Sculpting conducting nanopore size and shape through de novo protein design

Abstract

Transmembrane β-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane β-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded β-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (~0.5 nm pore diameter) to 430 pS (~1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand β-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies.

Fig. 1:

Sculpting β-barrel geometry. A. Pore diameter can be controlled through the number of β-strands in the β-barrel blueprint. B. β-barrel 2D interaction map. Strong bends in the β-strands (< 90° bend, right) is achieved by stacking several glycine kink residues (yellow spheres) along the β-barrel axis, as opposed to placing one kink (>90° bend, left). C-D. Cross-sections of explicitly assembled β-barrel backbones without (cylinder, C) and with (D) glycine kinks. The Cβ atoms of the residues facing the pore are shown as sphere’s and colored based on their respective repulsion energy. Glycine kinks positions are shown with arrows; placement at the corners of the embedded rectangular, oval and triangular shapes (dashed lines in D) generates the desired backbone geometries. E. Polar threonine residues are tolerated on the membrane-exposed surface of TMBs (right) as they can form a hydrogen bond to the backbone, mimicking the interactions with water molecules observed in similarly curved areas of water-exposed β-strands (left).

Fig. 2:

Biophysical characterization of designed nanopores. Top row: 10-stranded design (TMB10_163); Bottom row: 12-stranded design with a square cross-section (TMB12_3). Both designs elute as one major species with retention time consistent with a monomeric protein in complex with DPC detergent (A), show distinct negative maxima in far UV CD spectra at 215 nm (B) that remain stable up to >70°C (D), and cooperative and reversible folding/unfolding transitions in DUPC LUVs (obtained at 25°C) (D),

Fig. 3:

Experimentally determined nanopore structures closely align with the computational design models. A. Seven surface mutations differentiate TMB10_163 from TMB10_165. B-D. Crystal structure of TMB10_163 B. backbone superposition C. superposition of side-chains involved in key folding motifs in the lumen D. cross-sections superposition with the water-accessible pore shown as a gray surface. Water-molecules crystallized in the pore are shown as red spheres. E-G. TMB2_13 structure in LDAO micelles. E. 2D [¹⁵N,¹H]-TROSY NMR spectrum of [U-²H,¹⁵N]-TMB12_3 with sequence-specific resonance assignments. F. Long-range NMR NOE contacts mapped to the expected TMB12_3 hydrogen bonds (dashed black lines). Residues with amide assignment are shown in white and green, unassigned residues are shown in ash gray. Residues with β-sheet secondary structure are shown as squares, all others as circles. Bold outlines indicate available methyl assignments. NOE contacts are shown as red lines (long-range amide-amide, dashes indicating diagonal overlap) and blue lines (contacts involving side chain methyl groups). G. Ensemble of the 20 lowest energy solution NMR structures (β-sheets shown in brown).

Fig. 4:

Conductance of designed nanopores. A. TMB10_165, B. TMB12_3, C. TMB12_oval_4, D. TMB12_rect_8, E. TMB12_tri_12, F. TMB14_8. i) Top view cartoon representation. ii) Vertical cross sections of the pore. iii) single channel conductance (smallest observed conductance jump). iv) sequential insertions of designed pore in planar lipid bilayer membrane from detergent solubilised sample at low concentrations. v) histogram of smallest measured current jumps for each design up to 50 pA. The applied voltage across the bilayer was 100mV and experiments were performed in a buffer containing 500 mM NaCl. A gaussian fit was carried out for the single channel current histograms for each design. For TMB10_165, 38 independent single channel jumps were identified from 3 recordings to plot the histogram shown. Similarly, 44 single channel insertions were identified for TMB12_3 (4 recordings), 29 insertions for TMB12_oval_4 (3 recordings), 30 insertions for TMB12_rect_8 (3 recordings), 45 insertions for TMB12_tri_12 (5 recordings) and 32 insertions for TMB14_8 (3 recordings) to plot the above depicted histograms.