Abstract

Modifying the phi29 connector

In general, membrane pores and ion channels contain a hydrophobic domain which anchors the protein to the membrane. Analysis of the surface charge of the connector revealed that its central surface region exhibits slight hydrophobicity compared with the two flanking layers at the wide and narrow ends, respectively (Fig. 1a, b)^12,13. To facilitate connector purification, a C-terminus his or strep tag was inserted just downstream of a six glycine linker for improved affinity tag flexibility. Six glycine linkers were included to provide end-flexibility (Supplementary Fig. 1). After purification to homogeneity, it was found that the modified GP10 self-assembled into the dodecameric structure with similar morphology to the 12-fold symmetric wild type connector (Fig. 1f), as observed by TEM (Fig. 1d and e). The existence of a native and authentic motor configuration was verified through its competency to package the double-stranded DNA after incorporation into the procapsid (Supplementary Fig. 2) and to assemble the resulting DNA-filled capsid into the infectious phi29 virion (Supplementary Fig. 3).

Reconstituting the connector into liposomes

A procedure for reconstituting the connector into liposomes was developed by co-incubation of the connector with the lipid in the presence of sucrose. Such incubation provided an opportunity for the hydrophobic layer of the connector to interact with the hydrophobic domain of the lipid molecules. The dehydration–rehydration method⁴² led to the production of giant liposomes up to 50 µm (Fig. 2 a-c). The insertion of the connector protein into the lipid membrane was confirmed by fluorescence microscopy, filtration assay, and sedimentation analysis (Fig. 2 a-g). The presence of the connector in the membrane was visible with fluorescence microscopy, showing a clear fluorescent ring around the liposome (Fig. 2b). The fluorescent ring was very similar to the liposome generated with fluorescent lipids NBD-PE (Fig. 2a). No fluorescent ring was observed when the fluorescently-tagged connector was mixed non-specifically with the non-connector inserted liposome (Fig. 2c). The free connectors were removed by filtration using a membrane with a pore size of 0.45 µm or by 5–20% sucrose gradient ultracentrifugation (Fig. 2 d-f and 2g).

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Fig. 2

Images of giant liposome containing the connector

a-c, Epifluorescence images of liposome: lipid labeled with NBD-PE without connector (a); a proteoliposomes reconstituted by FITC labeled connectors (b); FITC-connector mixed non-specifically with liposomes (c). d-f, Membrane filtration isolated most of the free connectors. g, Separation of liposome/FITC-connector complexes by sucrose gradient sedimentation. Free connectors appeared in the top fractions while proteoliposomes remained in the lower fractions. Fractions #1–12 are not shown. h, Fluidity of fluorescent (red) lipid bilayer demonstrated by FRAP (Fluorescence Recovery After Photobleaching) showing that the fluorescence intensity of photobleached area (black) was gradually increased over time due to lipid diffusion. i, Schematic showing the insertion of the connector into a planar lipid bilayer.

Incorporating the connector into planar lipid membranes

Since none of the above experiments could distinguish between loose attachment of the connector to the bilayer surface and tight incorporation of it into the bilayer membrane to form a channel, a single channel conductance assay was performed. Results showed that direct incubation of the connector with liposomes or a planar lipid bilayer did not lead to channel formation in the bilayer membrane (Fig. 3a). Connector insertion into the bilayer only occurred when the connector reconstituted proteoliposomes were fused into the bilayer (Fig. 3b and 3c). The channel insertion was observed through a discrete step-wise increase in conductance as shown in a continuous current trace (Fig. 3b), under either positive or negative transmembrane voltage (Fig. 3c).

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Fig. 3

Conductance assay confirm the insertion of the connector into bilayer lipid membranes (BLM)

a, BLM with connector only (top) or liposomes only (bottom). b, Addition of connector-containing proteoliposomes resulted in multiple insertions. Reproduced in over 200 experiments. Inserts: Insertion of one (top) and two connectors simultaneously (bottom). c, One connector insertion at positive voltage (top) and at negative voltage (bottom). d, Distribution of current jump after multiple connector insertions. e, Current-voltage relationship of single connector channels. Error bars represent 3–5 insertions under each applied voltage from a total of 38 inserted connectors in 4 individual experiments. f-g, Slopes of current traces with 1, 2 or 3 connectors(f) and 1 connector in the presence of dsDNA (cis- chamber) (g). d-f, 5mM Tris buffer, pH 7.9 with 0.5M NaCl. g, TMS with 1M NaCl.

Normally, the insertion of one connector into the bilayer results in an increase in the current of approximately 65 pA (equivalent to 1.6 nS) at a potential of −40 mV in a 5 mM Tris (pH 7.9)/0.5 M NaCl. Occasionally a 130.9 pA jump, attributed to insertion of two connectors, was also observed (Fig. 3d). Similar results were obtained when the channel conductance was measured in TMS/1 M NaCl (Supplementary Fig. 4). In this case, the occurrence of simultaneous insertion of 2 connectors and 3 connectors was 4.7% and 1.9%, respectively.

For conductance measurements, a plot of the I-V curve was obtained under different voltages (Fig. 3e). The average conductance per single pore was 1.57±0.16 nS/pore (a total of 38 inserted connectors) in a 5 mM Tris/0.5 M NaCl (Fig. 3e) and 3.21±0.51 nS/pore (a total of 213 inserted connectors) in a TMS/1 M NaCl buffer (Supplementary Fig. 4). As a comparison, the conductance measurements were also performed for connector channels under a ramp voltage (Fig. 3f and 3g). The slopes from the fitted curves representing the conductance of formed channels in the 5 mM Tris/0.5 M NaCl were 1.59 nS/single pore, 3.40 nS/two pores, and 4.98 nS/three pores, respectively. When NaCl concentration increased to 1 M (TMS buffer with 1 M NaCl), the slope of the curve was 3.12 nS/ single pore. A buffer of 5 mM HEPES /1 M KCl was also used for conductance measurements (Table 1).

The channel conductance of the connector was compared with that of α-HL using solutions of different ionic strength (Table 1). It has been reported that the diameter of the narrow end of the connector channel is 3.6 nm, while the channel formed by α-HL has a diameter of only 1.5 nm³⁰. Therefore, the ratio of the cross-sectional area of the channels between the connector and α-HL is 5.7. The ratio of measured conductance of the connector to α-HL is 5.1 (Table 1). Since the conductance of a channel is proportional to its cross-sectional area, it can be concluded that the cross-sectional area of the connector in the buffer solutions was approximately 5.1 fold greater than that of α-HL, which compares well with the ratio of cross-sectional areas from the crystal data of both proteins. Moreover, compared with other transmembrane proteins or ion channel proteins with larger channels, e.g., Streptolysin⁴³, Kir⁴⁴, VDAC⁴⁵ and bacterial porins³², the connector channel has additional advantages. For example, the connector channels were stable and did not exhibit voltage gating under the reported conditions. The channel conductance was uniform, demonstrating a linear response to applied voltages between −100 mV and 100 mV (Fig. 3f and 3g).

Translocation of double-stranded DNA

Both linear and circular plasmid Cx43 DNA (5.5 kb) were used to examine the translocation of dsDNA through the connector channel. In the case of the linear DNA plasmid, DNA translocation induced numerous current blockades which led to the current jump of single connector insertion transiently reduced by 25–45% (Fig.4c). Similar results were also found in translocation experiments of a 35-bp dsDNA (Supplementary Fig. 5). However, when the linear Cx43 was added to the cis-chamber, no such blockades were observed until the voltage was switched to positive potential (Fig. 3g). The short-lived blockades could be attributed to the occurrence of DNA translocations. In contrast, in the absence of DNA, the current trace was quiescent (Fig. 4a and Supplementary Fig. 5b). Occasionally, unspecific blockades were observed with a minimum detectable time. These unspecific blockades rarely occurred compared to DNA translocation events. They were usually characterized with detectable time very close to the limit of sampling frequency. (Fig. 4b and Fig. 3g). When circular plasmid dsDNA Cx43 was used, no translocation of the circular plasmid was observed (Fig. 4b upper left). Interestingly, when the same amount of circular plasmid digested by DNase I was added to the chamber, a burst of transient blockades occurred (Fig. 4b lower left). The same results were also observed when the linear Cx43 digested by DNase I was used (Fig. 4b lower right). All the above results confirmed that only the linear dsDNA passed through the connector channels.

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Fig. 4

Translocation of dsDNA through connector channels in BLM

a, A typical current trace when BLM contains a connector but no DNA (Control). b, Representative blockades caused by 45pM double-stranded circular and linear plasmid DNA without and with DNase digestion in BLM containing 3 connectors. c, d, Histogram showing the percentage of current blockade (c) and dwell time (d) caused by linear plasmid dsDNA under −40 mV and −75 mV. DNA translocation experiment was repeated 45 times.

Occasionally, we also witnessed blockade events in the range of 5–15% (Fig. 4c right) with a dwell time from several to hundreds of milliseconds. The events were attributed to non-specific blockades other than DNA translocation because they were found to occur in the absence of DNA (Supplementary Fig. 5b). The non-specific blockade events could be due to interactions of connector pores with lipid or lipid micelles because their occurrence increased after the addition of more liposomes to the chamber (data not shown). The occurrence of the non-specific blockades can be minimized when the diluted connector reconstituted liposomes are used and/or lower transmembrane voltage is applied (Fig. 4c left). Interestingly, we occasionally observed simultaneous blockade events (insert in Fig. 4b upper right and Supplementary Fig. 5a). These events were recorded under multiple pore conditions. A continuous current trace recording events before and after addition of DNA was also shown (Supplementary Fig. 5b). On one occasion, a burst of DNA blockades was observed after the insertion of a third connector. In comparison, when DNA was premixed with buffer before connector insertion, the DNA blockades were observed immediately after the first insertion occurred (Supplementary Fig. 5a). This result indicates a lack of a stirring facility in the DNA chamber leading to a delay in DNA translocation.

The blockade rates were affected by two factors: DNA concentration and transmembrane voltage. In the presence of 45 pM DNA under 3 connector insertions, the blockade rate was approximately 0.8–1 blockades/s (Fig. 4b upper right). Under the same number of insertions, when 4 µM of DNA was placed in the chamber, the blockade rate was approximately 5–5.8 blockades/s (Supplementary Fig. 5a and 5b). For the linear Cx43 DNA, the blockade rate increased as the ramping voltage was applied (Fig. 3g).

To calculate the dwell time (τ_p) for DNA translocation events, we grouped blockade episodes greater than 32% since this percentage of blockade seemed consistent with the ratio of cross sectional area between dsDNA and the pore. A histogram of these events can be seen in Fig. 4d. It should also be noted that 6 (under −75 mV) and 20 (under −40 mV) individual outlying events scattered between 120 ms to 9800 ms were not included in the graph for clarity. The dwell time distribution under −40 mV seemed broader than that under −75 mV. The average dwell time for DNA blockades under the −75 mV and under −40 mV was 9.2 ms and 22.1 ms respectively (Note: only the events less than 50 ms under −40 mV were used for the calculation). As a comparison, the distribution of dwell time for the 35-bp dsDNA are also included (Supplementary Fig. 6b and 6c). The average of the dwell times in this case was 0.53 ms. Therefore, it can be concluded that the dwell time of DNA translocation was affected by applied voltage and the size of DNA.

To verify the passage of dsDNA through the connector channels, quantitative PCR (Q-PCR) was used to quantify the translocation of 141-bp DNA under a constant voltage. DNA was added to the trans- side and samples were taken from the cis- side for quantification at 30 minute intervals. For comparison, control experiments were performed in the absence of connectors (Fig. 5c). Experiments with connector insertions in BLMs showed an increase in the number of DNA molecules in the cis-chamber over time (N=9 experiments). In contrast, in the absence of connectors the number of DNA molecules in cis-chamber remained undetectable over the 90-minute time course (N=4 experiments). Moreover, the DNA translocation rate was affected by the number of inserted connectors (Supplementary Fig. 7a).

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Fig. 5

Quantitative PCR analysis of DNA translocation events

a, Q-PCR amplification curves of the dilution series run in triplicate. b, A standard curve with the C_T plotted against the log of the starting quantity of template for each dilution. c, Quantitative analysis of the total number of DNA passing through one of the connectors in the lipid membrane from the trans- chamber to the cis- chamber (top). Negative controls (bottom) were carried out under the same condition but without connectors. The error bars represent standard deviations of the mean from 9 independent experiments and 4 negative control experiments.

To verify that the increase of the DNA copy number in the cis-chamber was due to DNA translocation through the channels rather than membrane leakage, three additional experiments were carried out under the conditions of known leakage of BLM or partitions. When leaking occurred, the copy number of DNA per ul of solution in the cis-chamber was approximately 10⁴ – 10⁵ fold higher than those experiments without leakage (Supplementary Fig. 7b).

Conclusion

Herein, an engineered form of the GP10 connector has been incorporated into a lipid bilayer, forming a highly conductive nanopore. By evaluating the changes of the conductance and performing Q-PCR analysis, the translocation of the linear 5.5-kb plasmid DNA, 141-bp DNA and 35-bp DNA through the channel was confirmed. This work provides a system or a tool for future electrophysiology studies of the phi29 DNA packaging motor. Also, the connector is a biological nanopore that is extremely reproducible and easily engineered, making it ideal and suitable for future biomedical applications.

METHODS

Supplementary Material

ACKNOWLEDGEMENTS

Footnotes

REFERENCES