Robust properties of membrane-embedded connector channel of bacterial virus phi29 DNA packaging motor

Abstract

Biological systems contain highly-ordered macromolecular structures with diverse functions, inspiring their utilization in nanotechnology. A motor allows linear dsDNA viruses to package their genome into a preformed procapsid. The central component of the motor is the portal connector that acts as a pathway for the translocation of dsDNA. The elegant design of the connector and its channel motivates its application as an artificial nanopore (Nature Nanotechnology, 4, 765-772). Herein, we demonstrate the robust characteristics of the connector of the bacteriophage phi29 DNA packaging motor by single pore electrophysiological assays. The conductance of each pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by dsDNA are consistent with the dimensions of the channel and dsDNA. Furthermore, the connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for connector quantification. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of DNA passing through the channel. The fingerprint of DNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of DNA transportation, motion, and packaging.

Figure 1

(A–C) Structure and dimensions of the phi29 connector. (D) Illustration of a lipid membrane embedded connector channels. (E) AFM electron micrograph of purified connector channels after array formation.

Figure 2. A current trace showing continuous insertions of single connectors into BLM

(A) at 5 mM Tris (pH 7.8) with 1 M NaCl under −40 mV (1 insertion); (B) at 5 mM Tris (pH 7.8) with 1 M NaCl under −75 mV (2 insertions); (C) at 5 mM Tris (pH 7.8) with 2 M NaCl under 75 mV (3 insertions); (D) at 5 mM HEPES (pH 7.8) with 1 M KCl under 45 mV (4 insertions); (E) at 5 mM Tris (pH 7.8) with 0.5 M NaCl under −40 mV (5 insertions); (F) Histogram of conductance measurements for single insertion under a specific voltage −75 mV with 1 M NaCl from 40 independent experiments.

Figure 3. Analysis of DNA translocation through phi29 connector channel embedded in the membrane

(A) A current trace depicting passage of 141 bp ds-DNA through multiple phi29 connector channels. Insert a. A magnified view of a typical translocation event induced by one dsDNA molecule. Insert b: A histogram of current blockade percentage induced by linear dsDNA with a total 3264 events. (Note: The data were obtained in the presence of 1M NaCl, pH 7.8 under −75 mV holding potential). (B) A comparison of copy number of translocated dsDNA measured by Q-PCR with the relevant blockade events counted from current trace recorded in two independent experiments (Note: Only the events with 29–34% current blockades were counted). The error bars represent the standard deviation of three independent Q-PCR measurements.

Figure 4. Current traces from BLM inserted with one, two and three phi29 connector channels

at 5 mM Tris (pH 7.8) with 2 M NaCl under a ramp voltage from −100 mV to 100 mV.

Figure 5. Current-Voltage traces of single connector channel

(A) Different concentrations of NaCl with 5 mM Tris, pH 7.8. (B) Different concentrations of KCl with 5 mM HEPES, pH 7.8.

Figure 6. Relationship of measured conductance with number of connectors

(A) Different concentrations of NaCl with 5 mM Tris, pH 7.8. ( B) Different concentrations of KCl with 5 mM HEPES, pH 7.8. All the conductance measurements were from more than three individual experiments. Error bars represent standard deviations for the measurements. The regression equation for 0.5 M NaCl is: ^G_m/nS = 1.73 × N + 0.01; for 1.0 M NaCl is: G_m/nS = 2.77 × N + 0.26; for 1.5 M NaCl is: G_m/nS = 3.81 × N + 0.26; and for 2.0 M NaCl is G_m/nS = 4.62 × N–0.00. The regression equation for 0.5 M KCl is: G_m/nS = 2.60 × N + 0.11; for 1.0 M KCl: G_m/nS = 4.73 × N–0.10; for 1.5 M KCl is: G_m/nS = 6.35 × N–0.16; and for 2.0 M KCl is: G_m/nS = 7.86 × N–0.01, respectively.

Figure 7. Relationship of buffer conductivity vs. conductance of single connector

(A) at 5 mM Tris with 0.5–2.0 M NaCl and (B) at 5 mM HEPES with 0.5–2.0 M KCl.

Figure 8. Current traces showing connector insertion accompany by DNA translocation (20 bp dsDNA) through the channel under different pH conditions in presence of 1 M NaCl at a constant voltage of −75 mV

(A) pH 2; (B) pH 7; and (C) pH 12..