4.

CE

Pelosi, P, Iovinella, I, Zhu, J, Wang, G and Dani, FR (2017) Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects. Biological Reviews. Pelosi, P, Zhou, JJ, Ban, LP and Calvello, M (2006) Soluble proteins in insect chemical communication.

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Cell. Mol. Life Sci. 63: 1658-1676. René, T and Christensen, MH (2006) MolDock: a new technique for high-accuracy molecular docking. J. Med. Chem. 49: 3315-3321. Ross, PD and Subramanian, S (1981) Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 20: 3096-3102. Rusconi, B, Maranhao, AC, Fuhrer, JP, Krotee, P, Choi, SH, Grun, F, Thireou, T, Dimitratos, SD, Woods, DF, Marinotti, O, Walter, MF and Eliopoulos, E (2012) Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays. Biochim. Biophys. Acta 1824: 947-53. Sanchez-Gracia, A, Vieira, FG and Rozas, J (2009) Molecular evolution of the major chemosensory gene families in insects. Heredity 103: 208-16. 19

ACCEPTED MANUSCRIPT Schröder, R and Hilker, M (2008) The relevance of background odor in resource location by insects: a behavioral approach. Bioscience 58: 308-16. Schwede, T, Kopp, J, Guex, N and Peitsch, MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31: 3381-3385. Shahabadi, N, Hadidi, S and Feizi, F (2015) Study on the interaction of antiviral drug 'Tenofovir' with human serum albumin by spectral and molecular modeling methods. Spectrochim. Acta A Mol. Biomol. Spectrosc. 138: 169-175. Sreerama, N and Woody, RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded

PT

reference set. Anal. Biochem. 287: 252-60.

Sun, L, Zhou, JJ, Gu, SH, Xiao, HJ, Guo, YY, Liu, ZW and Zhang, YJ (2015) Chemosensillum

RI

immunolocalization and ligand specificity of chemosensory proteins in the alfalfa plant bug Adelphocoris lineolatus (Goeze). Sci. Rep. 5.

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Tumlinson, JH, Paré, PW and Lewis, WJ (1999) Plant production of volatile semiochemicals in response to insect-derived elicitors. Novartis Found Symp 223: 95-105. Verma, LR and Partap, U (1993) The Asian hive bee, Apis cerana, as a pollinator in vegetable seed

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production.

Ware, WR (1962) Oxygen quenchig of fluorescence in solution: An experimental study of the diffusion process. J. Phys. Chem. 66: 316-20.

MA

Williams, D and Westwell, M (1998) Aspects of weak interactions. Chem. Soc. Rev. 27: 57-64. Xu, J and Wang, Z (2006) Methods of fluorescence analysis. Science Press: Beijing, edn 3: 67. Yi, X, Wang, P, Wang, Z, Cai, J, Hu, M and Zhong, G (2014) Involvement of a specific chemosensory protein from Bactrocera dorsalis in perceiving host plant volatiles. J. Chem. Ecol. 40:

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267-275.

Yi, X, Zhang, Y, Wang, P, Qi, J, Hu, M and Zhong, G (2015) Ligands binding and molecular simulation:

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the potential investigation of a biosensor based on an insect odorant binding protein. Int. J. Biol. Sci. 11: 75-87.

Zhang, ZK and Lei, ZR (2015) Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the Western flower thrips, Frankliniella

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occidentalis. PLoS One 10: e0117726. Zhuang, X, Wang, Q, Wang, B, Zhong, T, Cao, Y, Li, K and Yin, J (2014) Prediction of the key binding site of odorant-binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida).

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Insect Mol. Biol. 23: 381-90.

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ACCEPTED MANUSCRIPT Figure Captions Fig.1. SDS-PAGE of purified recombinant CSP1 protein. M represents protein molecular weight marker. Lane 1 and lane 2 shows that the whole of lysis bacterial proteins including pET32-CSP1 plasmid without and with induction of 1 mmol∙L-1 IPTG, respectively. Lane 3 shows that the purified recombinant CSP1 protein, which is pointed by a black arrow on the right of the figure.

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The molecular weight is approximately 28 KD.

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Fig.2. Fluorescence quenching spectra and chemical structure of β-ionone. As β-ionone titrated in

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fluorescence intensity of CSP1 obviously decreased.

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from 1 to 9 (final concentration is 0, 5, 15, 25, 35, 45, 55, 76, and 109 µmol∙L-1, respectively), the

Fig.3. Synchronous fluorescence spectra of CSP1 and β-ionone. (A): ∆λ=15nm (tyrosine), when

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β-ionone is titrated, the fluorescence intensity decrease slightly. The maximum emission wavelength has a slight blue shift from 303 to 300 nm. (B): ∆λ=60nm (tryptophan), when

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β-ionone is titrated, the fluorescence intensity also decrease sharply.

Fig.4. The Stern-volmer plots of fluorescence quenching of CSP by β-ionone at 284K and 294K. The concentration of the CSP1 proteins was 1 µmol∙L-1, and the tittering concentration of β-ionone

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was 10 mmol∙L-1.

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Fig.5. UV spectra of CSP1 and that with β-ionone 1:1. Scanning range from 230 to320 nm.The maximum absorption wavelength did not change significantly after β-ionone added. c (CSP1) = 1.0 µmol∙L-1, pH = 7.4.

Fig.6. Circular dichroism (CD) spectra of CSP1 and β-ionone. As β-ionone added in (final concentration of β-ionone from a to c is 0, 10, 50 µmol∙L-1 respectively), the typical shoulder peaks of α-helix increased at 208 and 222 nm.

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ACCEPTED MANUSCRIPT Fig.7. Molecular docking of CSP1 with β-ionone. (A): β-ionone interacts with residues located on α-helices as well as N terminal flexible tail region. Red represents residues that provide hydrophobic bonds. (B): shows the detailed contributions of the residues that interact with β-ionone. (C): The sequence alignment between CSP1 and 1n8v (CSPMbraA6). The 12 interacting residues of CSP1 with β-ionone are labeled by blue letters. Two direct mutation sites in CSP1

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residues are labeled by red letters and pointed by red arrows. 4 conserved cysteines are shown as

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yellow shadow.

Fig.8. SDS-PAGE of purified recombinant CSP1 proteins of wild type and mutants. M represents

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protein molecular weight marker. Lane 1, lane 2, and lane3 show that the purified recombinant

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CSP1 protein of wild CSP1, CSP1-F44G, and CSP1-Q63G, respectively.

Fig.9. The Double-Log plots of the fluorescence of wild CSP1 wild type, CSP1-F44G and

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CSP1-Q63G quenching by β-ionone. The concentration of the three proteins were 1 µmol∙L-1, and

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the tittering concentration of β-ionone was 10 mmol∙L-1 (final concentration is 0, 5, 15, 25, 35, 45,

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55, 76, and 109 µmol∙L-1, respectively).

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ACCEPTED MANUSCRIPT Table 1. Apparent association constant (KA), the binding interaction and the Stern-Volmer dynamic quenching constant (Ksv) of CSP1-WT, CSP1-F44G, CSP1-Q63G at 284K and 294K.

mutants

Thermodynamic parameter T/K

-1

KA/(L mol )

284

3.994×104

294

4

CSP1Wild type

binding force ΔH

ΔS

-25.02 -20.33

2.980×10

ΔG hydrophobic

Ksv/(L mol-1)

-25.18

electrostatic

characte ristic

2.56×104

and

16.49

binding

dynamic

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Proteins/

3.96×104

force -22.81

hydrophobic

2.18×104

294

2.354×104

-24.61

interaction

2.41×104

284

2.125×104

-23.53

294

4

CSP1-F44G

28.34

CSP1-Q63G

-25.49

-23.46

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hydrogen

dynamic 2.61×104

bonds and Van

-6.90

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1.472×10

180.09

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1.565×104

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284

der Waals force

2.31×104

static

ACCEPTED MANUSCRIPT Table 2. Individual residue contribution in CSP1 that interacts with β-ionone Site Number

Epaira

Phe Gln Phe Tyr His

44 63 43 4 47

-20.582 -18.301 -13.239 -8.554 -8.162

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Epair: hydrophobic and electrostatic forces energy between a ligand atom and a receptor atom.

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a

Residues interacted

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ACCEPTED MANUSCRIPT Table 3. Apparent association constant (KA) and the number of binding sites (n) of CSP1, CSP1-F44G and CSP1-Q63G at 284K Double logarithm equation Proteins

Decline

KA/(L·mol-1)

n

R2

CSP1

3.994×104

0.9264

0.9890

—

CSP1-F44G

1.565×104

1.1027

0.9948

60.82

CSP1-Q63G

2.125×104

1.0636

0.9969

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rate (%)

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46.80

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ACCEPTED MANUSCRIPT Highlights 1. Revealing how antenna-specific CSP1 interacts with plant semiochemical in detail. 2. Twice thermodynamic analysis combined with multispectral method

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was developed.

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3. Site-directed mutagenesis verified Phe44 maintained an electrostatic

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It also verified Gln63 contributes hydrophobic and electrostatic

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forces.

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4.

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interaction.

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Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9