Single-molecule protein unfolding in solid state nanopores

Abstract

We use single silicon nitride nanopores to study folded, partially folded, and unfolded single proteins by measuring their excluded volumes. The DNA-calibrated translocation signals of beta-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside the pore when translocation stalls due to the primary charge sequence. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation and that the electrical forces present under physiologically relevant potentials can unfold proteins. Our results show that the nanopore translocation signals are sensitive enough to distinguish the folding state of a protein and distinguish between proteins based on the excluded volume of a local segment of the polypeptide chain that transiently stalls in the nanopore due to the primary sequence of charges.

Figure 1

A) Schematic diagram of a nanopore translocation experiment. The three-dimensional structure of βLGa dimer derived from PDB file 2AKQ is shown approximately to scale with typical SiN_x nanopores. Positive and negative residues at pH 7 are colored blue and red respectively. B) Several recorded βLGa current blockage events (event driven mode). C) The primary sequence of βLGa.

Figure 2

Event distributions. βLGa in: (A) 2 M KCl with no urea at pH 7 120 mV, (B) 2 M KCl 5 M urea at pH 7 120 mV, (C) 2 M KCl 8 M urea at pH 7 120 mV, (D) 2 M KCl with no urea at pH 7 60 mV inset: Sample events, (E) 2 M KCl with no urea at pH 4.6 –120 mV inset: Sample events. (F) 2.7 kbp dsDNA in 1 M KCl 120 mV. The solid curves on the right axes are fits the marginal distributions (circles)of ΔI_b with individual Gaussian contributions shown. The solid curves on the bottom axes are fits to the marginal distributions (circles) of t_d. using the biased diffusion model. Circled numbers annotate clusters in the joint and marginal distributions as discussed in the text and Table 1. The scatter plots of the event joint distributions are colored by statistical contribution to the different clusters fit in the marginal current blockage distributions. All plots share the same excluded volume axis in the center. The diameter of nanopore used for the series measurements was D_p=8±2 nm as imaged by TEM. (See SI) The open pore current I₀ was 15.5, 12, 7.8, 7.9, 6.5, and 7.5 nA from panels A to F respectively.

Figure 3

HPr (right panels) and βLGa (left panels) measured in the same nanopore. The nanopore had an average diameter of D_p=4±1 nm and the open pore current was I₀=3.2 nA in 8 M urea and 2 M KCl.

Figure 4

A) The net charge at pH 7.0 of βGLa and HPr as predicted by treating the ionization of the individual amino acids as independent as a function of the number of residues translocated. B) The calculated Λ assuming a contour length equal to H_eff=20nm for βGLa and HPr as a function of number of amino acids translocated. The octagons mark the location of stall points in the translocation. C) The electrostatic contribution to the potential energy of βGLa as a function of the number of amino acids translocated through the nanopore for various values of H_eff as labeled in the figure. This potential is for the translocation of the C terminus first. D) A schematic of the linear translocation geometry for βGLa with positive and negative residues colored blue and red, respectively.

Figure 5

(A, B) Possible looped translocation geometries for the unfolded protein with no disulfide bonds intact. These loops are pre-formed in the native structure as shown in Fig. 1. (A) The E51 loop insertion has two stall locations with different excluded volumes. (B) Two stall points for the L133 loop occur at similar excluded volumes. (C, D, E, G) Possible translocation geometries for the partially unfolded protein with native disulfide bonds intact. (C) Two stall points for the C106-C119 loop occur at different excluded volumes. (D) The C66-C161 insertion has only a shallow stall point near the exit and is expected to translocate more rapidly. (E) If the negatively charged loop at E51 inserts first the result is a broad stall with transloation volume close to that of the full protein. (F) Diagram of βLGa showing the location and relationship of secondary structural elements. The small circles show the charge of residues as red for negative and blue for positive. The large red circles highlight clusters of negatively charged (at pH 7.0) amino acids present on turns at the surface of the protein that may be “hooks” for unfolding translocation with a positively biased trans chamber. Native disulfide bonds between C106-C119 and C66-C161 are shown in yellow. (G) Disulfide linked dimers will have more complicated translocations patterns with peak volumes ranging from 4–6 strands. These translocation diagrams were calculated for a H_eff=20nm pore. (H) The primary sequence of βLGa.