Sensing Native Protein Solution Structures Using a Solid-state Nanopore: Unraveling the States of VEGF

Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely corresponding to monomer, dimer, and higher oligomeric states. Upon changing from a non-reducing to a reducing condition, ion-current blockage events from the monomeric state dominate, consistent with the expected reduction of the two inter-chain VEGF disulfide bonds. Cleavage by plasmin and application of either a positive or a negative NP bias results in nanopore signals corresponding either to the VEGF receptor recognition domain or to the heparin binding domain, accordingly. Interestingly, multi-level analysis of VEGF events reveals how individual domains affect their translocation pattern. Our study shows that careful characterization of ssNP results elucidates real-time structural information about the protein, thereby complementing classical techniques for structural analysis of proteins in solution with the added advantage of quantitative single-molecule resolution of native proteins.


Evidences of VEGF dimer and monomer in bulk solution:
Full-length gel electrophoresis image of VEGF before and after reduction with TCEP Figure SI1 shows a denaturing gel (SDS-PAGE) of the recombinant VEGF used in this experiment. The stock solution of 20 µM VEGF was diluted to final concentration of 0.5 ug in 10 ul phosphate buffer saline solution at pH 7.5. In one sample, pH-neutralized TCEP was added into the solution to obtain the final concentration of 10 mM and allowed the disulfide reaction to occur for 15 min. Both solutions were then mixed with 5x sample buffer containing 10% SDS, 20% glycerol, and small trace of bromophenolblue and boiled for 5 min.
The gel electrophoresis was performed using 4-20% TGX precast gel (Biorad, USA) with Tris/Glycine/SDS running buffer in electrophoresis gel apparatus (Biorad, USA). The voltage was set at 200 V and run for 30 min. The gel was then stained using a silver-stain (Pierce, ThermoFisher Scientific, USA).

Evidences of VEGF dimer and monomer in bulk solution: Mass spectroscopy result of VEGF
Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was performed to detect the presence of monomer and dimer of VEGF at pH 6.8 and 8.0, as shown in figure   SI1. Roughly, 1 ug of VEGF was diluted into 1 ul of 50 mM phosphate buffer at pH 6.8 and 8.0 and was equilibrated for 15 min (final concentration, 50 uM). The VEGF solution was then deposited onto a HCCA/1% TFA solution on a target plate and quickly dried using heating gun. The sample was then analyzed the mass spectra using MALDI-TOF MS technique (Bruker autoflex Speed, at 10-50 kDa window). The mass spectra consistently showed a mixed population of monomer, at 19-kDa peak and dimer at 38-kDa peak. We also observed that the ratio between two peaks were not the same in two different pH. Although we realized that the ionization process might artificially introduce more monomer, this result provided a supported evidence of a presence of monomer and dimer in a solution, and the ratio was varied as a function of pH's solution.

Determination of a nanopore's geometry
We evaluate the pore's geometry (thickness and diameter) by first using the measured parameters obtained from the fabrication process and later on confirmed with the conductance measurement. Initially, the film thickness was determined by the elipsometer. With a known etching rate, the locally thinned area thickness was calculated. After the nanopore was formed by mean of the transmission electron microscope (TEM), the electron micrograph of the pore was taken and used to measure the diameter of the pore. We anticipate that after the pore went through intensive cleaning with piranha solution, the final geometry might changes, thus the conductance measurement was performed before the protein translocation experiment. The dsDNA was translocated through a nanopore and the open pore current as well as the blocked current were measured. The two set of equations, which derived from a geometrical model of pore conductance, were used to fit the two unknowns (thickness and diameter) 1 . is the effective diameter of the pore when the pore diameter d < is partly occupied by the molecule diameter 2 7 . $ is the buffer solution conductivitiy (10.5 nS nm -1 for 1M KCl, 25°C). Using this model, the pore we used in this study was found to have a thickness, : = 12 nm and 2 5 =6.0 nm.

Translocation of VEGF in comparison with DNA
As a control, and to ensure that the nanopore itself did not bias the translocation pattern of the VEGF, DNA translocation experiment was performed to verify the pore's geometry and its performance. After

Estimation of the linearized peptide's event amplitude
If VEGF subjected to be unfolded and linearized, it would have a length of 85 nm (estimated by bond length of 165 amino acids) and approximate cross section 0.45 nm 3 . Since unfolded peptide translocated as a linear chain, the relationship between the event amplitudes and cross section of the molecule is followed 4 : This equation give an expected block current of a linearized peptide of 0.2 nS, which is smaller than the value obtained in the experiment by a factor of 10. Therefore, it is unlikely that VEGF is translocated through the nanopore as a fully linearized peptide under our experimental condition.

Estimation of the hydrodynamic diameter of the VEGF protein.
To determine the hydrodynamic diameter of the VEGF protein, 2 E , we first estimate the theoretical smallest size of the protein assuming that it is a closed-pack spherical particle according to Erickson

Voltage-dependent VEGF translocation
In order to verify that most of the ion current blockade events correspond to protein translocation and not collisions, we measured the dwell-time histogram of VEGF under different voltages using the same nanopore. Briefly, VEGF was introduced into a nanopore chamber containing 1 M KCl, 50 mM phosphate buffer at pH 7.2 and 5 mM EDTA such that the final concentration of the protein is 20 nM. The voltage bias was set at -300 mV on the trans (opposite) chamber to attract positively charged VEGF. After 10 minutes of data collection, the voltage bias was changed to -500 mV, data was collected for another 10 minutes and same procedure was repeated at -700 mV. Figure SI5 shows the result of this voltagedependent experiment.
As the absolute value of voltage increases from 300 to 700 mV, the mean residence time (obtained from fitting the dwell time histogram with the exponential functions) decreases from 391±55 µs to 56±4 µs.
Notably, if the observed events were due to protein collisions, increasing voltage should result in longer dwell time 4 . This experiment thus negates this possibility, supporting the hypothesis that the observed events likely result from the translocation of VEGF.

Example event traces of VEGF at pH 7.6 before and after TCEP
Here we showed additional translocation events from VEGF at pH 7.6 before introducing TCEP.

Example event traces of VEGF at pH 7.2 before and after TCEP
Here we showed additional translocation events from VEGF at pH 7.2 (figure SI4a). One can see that the event that exhibits multiple steps mostly in B to A transition pattern. Different level is clearly distinguished and larger than the noise floor. After introducing TCEP (figure SI4b), the majority of the events only show single level pattern.

S15
Determining the turnover rate of plasmin-digested VEGF reaction using nanopore