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. 2017 Dec 26;11(12):11931-11945.
doi: 10.1021/acsnano.7b06980. Epub 2017 Nov 15.

Porphyrin-Assisted Docking of a Thermophage Portal Protein Into Lipid Bilayers: Nanopore Engineering and Characterization

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Porphyrin-Assisted Docking of a Thermophage Portal Protein Into Lipid Bilayers: Nanopore Engineering and Characterization

Benjamin Cressiot et al. ACS Nano. .
Free PMC article

Abstract

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.

Keywords: electrical detection; electroosmosis; lipid bilayer; porphyrin; portal protein; protein nanopore; single molecule.

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Route to studying transport through the G20c portal protein. The bacteriophage DNA translocation motor is formed when the large terminase (magenta) is assembled onto the portal protein (dark blue) vertex of the viral capsid (example shown for T4 phage EMD 1572). The motor translocates viral genomic DNA (graphical representation not to scale, light blue) into the capsid. The thermostable portal assembly from bacteriophage G20c (PDB code 4zjn; dark blue) is shown schematically with inserted DNA (gray, not to scale). Protein engineering through a point mutation (mutated protein shown in light blue with introduced cysteine residues in orange) allows attachment of a maleimide–porphyrin lipid anchor (orange) to facilitate insertion into a lipid membrane and single-channel measurements. Internal point mutations (not shown) afford pore size/shape control.
Figure 2
Figure 2
Structure of G20c portal proteins and stability. (a) Side view of a cartoon depiction of the G20c portal protein (PDB 4zjn). (b) Slice through the middle of the G20c diagram showing the electrostatic potentials inside the tunnel from −1 (red) to +1 kT/e (blue) represented by the scale bar and, (e) on the outside of the pore. (d) Top view of a cartoon depiction of the G20c portal protein. (c) Transmission electron microscopy of negatively stained wt, (f) single mutant 49C, (j) and triple mutant CGG portal proteins. (g) 12% SDS-PAGE of purified recombinant 49C and CGG portal proteins. (h) wt (red), 49C (blue), and GG (double mutant, green) unfolding equilibrium transition assessed by measuring the change in tryptophan fluorescence emission ratio of 335/350 nm (excitation wavelength: 280 nm) as a function of GdnHCl concentration in 1 M NaCl, 20 mM Tris pH 7.5. (i) Melting temperatures of wt (red), 49C (blue), and CGG (green) portal proteins deduced by Thermofluor assay in 1 M NaCl, 20 mM Tris pH 7.5.
Figure 3
Figure 3
Modification of portal protein with maleimide–porphyrin conjugate for incorporation into lipid bilayers. Analytical size-exclusion chromatography of 49C (a) and CGG (b) mutant portal proteins before (1,2 dashed lines) and after (3,4 solid lines) conjugation with maleimide–porphyrin followed at 280 (1,3 black) and 410 nm (2,4 red). (c) UV–vis absorbance spectra of the main peak fractions for 49C (green) and CGG (blue) mutants after maleimide–porphyrin conjugation and analytical gel filtration chromatography. (d) Negative-stain transmission electron micrographs of the 49C (d) and CGG (e) mutant proteins after maleimide–pophyrin conjugation.
Figure 4
Figure 4
Electrical properties of the bacteriophage G20c portal inserted into a lipid bilayer. (a) Schematic of the ion–current measurement setup. One G20c portal protein is inserted into a suspended lipid bilayer via maleimide–porphyrin tags (red). An electrical potential is applied via two Ag/AgCl electrodes, which induces a current of Na+ and Cl ions through the nanopore (1 M NaCl, 20 mM Tris pH 7.5). (b) Typical current trace and the current histogram showing insertion of individual CGG portal channels into a lipid membrane. Data were collected at +100 mV. The average current value is 107.4 ± 4.5 pA for a single pore insertion, 222.8 ± 6.5 pA for two pores, and 325.7 ± 8.1 pA for three pores. (c) A typical current trace recorded through a single 49C portal at ±100 mV, showing pore expunction from the lipid membrane at ~57 s. The average current value is −98.0 ± 3.2 pA at −100 mV and 96.4 ± 3.7 pA at +100 mV. (d) Current–voltage (IV) curves of 49C (red) and CGG (blue) portals fitted to average data from eight independent recordings. The error bars represent a standard deviation from the mean curve.
Figure 5
Figure 5
Molecular dynamics simulation of G20c portal ionic conductance. (a) Simulation system consisting of the protein channel, shown as a cut-away molecular surface, embedded in a lipid bilayer membrane (cyan) via porphyrin moieties (orange). A white semitransparent surface shows the extent of the solvent (1 M NaCl); green and purple spheres represent the chloride and sodium ions, respectively. The system contains 792391 atoms. A bottom panel shows a zoomed-in view of the equilibrated lipid–protein interface, where water molecules are shown explicitly as red (oxygen) and white (hydrogen) spheres. (b) The root-mean-square deviation (RMSD) of the protein Cα atoms from their crystallographic coordinates during the equilibration simulations. The black and red lines correspond to simulations carried out in bulk electrolyte and lipid bilayer environments. The data were sampled every 4.8 ps. (c) A set of cross sections illustrating development of the lipid–protein interface during the equilibration simulation. Blue and green color maps specify local density of the protein’s α-carbon and lipids’ phosphorus atoms, respectively. Each cross-section represents a time average of 4.8 ps sampled coordinate frames. (d) Total charge transported through the channel by various ionic species versus simulation time. The slope of each line gives the average ionic current. The simulations were performed under a transmembrane bias of ±100 mV. Solid and dashed lines illustrate the simulated currents for 49C and CGG portal channels. The plots were obtained by integration of the ionic current versus simulation time; the ionic current data were sampled every 4.8 ps and averaged in 2.4 ns blocks prior to integration. (e) Simulated conductance of 49C and CGG channels. The conductance values were scaled by the ratio of the experimentally measured (7.43 S/m) and simulated (11.56 S/m) bulk conductivity of 1 M NaCl. Error bars represent standard errors. (f) Ionic selectivity of 49C and CGG variants of the channel defined by the ratio of chloride to sodium currents. (g) Steady-state local densities of lipids (all non-hydrogen atoms, green color scale), protein (all non-hydrogen atoms, blue color scale), and ionic current (streamlines, purple-red-yellow color scale). The arrows indicate the direction of the local ionic current flux, and the color indicates the flux’s magnitude. The maps were computed from a 30 ns long MD trajectory at a + 100 mV bias sampled with a frequency of 48 ps, radially averaged about the z-axis to improve the resolution.
Figure 6
Figure 6
Interaction of α-, β-, and γ-CDs with the CGG portal protein. (a) Current vs time trace recorded through a single CGG portal pore at +100 mV in the presence of 0.16 mM α-CD in both chambers. (b) Scatter plot of fraction blockade versus time for α-CD at +100 mV. (c, d) Same as in panel a and b, respectively, but in the presence of β-CD. The calculated standard deviation for the noise is 8.3 pA (see Methods). (e, f) Same as in panel a and b, respectively, but in the presence of γ-CD. Arrows represent the population of longer-lived events in each respective experiment. (g) Steered MD simulation of CD transport through the CGG portal. The protein channel (gray) is shown as a cutaway molecular surface, the α-CD is in orange, chloride and potassium ions are in green and purple, respectively, water molecules not shown for clarity. CD molecules were pulled along the axis of the channel using the constant velocity SMD protocol (see the Methods). (h) Blockade current through the portal channel for different placements of the CD variants. The currents were computed using a theoretical model based on the position dependence of the electrolyte conductivity (see the Methods for details).
Figure 7
Figure 7
Electroosmotically driven β-CD transport through the CGG portal. (a) Current vs time trace of a single CGG pore at +100 mV in the presence of 0.16 mM β-CD in cis compartment only, and scheme showing the direction of the electro-osmotic flux from trans to cis compartment. (b) Current vs time trace of a single CGG pore at −100 mV, in the presence of 0.16 mM β-CD in cis compartment only and scheme showing the direction of the electro-omotic flux from cis to trans compartment. Experiments were conducted in 1 M KCl, 20 mM Tris pH 7.5. The calculated standard deviation of the noise in these experiments is 8.3 pA (see the Methods).
Figure 8
Figure 8
Asymmetric interactions of β-CD with the CGG portal protein (a–f) Scatter plots of fraction blockade versus dwell time (bias values indicated in legends). (g) Mean fraction blockade of the long-lived events (>200 μs) as a function of the applied voltage. The error bars represent error over one recording. (h) Characteristic dwell times of the long-lived events as a function of the applied voltage in the presence of 0.16 mM β-CD in both chambers, cis side entrance for negative bias and trans side entrance for positive bias. The error bars represent error over one recording. (i) Force applied to β-CD during constant velocity SMD simulation of β-CD transport through the CGG portal in the cap-to-stem and stem-to-cap directions. The force plots were obtained by differentiating the work plots shown in the Figure S8. The z-coordinate is defined graphically in Figure 6g.

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