Characterization of Hepatitis C Virus Core Protein Dimerization by

Mar 6, 2018 - (49) It is noted that what we obtained from SMFS is the rupture force related to koff (dissociation rate constant), which is different f...
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Characterization of Hepatitis C Virus Core Protein Dimerization by Atomic Force Microscopy Wenhui Li, Xiaolong Kou, Jiachao Xu, Wei Zhou, Rong Zhao, Zhen Zhang, and Xiaohong Fang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05070 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Analytical Chemistry

Characterization of Hepatitis C Virus Core Protein Dimerization by Atomic Force Microscopy Wenhui Li,1, 2 Xiaolong Kou,1, 2 Jiachao Xu,1, 2 Wei Zhou,1, 2 Rong Zhao,1, 2 Zhen Zhang,1, 2 Xiaohong Fang, * 1, 2 1 Key Laboratory of Molecular Nanostructure and Nanotechnology, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China 2 University of Chinese Academy of Sciences, Beijing 100049, China ABSTRACT: Dimerization of core protein is a crucial step in the formation of the hepatitis C virus (HCV) nucleocapsid and inhibition of dimer formation is regarded as an attractive approach to design anti-HCV drugs. In this work, we developed the atomic force microscopy based single-molecular force spectroscopy (AFM-SMFS) method for the characterization of core protein dimerization with the advantages in small amount of sample consumption and no need of labeling. Interaction force of the core protein with its antibody or aptamer was analyzed to investigate its stoichiometry and binding property. The two specific binding forces were detected due to the probing of dimeric and monomeric of core protein respectively. Moreover, the binding property of protein dimer was different from the monomer. Our work offers a new approach to study the dimerization of core protein, as well as other proteins, and to screen the HCV candidate inhibitors.

INTRODUCTION Hepatitis C virus (HCV) is a major cause of chronic hepatitis, cirrhosis and hepatic carcinoma, thus represents a substantial clinical problem.1-3 Its most conserved protein, core protein, has been regarded as a drug target for HCV combination treatment since the core protein plays significant roles in not only viral nucleocapsid formation but also HCV pathogenesis by interacting with a variety of cellular proteins in host cells.4,5 As a virus capsid protein, core protein is normally homooligomerized.6-8 Previous studies indicated the C-terminal domain of core protein contains certain peptide sequences for self-association initiating.9 The cysteine residue at amino acid position 128 contributes to stable the disulfide-bonded core protein dimer.10 Dimerization of the core protein is an essential step in its oligomerization, which functions as the organizer of HCV nucleocapsid assembly and viral production.11-13 Blocking core protein dimerization becomes a useful approach to develop new anti-HCV drugs. Several peptides and chemical compounds which could disrupt the core protein dimerization were reported as potent HCV inhibitors.11,14-17 Therefore, the development of highly sensitive analytical methods to characterize core protein dimerization is of great importance. The commonly used methods for the investigation of core protein oligomerization can be summarized into two types. The first one is based on protein-protein interaction such as yeast two-hybrid system,9 enzyme-linked immunosorbent assay (ELISA),11 time resolved-fluorescence resonance energy transfer (TR-FRET)16 and amplified luminescent proximity homogeneous assay (AlphaScreen).18 For example, some inhibitors blocking the core protein dimerization were identified using TR-FRET assay.16 However, these methods require the preparation of core protein with two different tags, which may change the protein natural characteristics. The second ap-

proach is based on the molecular weight difference, which includes density-gradient ultracentrifugation,19 gel-filtration chromatography,9 native-PAGE.10 Fragment analysis by density-gradient ultracentrifugation and gel-filtration chromatography suggested that core protein residues 1-169 and 119-162 tended to self-associate into dimeric form.12 These methods consume a relatively large amount of sample (more than a few micrograms), and are often not easy to analyze the full-length proteins with high molecular weight. Atomic force microscopy (AFM) is a rapidly developing analytical technique to image macromolecules with high resolution and to measure bimolecular interaction force at the single-molecule level under physiological conditions.20-22 Especially, AFM single-molecule force spectroscopy (SMFS) has been widely used to study protein unfolding,23-25 and binding of ligand-receptor,26-28 antibody-antigen,29-31 DNA-protein,32 aptamer-protein,33,34 and DNA-DNA.35,36 In this work, we demonstrated, for the first time, the investigation of protein dimerization by quantifying the intermolecular binding forces from SMFS. In contrast to observe one peak in the normal rupture force histogram from previous reports, we found two forces in single-molecule force measurement between HCV core protein and its antibody. This was used to confirm that core protein was in forms of both monomer and dimer in solution. Further SMFS analysis indicated different binding properties of monomeric and dimeric core proteins. Comparing with the conventional dimerization assays, our SMFS method is advantageous in less sample consumption (less than 0.5 µg), label-free and the ability to study monomer/dimer binding property simultaneously. Thus, AFM-SMFS is expected to be a new tool to characterize the dimerization of HCV core protein as well as other proteins for its function study and therapeutic drug screen.

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MATERIALS AND METHODS Materials. Recombinant hepatitis C virus core antigen protein and antibody were purchased from Abcam (Shanghai, China). Aptamer C7 (5’-NH2-ACG CTC GGA TGC CAC TAC AG ACT ATA CAC A AA AAT AAC ACG ACC GAC GAA AAA ACA CAA C CT CAT GGA CGT GCT GGT GAC-3’), aptamer C97 (5’-NH2-ACG CTC GGA TGC CAC TAC AG TAA CAC ACA CAA CTT AAA ATC ATA CAA AAA AGA GTA AAT G CT CAT GGA CGT GCT GGT GAC-3’), and aptamer C103 (5’-NH2-ACG CTC GGA TGC CAC TAC AG TAC CAC ACA TGC AGA CCC ACA CAA ATA CAT ACT AGA GAC ACT CAT GGA CGT GCT GGT GAC-3’) were purchased from Life Technology (Shanghai, China). Peptide SL173 (LYGNEGCGWAGWLLSPRG) was synthesized from the GenScript Corporation. (Nanjing, China). Nhydroxysuccinimide-polyethylene glycol-maleimide (NHS-PEG-MAL, Mw3400) was purchased from Laysan Bio (Arab, Alabama, USA). 3-aminopropyltriethoxysilane (APTES), 3-mercaptotrimethoxysilane (MPTMS), toluene and glutaraldehyde (99.99%, HPLC grade) were purchased from Sigma (Shanghai, China). Other reagents used were of analytical grade. Milli-Q-purified water (18.2 MΩ/cm) was used for all experiments. Preparation of Tip and Silicon Substrates. AFM silicon nitride (Si3N4) tips (DNP-10, Veeco, California, USA) with ∼0.06 N/m nominal spring constant were used. Tips were chemical modified with antibody or aptamer, according to procedures described in the previous reports.37,38 Two modification methods were utilized. The first one was using glutaraldehyde to conjugate antibody/aptamers. Tips were cleaned by plasma cleaner (Mycro Technologies, shanghai) with highly purified oxygen gas for 5 min, immediately transferred to 1.0% APTES/toluene solution (v/v) for 2 h, and successively cleaned by toluene, acetone and ethanol. The animated tips were incubated in 0.1% (v/v) glutaraldehyde PBS solution for 45 min and washed with PBS buffer. The activated tips were incubated in 5.0 µg/mL antibody solution or 10-5 M aptamers solution at 4 ℃ overnight, then washed with buffer, and stored in 4 ℃ until use. For the second immobilization method, a PEG linker was introduced to couple antibody. After cleaned by plasma cleaner, tips were quickly transferred to 1% MPTMS/toluene (v/v) solution for 2 h, and successively cleaned by toluene, acetone and ethanol. The salinized tips were immersed in 1.0 mg/mL MAL-PEG-NHS in dimethyl sulfoxide (DMSO) solution for 3 h, and rinsed with DMSO. The activated tips were incubated in 5.0 µg/mL antibody solution at 4 ℃ overnight, then washed three times, and stored in 4 ℃ until use for a few days. Single crystal silicon substrates with 1.0 cm×1.0 cm were used for modification. The substrates were soaked in chloroform by ultrasonic oscillation for 30 min, immersed in 40% HF solution for 1 min, placed in NH4OH/H2O2/H2O (1/1/5, v/v/v) solution for 30 min, and incubated in piranha solution (98% H2SO4/H2O2 = 7/3, v/v) at 90 ℃ for 45 min. After dried by nitrogen gas, substrates were transferred to 1.0% MPTMS/toluene solution (v/v) for 2 h, and successively cleaned by toluene, acetone and ethanol. Then, the animated substrates were immersed in 1.0 mg/mL MAL-PEGNHS/DMSO solution for 3 h, and rinsed with DMSO. The activated substrates were incubated in core protein solution

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(5.0 µg/mL, 1.0 µg/mL, 0.5 µg/mL) at 4 ℃ overnight. For the inhibition of dimerization of core protein, the core protein solution was treated by 5% β-mercaptoethanol for reducing condition and boiled for 10 min, or the core protein solution was mixed with peptide SL173 inhibitor (10-6 M, 10-5 M, 10-4 M) before it was dropped on the activated substrate. After washing with buffer, the functionalized substrates were stored in buffer at 4 ℃ for force measurement. For AFM image, the functionalized substrates were dried by nitrogen gas. AFM Force Measurements. Force measurements between antibody/aptamer-modified tips and core protein functionalized silicon substrates were performed in a liquid cell by AFM (Multimode 8, Bruker, USA). The spring constant of cantilever was calibrated in buffer using the thermal fluctuation method.39,40 Each tip was calibrated for three times to obtain an averaged value. Before force measurements, the modified tip and functionalized substrate were thermally equilibrated to room temperature. The AFM stylus was pushed onto the substrate at a force of ∼1 nN, a ramp size of 1 µm, and surface delay time of 1 ms. Then, the stylus was withdrawn at 1 µm/s pulling velocity. Force measurements were carried out at several pulling velocities from 25 to 12000 pN/s. In each experiment, 5000 force curves were measured in scan area of 1 µm x1 µm from 10 different areas of the substrate. AFM Imaging. The AFM (Multimode 8, Bruker, USA) was operated in the scanAsyst air mode. Rectangular Si3N4 cantilever with nominal spring constants of ∼0.4 N/m in air was chosen (scanAsyst-air, Veeco, California, USA). The sample was scanned using a line frequency of 1 Hz with a resolution of 512×512 pixels. Images were analyzed using the Nanoscope analysis software v1.50 (Bruker). Data Analysis. The force-distance curves were fitted by modified freely jointed chain (M-FJC) model based on the extended Langevin function. The polymer chain PEG linker was considered as a chain of independent rigid Kuhn segments with a length of lk (Kuhn length) in M-FJC model. The segments occurred deformation under external stress were freely connected without any long-range interactions. The extended Langevin function is displayed below:41,42   Fl χ ( F ) =  coth  k  k BT 

 k BT   nF  L +  Fl k   c K s

  

Here, χ is the extension of a polymer chain, lk is the length of independent segment, F represents the applied force on an individual polymer chain, kB is the Boltzmann constant, Lc is the contour length of polymer chain, n is the number of segments, T is the temperature in Kelvin, and Ks is the segment elasticity. A user-defined program in Matlab was used to analyze the force curves. The histograms of specific forces were simulated by Gaussian function. According to Bell model, the relationship between the rupture force F and loading rate γ is described by43,44 F=

 kBT γχβ • ln  χβ  koff (0)kBT

 k BT  kBT γχβ • ln γ + • ln  = χβ χβ   koff (0)kBT

  

Where kB is the Boltzman constant, T is Kelvin temperature, koff (0) is dissociation rate constant at a zero external force. χβ is the distance to the transition state separating bound and unbound state. The energy barrier height (∆G0) calculated from an Arrhenius equation in the absence of external force was evaluated as45

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Analytical Chemistry ∆ G β , 0 = − k B T • ln ( τ D ⋅ k off(0) )

where τD denotes the diffusive relaxation time,τD = 10-9 s. The most probable interaction forces were calculated by the Gaussian fitting of the force distribution histograms, as the rupture force measured by AFM was affected by the orientation of biomolecules.26, 28 By averaging the values of the most probable interaction forces obtained from three independent experiments, the interaction force was determined. The data were presented as mean ± standard error of the mean (S.E.M). To compare binding force between different binding partners, six rupture force histograms of core protein-antibody and that of core protein-aptamer were obtained in three independent experiments from three silicon substrates, with two histograms from each substrate. Therefore, six force values were determined for each binding partners. Statistical analysis was performed for comparison using Student’s t-test, and p > 0.05 was regarded as no statistically significant difference. RESULTS AND DISCUSSION Two specific binding forces in the SMFS of core proteinantibody. We firstly applied SMFS to measure the interaction force between HCV core protein and antibody (Figure 1a). The core protein was coupled to the substrate with a PEG linker, which maintained protein free conformation and facilitated the identification of specific binding force in the forcedistance curve.37 The antibody was tethered to the AFM tip by glutaraldehyde. Low-concentration solutions of core protein and antibody (5 µg/mL) were used for their immobilization, so that their densities were low enough to make sure that only one pair of core protein-antibody molecule was formed and ruptured in the force measurement. Typical force-distance curves were shown in Figure 1b with the peak represented rupturing of core protein-antibody. The peak disappeared after adding antibody solution, indicating it was caused by the specific core protein-antibody interaction. The most probable rupture forces of core protein-antibody were derived by the Gaussian fitting from 5000 force-distance curves in one experiment (Figure 1c). Unlike our previous SMFS studies which observe only one most probable force,26,27,30,32 we found two forces in this work with doubleGaussian fitting (R2=0.9885). By averaging the force values from three independent experiments, the two rupture forces of core protein-antibody were determined as 38.3±1.8 pN and 67.5±5.9 pN at the retract speed of 1.0 µm/s. The binding probability of 20.2%±2.5% was lower than 30%, which met the criteria for single-molecule force measurement (Figure 1d).46 Considering that the measurement of biomolecular interaction force could be influenced by molecular orientation, we changed to use a more flexible PEG linker for antibody coupling to exclude the immobilization effect, and confirmed the two specific forces of core protein-antibody (Figure S1a). Similar results were obtained with the forces of core proteinantibody as 41.7±2.4 pN and 70.5±3.1 pN (Figure S1b), and binding probability as 16.2%±2.1% (Figure S1c).

Figure 1. Detection of core protein-antibody interaction force. (a) Schematic illustration of the antibody/core protein immobilization on the AFM tip and substrate by glutaraldehyde and PEG linker respectively. (b) The typical force curves of core proteinantibody (i, ii) and the one after antibody blocking (iii). (c) Histogram of the forces of core protein-antibody. The concentrations of core protein and antibody were 5 µg/mL for immobilization. (d) Binding probability of core protein and antibody without (left) or with (right) antibody blocking.

Then we performed SMFS by replacing the antibody with DNA aptamer, a new type of molecule probe. Aptamers are ssDNA or RNA molecules that functions as antibody for specific protein recognition.47,48 The core protein aptamer C7 we previously selected was used to measure the single-molecule binding force with core protein.49 Two specific forces of core protein-aptamer C7, 41.4±2.1 pN and 71.5±1.6 pN, were also obtained (Figure S2). Thus we confirmed the two specific binding forces for interaction of core protein with different immobilization strategies and different ligands. Two binding forces caused by monomeric and dimeric core protein. Since the mature core protein had been reported to form dimeric protein due to its self-association tendency,10 we expected that the two specific forces of SMFS in Figure 1 were attributed to the binding of core protein monomer and dimer, as shown in Figure 2a. However, there were other possibilities need to be excluded, such as simultaneously binding of two core protein monomers to one antibody or formation of two pairs of core protein monomer-antibody (Figure 2b, c). In order to clarify this issue, several control experiments were carried out and discussed below. First of all, the force curves were fitted by a modified freely jointed chain (M-FJC) model based on the extended Langevin function as the PEG linker was extended during the rupture of core protein-antibody complex.41,42 The two force-distance curves we chosen, in which the rupture peaks displayed a typical high force value and low force value in Figure 1c, were superimposed well after normalization (Figure S3a). In addi

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Figure 2. Detection of core protein-antibody interaction force under different modification concentrations. (a-c) Schematic diagram of the possible interaction model between core protein and antibody during the force measurements. (a) The rupture of dimer-antibody (left) and monomer-antibody (right). (b) The rupture of two monomers binding to one antibody (left), and monomer-antibody (right). (c) The rupture of two pairs of monomer-antibody (left) and monomer-antibody (right). (d-e) Histogram of the forces of core protein-antibody, when the concentrations of core protein and antibody were 1 µg/mL (d) and 0.5 µg/mL (e). (f) The binding probabilities of core protein and antibody under different modification concentrations (5 µg/mL, 1 µg/mL and 0.5 µg/mL).

tion, all of the force curves exhibited similar deformational parameter (Ks) by fitting with the M-FJC model. This suggested that single-chain PEG was stretched,50 thus only one pair of molecules was ruptured, which was against the situations in Figure 2b and 2c. Next, we checked the immobilization density of HCV core protein to verify our expectation. According to the calculation of AFM tip-substrate contact area and protein density from AFM imaging of core protein, the average number of core protein covered by the tip was 0.3 molecule (Figure S3b-f), when 5 µg/mL core protein concentration was used for immobilization. Therefore, the possibility of probing two core proteins simultaneously during the force measurement in Figure 1c should be quite low, and could not be higher than that of one core protein. However, from the SMFS histogram in Figure 1c, the population of the component with the high force value was higher than that of the low force value. Therefore, the second peak with high force in the histogram could not be caused by rupturing two molecule pairs. In addition, we did not observe multiple rupture peaks from more than one pair of biomolecules in the force-distance curves. This indicated that the rupture of the antibody-core protein dimer performed simultaneous unbinding, which was similar to SMFS of thrombinaptamer dimer and macrocyclic hexazole dimer-telomeric DNA in the previous reports.51,52 We further carried out experiments under the reduced modification concentrations of core protein as well as the antibody. The results of SMFS were still characterized by two specific forces (Figure 2d,e), even when the modification concentration of core protein and antibody was both reduced to 0.5 µg/mL, and the binding probability of core protein-antibody was decreased from 20.2±2.0% to 7.9%±1.3% (Figure 2f). The decreased binding probability was caused by the decreased density of immobilized core protein and antibody, thus possibility of complex forming between AFM tip and substrate

during AFM force measurement was also reduced. Under such low modification concentrations and low binding probabilities, the population of the component with the high force value was still quite high, suggesting the high force value was not caused by simultaneously rupturing two pairs of biomolecules. All together, the two specific forces were attributed to rupture monomer-antibody and dimer-antibody (Figure 2a). Our work suggested the co-existence of core protein monomer and dimer in solution, which was in agreement with the previous report.10 It has been reported that the HCV core protein with different tags (Flag tag vs Myc tag) and from different sources (microsomal membrane vs cell lysate) exhibited different relative proportion of dimer and monomer. 10 In this work, the SMFS results indicated the higher population of dimer than monomer for the recombinant GST tagged core protein in PBS solution. Therefore, SMFS could be used to distinguish the monomeric and dimeric HCV core protein and pave a new way to study the dimerization of core protein with high sensitivity. One binding force of monomeric core protein and antibody after blocking dimerization. According to the previous reports, the dimer of core protein was stabilized by intermolecular disulfide bond, and β-mercaptoethanol treatment could result in monomeric proteins.10 Hence, we performed the SMFS experiment after the addition of β-mercaptoethanol in the core protein solution before immobilization. As expected, the Gaussian function of the rupture force histogram could only be fitted with one peak (Figure 3a). And there was no significant change in force distribution among different core protein modification concentrations (Figure 3b,c). The most probable force values from these histograms were, 42.0±2.7 pN, close to that of the low peak force value, 38.3±1.8 pN, in Figure 1b. This indicated that the low force value in Figure 1b

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Analytical Chemistry

Figure 3. Histograms of rupture force of core protein-antibody under different modification concentrations by β-mercaptoethanol treatment. The concentration of β-mercaptoethanol was 5% (v/v). The concentrations of HCV core protein and antibody were 5 µg/mL (a), 1 µg/mL (b), 0.5 µg/mL (c).

Figure 4. Histograms of rupture force of core protein-antibody with peptide SL173 blocking under different conditions. The concentrations of HCV core protein and antibody were 5 µg/mL, and the concentration of peptide SL173 was 10-6 M (a), 10-5 M (b) and 10-4 M (c).

was caused by the rupture of monomer-antibody, and the high force value was caused by the rupture of dimer-antibody, which, in turn, indicated that core protein was in forms of monomer and dimer without β-mercaptoethanol treatment. Since blocking core protein dimerization is a practical strategy to develop anti-HCV drugs, the SMFS experiment was also performed under the condition of peptide SL173, a wellknown inhibitor of core protein dimerization.11 With peptide SL173 blocking, the population of rupturing dimer-antibody was dramatically reduced (Figure 4a). The dimerization of core protein was vanished when the concentration of peptide SL173 was up to 10-5 M (Figure 4b, c). The results demonstrated the ability of SMFS for dimer inhibitor screen. Different binding properties of dimer and monomer of core protein. SMFS can not only be used to characterize the monomeric and dimeric HCV core protein under different conditions, but also investigate their binding properties. It is known that aptamers are new molecular probes that compete with antibodies in disease diagnosis and therapy.47,48 The affinity of aptamer to target protein can be higher, similar or lower than that of antibody. Beside aptamers C7 mentioned above, other two aptamers C103 and C97 we previous selected were studied with SMFS. The interactions forces of the core protein-aptamer (C7, C103, C97) also displayed double-Gaussian fitting with different binding strengths, which were 41.4±2.1 pN and 71.5±1.6 pN, 27.9±2.5 pN and 55.1±6.2 pN, and 32.7±1.9 pN and 66.7±3.9 pN, respectively (Figure S2, S3a,b). SMFS analysis at the single-molecule level showed that aptamer C7 had the highest binding strength and C97 the lowest, which was consistent with the results by ELISA.49 It is noted

that what we obtained from SMFS is the rupture force related to koff (dissociation rate constant), which is different from the commonly used kd (dissociation constant, koff / kon,) for affinity. Besides binding force, more binding information on core protein-aptamer could be derived from SMFS. As shown in Figure 5, while C7 has comparable binding strength as antibody for both monomeric and dimeric HCV core protein, C103 has lower binding strength than antibody for both monomeric and dimeric HCV core protein. However, the binding strength of C97 was comparable to antibody for the core protein dimer but lower than antibody for monomer.

Figure 5. Comparison of the rupture forces of core proteinantibody and core protein-aptamer. (a) The rupture forces of monomeric core protein-antibody/aptamer. (b) The rupture forces of dimeric core protein-antibody/aptamer. The concentration of core protein and antibody was 5 µg/mL, and the concentration of aptamer was 10-5 M. Data shown were mean±S.E.M of six independent experiments. P > 0.05 in the Student’s t-test, there was no significant statistical difference.

The force measured by SMFS in the studies above was performed at a constant rate. According to the Bell model, the rupture force strength is affected by the force-loading rate of

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the cantilever.43 By plotting the most probable force against the logarithm of AFM loading rate to obtain the dynamic force spectroscopy (DFS), the height and width of free energy barriers along dissociation pathway of the binding complex can be quantified, as well as dissociation rate constant.43,44 Here we used DFS to analyze the dissociation processes of monomerantibody and dimer-antibody. With the force-loading rates changed from 25 pN/s to 11000 pN/s, the force of the monomer-antibody and dimer-antibody increased with the logarithm of force-loading rate in two linear regimes (Figure 6), which indicated the complex experienced an intermediate state during dissociation.45 However, the slopes of plots for dimer-antibody and monomer-antibody were obviously different, suggesting two different dissociation pathways (Figure S5). The parameters describing their energy landscapes were shown in Table 1. The parameter xβ represents the distance between bound state and transition state. At low loading rates, the outer barrier was probed, and at high loading rates, the inner barrier governs the unbinding rate. Short xβ sustains high forces but small deformations.53 The dissociation off-rate constant (koff (0)) of dimer-antibody and monomer-antibody were 0.12 s-1 and 0.20 s-1, respectively at low loading rates, and were 143.35 s-1 and 232.90 s-1 at high loading rates. The higher koff (0) value indicated a tendency to rapidly dissociate between core protein and antibody, thus proving the complex of dimer-antibody was more stable.

protein antibody and aptamer. DFS experiments revealed monomer-antibody and dimer-antibody had different energy landscapes during dissociation process. The results provide useful information on HCV core protein dimerization and binding property for drug development. Our work offers an analytical framework to study biomolecule stoichiometry and screen pharmacological inhibitors.

ASSOCIATED CONTENT Supporting Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone: 86-10-62650024. Fax: +8610-62650024.

ORCID Xiaohong Fang: 20000-0002-2018-0542

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (No. 21127901, 91413119, 91213305), and Chinese Academy of Sciences.

REFERENCES Figure 6. Plot of the binding force for the interactions of monomer-antibody and dimer-antibody against the logarithm of the loading rate. Each data point represented the mean value of three independent experiments.

Table 1. Parameters characterizing energy landscapes of dimer-antibody and monomer-antibody. Parameters

dimer-Antibody

monomer-Antibody

Loading rate (pN/s)

High rate 350010000

Low rate 25-3500

High rate 450011000

Low rate 30-4500

χβ (nm)

0.05

0.48

0.06

0.82

koff (0) (s-1)

143.35

0.12

232.90

0.20

∆G0 (kBT)

15.9

23.1

15.1

22.2

CONCLUSION In summary, we have established a new method to detect the dimerization of core protein with high sensitivity using AFM-SMFS. By analyzing the distribution of measured forces, monomeric and dimeric of core protein were distinguished. The forces of dimer-antibody and monomer-antibody were determined as 38.3±1.8 pN and 67.5±5.9 pN, respectively. Binding of dimer was different from monomer for both core

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