Identification of spherical and non-spherical protein by solid-state

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Identification of spherical and non-spherical protein by solid-state nanopore Jingjie Sha, Wei Si, Bing Xu, Shuai Zhang, Kun Li, Kabin Lin, Hongjiao Shi, and Yunfei Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04136 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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Analytical Chemistry

Identification of spherical and non-spherical protein by solid-state nanopore Jingjie Sha*ab#, Wei Si ab#, Bing Xuab, Shuai Zhangab, Kun Li ab, Kabin Linab, Hongjiao Shi ab and Yunfei Chen*ab aSchool

of Mechanical Engineering, Southeast University, Nanjing 211189, China.

bJiangsu

Key Laboratory for Design and Manufacture of Micro-nano Biomedical Instruments,

Southeast University, Nanjing 211189, China # These authors contributed equally to this work. Email: [email protected], [email protected] Abstract: Three-dimensional structure of a protein plays an important role in protein dynamics in biological system of human. By now, it remains challenge to characterize and quantify the shape of a protein at single-molecule level. Nanopore, as a novel single-molecule sensor, has been widely applied in many fields such as DNA sequencing and human diseases diagnosis. In this paper, we investigated the translocation of sphere-like con.A and the prolate Bovine Serum Albumin (BSA) under electric field by solid-state nanopore. By analyzing the ionic current, the con.A and the BSA could be characterized and differentiated due to their intrinsic shape difference. Because the prolate BSA will have the preferred orientations for higher electric field when it is residing inside the nanopore, thus multiple ionic current blockade levels will be observed. While for the spherical con.A, there is only one ionic current blockade level. This method presented here will be potentially applied to fingerprint a single protein as a new method having features of low-cost and high-throughput in the near future.

Keywords: protein, shape, orientation, ionic current, solid-state nanopore, electric field

1 Introduction

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

An amino acid chain, the primary structure of protein, is generally considered to be inactive. Upon the linear amino acid sequence folds into a three-dimensional structure, protein would play a significant role in biological system. Fully folded proteins have a certain shape and function in all aspects of human life. It controls the metabolism, regulates life activities of organisms and protects human from outside attack. To understand proteins’ working mechanism in human bodies, protein characterization is pretty essential, which includes the protein size measurements, protein conformational changes detection and shape approximation. Probing the shape of a protein is a part of the characterization. It could provide the three-dimensional structure data and then help researchers monitor the dynamics of protein. Optical methods are traditional for protein characterization, such as Scanning Electron Microscope (SEM) 1, Fluorescence Resonance Energy Transfer (FRET) 2 and Laser-induced fluorescence (LIF) 3. Likewise, biomechanical tools are widely used in recent years, such as Optic Tweezer4, Magnetic Tweezer5 and Atomic Force Microscopy (AFM) 6. These methods are usually reliable and useful, but the limits are also obvious. Pre-treatments for samples may impact the native proteins’ states. Complicated experimental procedures are challenging for researchers. Expensive equipment and materials may limit its development.

Nanopore technology is one of the single–molecular methods7-25. Comparing to the mentioned methods before, more practical benefits are listed as follows: (1) simple preparation for samples: label-free or chemical modification-free; (2) rapid detection; (3) high throughput. Many efforts have been devoted to the research of nanopore. DNA sequencing is the first hot research area. Later, with the development of solid-state nanopore, molecules with size comparable or larger than DNA, such as short peptides, small proteins as well as


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protein-protein complexes26-32, have been investigated in recent years. When charged proteins are driven through the pore under applied voltage, A series blockage ionic current signals are recorded ， and the researchers can obtain the information of the proteins by analysing the data. Using nanopores, Pradeep Waduge found a correlation between the shape of the current signal amplitude distributions and the protein fluctuation as obtained from molecular dynamics simulations33. Gautam V. Soni detected nucleosomes and its histone subcomplexes34. A systematic dependence of the conductance blockade and translocation time on the molecular weight of the nucleosomal substructures was observed. Erik C. Yusko estimated the shape and volume of single proteins from individual resistive pulses by analysing current values 35.

Recently molecular dynamics simulations have demonstrated that the nanopore ionic current can be potentially used to characterize the conformation changes of a protein inside the nanopore with high resolution36.

And a lot of pioneering work37-40 have validated these

theoretical observations. Besides investigation of folding and unfolding of the protein, characterization of three-dimensional shape of a protein is also significant as it plays an important role in human bodies. Unfortunately, seldom work were reported to successfully characterize the unlabelled and folded protein in aqueous environments at a single-molecule level 35.

In this paper, we report that proteins with different shape could be characterized and differentiated by the ionic current through the solid-state nanopore. The study performed here can be potentially used to identify single proteins in real time and may ultimately reveal biochemically or clinically relevant static or dynamic heterogeneities, allow monitoring an



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individual’s proteome1 and enable single-molecule sorting in the future.

2 Experimental section

2.1 Samples preparation BSA

41-43(66.43

KDa, Shanghai Sangon Biotech Co., Ltd., Shanghai, China) as an

example

of

prolate

protein

and

Con.A (104 KDa, Shanghai Sangon Biotech Co., Ltd., Shanghai, China) as an example of the spherical protein were used in the research. BAS has 583 amino acids residues and its isoelectric point is around 4.7 in water at 25℃. Native state BSA is an ellipsoid with

dimension

of

4×4×14

nm

in

pH

7.4.

Con.A has 948 amino acids residues and its isoelectric point is 4.5~5.5 in water at 25℃. Native state con.A is like

a Sphere with diameter of 8 nm in pH 8.0.

The

native

structures of BSA and con.A are shown in Figure 2b and Figure 4b, respectively. Potassium chloride (KCl, Shanghai Sangon Biotech Co., Ltd., Shanghai, China) was used as the electrolyte solution. Phosphate buffered saline (PBS, Shanghai Sangon Biotech Co., Ltd., Shanghai, China) was used to make the protein more stable in the electrolyte solution. All solutions were prepared with ultrapure water from milli-Q water purification system (Millipore Co., Billerica, MA, USA).

2.2 Solid-state nanopore fabrication A 100-nm thick, low-stress Si3N4 window (75 × 75 μm2) supported by a silicon chip was firstly fabricated by lithography and wet etching method. Then, the membrane in a small window with size of 500 × 500 nm2 was milled to reduce the membrane


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thickness to approximately 20 nm. A solid-state nanopore was eventually drilled on the milled region of Si3N4 film. Both the milling and drilling processes were completed by focused ion beams in a dual beam microscope (Helios 600i NanoLab, FEI Co., Hillsboro, USA)44-50. Prior to experiments, the nanopore was immersed into piranha solution (H2SO4 : H2O2 = 3:1) for 30min at 200℃ to make it clean.

2.3 Simulations of BSA dynamics All the molecular dynamics (MD) simulations in this work were conducted by using the MD program NAMD2

51

with a 2 fs time step. Periodic boundary conditions were

applied along the x, y and z directions, respectively. The CHARMM36 force field52 was used to describe proteins, TIP3P water and ions. Covalent bonds involving hydrogen atoms in proteins and water molecules were described by applying RATTLE 53and

SETTLE54 algorithms. The description of the interactions between a charge and

another

charge

were

improved

by

using

the

previously

reported

CUFIX

corrections55,56. The system was divided into grids with 1 Å resolution and the long-range electrostatic interaction was evaluated by Particle-Mesh-Ewald (PME)57 algorithm. A cutoff of smooth 10−12 Å was used to evaluate van der Waals interactions. The full electrostatics was calculated for every three timesteps. Atomic coordinates of the protein BSA (PDB entry 3v03) was obtained from the Protein Data Bank. The systems were solvated using the VMD’s Solvate plugin. Water molecules overlapping with the proteins were removed. Potassium and chloride ions were evenly added to neutralize the simulation system and bring the ion concentration to 1 M. The final system contained approximately 111,000 atoms. By using the conjugate gradient



method each simulation system was firstly minimized for 5000 steps. After that, the system was further equilibrated for 1 ns in the constant number of atoms, pressure and temperature (NPT) ensemble. The pressure of the simulation system was maintained at 1 atm by adjusting the system’s dimension using the Nose-Hoover Langevin piston pressure control58,59 . Finally that the equilibrated system was further simulated in NVT ensemble with a constant volume determined by the dimension averaged using the equilibration trajectory. The system temperature was maintained by using Langevin thermostat which applied a damping coefficient of 0.1 ps−1 to all the heavy atoms of the system . An external electric field along z direction was applied to induce the movement of ions or the rotation of the protein. All trajectories were analyzed by using VMD60.

2.4 Theoretical calculation of the blockade current

When the protein was presented in the 1 M KCl solution, the corresponding amplitude of ionic current blockade (IB) was calculated by using a previously reported theoretical model36,61. 200 similar conformations of protein were randomly selected for each orientation,, a 3 dimensional distance map composed of cubic grids with 1 Å resolution was computed at to obtain the nearest distance from the grid center to the protein surface. This distance map was then used to calculate local ionic conductivity in proximity to the surface of protein. IB was then computed using the Ohm law by combing all the grids in series and/or parallel.


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3 Results and discussion 3.1 Characterization of fabricated nanopore

Figure 1. Principle of nanopore technology. (a) Device of nanopore technology, the solid-state nanopore structure is enlarged in the circle; (b) Schematic of BSA passing through the nanopore; (c) Characterization of fabricated nanopore with diameter of 42nm. Current-voltage (I-V) curve with an inserted SEM image.

Device of the experiments and three-dimensional structure of solid-state nanopore is displayed in Figure 1a. Si3N4 membrane with nanopore separated two chambers: Cis side and Trans side, which were filled with 1 M potassium chloride (KCl) and 10 mM phosphate buffered saline (PBS). The cis chamber was electrically grounded and added with protein samples. A pair of Ag/AgCl electrodes were placed into both chambers and connected to a



patch-clamp amplifier (EPC 10 USB, HEKA Instruments), which is used to apply constant voltage and to acquire ionic current signals. All these signals were measured at a 200 kHz sample rate with a low-pass filter of 10 kHz. The entire device was placed in a dark Faraday cage to reduce electrical noise. Figure 1b presents the schematic of BSA translocation through the nanopore. Charged proteins in the solution were driven to pass through the nanopore while the voltage was applied onto the electrodes. A typical current-voltage (I-V) curve of the nanopore in the buffer solution of 1 M KCl (pH=8) is presented in Figure 1c. The open pore ionic current and the applied bias voltage have a linear relationship, indicating the nanopore is highly symmetric.

Inserted graph in Figure 1c displays the SEM image of the fabricated

nanopore by focused ion beam (FIB), the diameter of the nanopore is about 42 nm and the membrane thickness is about 10 nm.

3.2 Detection of non-spherical BSA

Figure 2. Non-spherical BSA translocation through the nanopore. (a) Examples of the original ionic


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current trace as a function of time, the applied bias is +400 mV (above), +500 mV (middle) and +600 mV (below), respectively. (b) The three dimensional conformation of BSA with atomic resolution from the Protein Data Bank. The dark green ellipsoid is used to mimic the approximate shape of the protein. (c) The histogram of the relative current blockade ( △ Ib/I0) for different applied bias voltage ranging from +200 mV to +700 mV with 100 mV increment. 1521, 1338, 1421, 1029, 913, 795 Events were observed for 200 mV, 300 mV, 400 mV, 500 mV, 600 mV and 700 mV, respectively.

Native BSA has an isoelectric point about 4.7 in water at 25 ℃ , thus the protein carries negative charge at pH 8.0. Before recording translocation events, BSA samples were injected into the negatively biased cis chamber, then the potential was applied across the nanopore with diameter of 42 nm. Figure 2b shows the three dimensional (3D) conformation of BSA with atomic resolution obtained from the widely used Protein Data Bank. Obviously the 3D shape of BSA is close to an oblate ellipsoid which has also been considered by Yusko et al. 35. In the absence of samples, the current signal was a straight line with less fluctuations caused by noises. Upon addition of BSA, ionic current trace started to change. Downward spikes occurred one by one due to the translocation of BSA through the nanopore, see Figure 2a. Here, for the non-spherical BSA, experiments were further conducted in a solution of 1 M KCl at pH 8 with applied voltage ranging from 300 mV to 700 mV with increment of 100 mV. The trace for BSA passing through the nanopore under the different potentials (400 mV, 500 mV and 600 mV) are shown in Figure 2a. The scatter plots of the translocation time versus the relative ionic current drop (∆Ib/I0) and the relative current drop histograms for BSA at six different applied voltages are showed in SI Figure S1 and Figure 2c, respectively.



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Gaussian distribution is used to fit the histogram, clearly to show the concentration of data. Different from the results obtained from the data for 200 mV, which has only one Gaussian peak for the histogram of the relative current blockade, there are two Gaussian peaks when the applied bias voltage is larger than 300 mV. The phenomenon observed here is very interesting and should not be related to the protein unfolding as the electric force in our experiments cannot unfold a stable stacked protein structure, which have been validated by Bekard et al.

62

that a 780 mV bias can unfold a protein which is larger than the largest bias

700 mV applied in our experiments. No correlation between dwell time of BSA and

applied

bias voltage further validate the conclusion (SI Figure S2), as it was reported previously that dwell time will change if the protein unfolds63. Thus there must be some other reasons that will induce two current blockade levels.


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-90°

Identification of spherical and non-spherical protein by solid-state

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