Use of a solid-state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein

Abstract

A nanopore device is capable of providing single-molecule level information of an analyte as they translocate through the sensing aperture-a nanometer-sized through-hole-under the influence of an applied electric field. In this study, a silicon nitride (Si_x N_y )-based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid-state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture.

Keywords: dielectric breakdown; human serum transferrin; nanopore; protein unfolding; transferrin receptor protein.

Figure 1.

Structure of human serum transferrin receptor (TfR) and experimental setup. (A) Protein Data Bank (PDB) structure of transferrin receptor protein. The serum form of transferrin receptor is a 659 amino acid receptor that acts as the cellular gatekeeper of cells. PDB ID: 1CX8 [12]. (B) and (C) show the interaction interface of a monomeric TfR molecule, where it binds with another TfR molecule to form a dimer. (B) Interface of the monomeric TfR as colored by its accessible surface area (area accessible by water). Shades of blue represent inaccessible regions while shades of orange are accessible. The binding site for TfR is mostly blue. (C) Interface of the monomeric TfR as colored by hydrophobicity. Hydrophobic residues are colored in shades of green and hydrophilic residues are colored in shades of red. The interface is mainly hydrophobic. (D) Experimental setup for nanopore experiments. An external voltage is applied through the headstage (blue square in the figure) while the current is measured, and proteins are electrophoretically or electroosmotically driven across a nanopore from the cis side to the trans side. Upon transiting the nanopore, the protein causes a perturbation in the open pore current, which is amplified by the signal amplifier and is digitized and recorded by a digitizer. The real-time ionic current trace is then displayed, recorded, and fit with an event analysis software. Real-time ionic current traces from experiments can be found in the supplementary information (SI-7).

Figure 2.

Both holo-hSTf and TfR assume similar current blockage trends as voltage is increased, consistent with voltage-induced unfolding of proteins. (A) Peak current blockage with increasing voltage. TfR and hSTf are distinguishable from each other at all voltages other than 200mV. (B) Exemplary histogram of TfR at 100mV fitted with a Gaussian Mixture Model consisting of three components, which are attributed to its unfolded monomeric form, pseudo-folded monomeric form, and dimeric form, respectively. (C) Current blockage histograms of TfR and hSTf with increasing voltage, showing that at voltages ≤ 200mV, an additional peak exists in the TfR distribution that is not present in hSTf.

Figure 3.

The amount of unfolded TfR and hSTf proteins in each sample increases exponentially as voltage is increased. (A) Percentage of unfolded hSTf and TfR as voltage is increased. Error bars represent 95% confidence intervals on the estimations. (B) Normalized distribution of TfR (black) and hSTf (pink) at 100mV. Three peaks are evident for TfR and are labelled “1”, “2”, and “3”. Peaks 1, 2, and 3 correspond to (1) unfolded monomeric TfR, (2) pseudofolded monomeric TfR, and (3) dimeric TfR, respectively. No such third peak is seen in hSTf. (C) Histograms of TfR from 100–500mV. The distributions were fit with either a mixed Gaussian or an exponentially-modified Gaussian. The yellow-shaded region represents the FWHM cut-off value for classifying a protein as pseudo-folded or unfolded. The purple shaded region represents translocations that were considered to represent dimeric TfR and thus were not considered in this section. Similar histograms for hSTf can be found in SI-6 of the Supplementary Information.

Figure 4.

(A) Both hSTf and TfR increase in the fraction of events with a translocation time less than 50 μs as voltage is increased. Error bars represent 95% confidence intervals, and the dashed black line is an exponential fit to TfR’s translocation data. To minimize obscuring the graph, the fit for hSTf is not shown. (B) Histograms of the translocation times of TfR and hSTf at pH 8. TfR is indistinguishable from hSTf based on their peak translocation times alone. (C) Both holo-hSTf and TfR assume similar and rapid peak translocation times at a pH of 8. Error bars represent 95% confidence intervals for peak translocation time.

Figure 5.

Excluded Volume analysis of TfR (A) Monomeric and dimeric excluded volumes of TfR as a function of voltage. Both dimeric and monomeric TfR have similar excluded volumes to their geometrical volumes (SI-3) at lower voltages. As voltage increases, monomeric TfR experiences a decrease in excluded volume, and dimeric TfR disappears entirely. (B) Percentage of overall TfR population that is in its dimeric state. Boundaries for classifying the dimers were determined from the excluded volume distribution of TfR at 50mV, where the protein is expected to be mostly in its native state. The black dotted line is an exponential fit to the data (RMSE = 1.258). Increasing voltage causes an exponential drop in the percentage of dimeric proteins.