Channel of viral DNA packaging motor for real time kinetic analysis of peptide oxidation states

Abstract

Nanopore technology has become a powerful tool in single molecule sensing, and protein nanopores appear to be more advantageous than synthetic counterparts with regards to channel amenability, structure homogeneity, and production reproducibility. However, the diameter of most of the well-studied protein nanopores is too small to allow the passage of protein or peptides that are typically in multiple nanometers scale. The portal channel from bacteriophage SPP1 has a large channel size that allows the translocation of peptides with higher ordered structures. Utilizing single channel conductance assay and optical single molecule imaging, we observed translocation of peptides and quantitatively analyzed the dynamics of peptide oligomeric states in real-time at single molecule level. The oxidative and the reduced states of peptides were clearly differentiated based on their characteristic electronic signatures. A similar Gibbs free energy (ΔG⁰) was obtained when different concentrations of substrates were applied, suggesting that the use of SPP1 nanopore for real-time quantification of peptide oligomeric states is feasible. With the intrinsic nature of size and conjugation amenability, the SPP1 nanopore has the potential for development into a tool for the quantification of peptide and protein structures in real time.

Keywords: Bacteriophage assembly; Biomotor; Nanobiotechnology; Nanopore sensing; Peptide identification; Viral motor.

Figure 1. Structure of the SPP1 DNA packaging motor channel

(a) Side and (b) top views showing hydrophilic (red), hydrophobic (blue) and neutral (white) amino acids; and dimensions of the channel. PDB: 2JES. This crystal structure was solved by Antson group [9]. (c) Coomasie-blue stained SDS PAGE showing the purified SPP1 channel subunits gp6.

Fig. 2. Electrophysiological properties of membrane-embedded SPP1 connector

(a) Current trace showing the insertion of SPP1 connector into the planar membrane with a characteristic step size of ~200 pA at −50 mV. (b) Conductance distribution based on 104 insertion events. (c) Current-Voltage trace acquired from −50 → +50 mV. Buffer: 1 M KCl, 5 mM HEPES, pH 8.

Fig. 3. Peptide translocation through SPP1 connector

(a) Current trace showing a burst of current blockage events with characteristic current amplitude and dwell time indicating the translocation of TAT peptides. Representative magnified events are shown in the box. [TAT peptide] =0.5 μg/mL (b) Rate of peptide translocation as a function of peptide concentration (n = 3). (c) Histogram of current blockage percentage from 1939 events. (d) Dwell time of peptide translocation events fitted with a single exponential function from 1939 events. Applied voltage: 50 mV; Buffer: 1 M KCl, 5 mM HEPES, pH 8.

Fig. 4. Single molecule fluorescent images validating TAT peptide translocation

(a) The upper row is the image showing the detection of Cy3-labeled TAT peptide from the fractions collected from patch clamp at 0, 20, 40 and 60 mins. Excitation λ: 532 nm; laser power: 5 mW; 60× objective (N.A. = 1.4, oil immersion); Exposure time: 500 ms. (b) Quantitative analysis showing the increase in Cy3-TAT peptide signal in presence of SPP1 connector compared to control without connector. The errors represent mean ± standard deviation from three independent imaging from one experiment. Three independent experiments were performed and similar trend was observed. Applied voltage: 50 mV; Buffer: 1 M KCl, 5 mM HEPES, pH 8.

Figure 5. Determining the conformational states of TAT peptide

Current trace (left), current blockage distribution (middle) and conformation (right) for (a) Dimer state of TAT peptide; (b) Monomer state of TAT peptide; and (c) Cy3-conjugated TAT monomer. Applied voltage: 50 mV; Buffer: 1 M KCl, 5 mM HEPES, pH 8. Total number of events: 858 in A; 367 in B and 1128 in C.

Fig. 6. Real-time assessment of the conformational states of TAT peptide

(a) Continuous current trace showing transition of oxidized dimer states to reduce monomer states after addition of reducing agent TCEP. (b) Quantitative analysis showing the fraction (γ) of dimer and monomer states as a function of reaction time. (c) Current blockage vs. dwell time distribution over the course of reaction time. Applied voltage: 50 mV; Buffer: 1 M KCl, 5 mM HEPES, pH 8. (d) Quantitative analysis showing the reaction quotient Q_r as a function of reaction time.