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 pass 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 conduction 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.

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

“…To date, the main ring-shaped portal assembly studied using single-channel ion current measurements is the heavily mutated portal protein (also called “connector”) from bacteriophage phi29, , in which the loops that make up the pore constriction are either flexible and/or exhibit conformational variability . Recent work , has also explored the use of the portal assembly from bacteriophage SPP1 which can insert spontaneously into lipid bilayers to form a pore with a well-defined internal structure . However, this protein exhibits oligomeric state variations where 12-, 13-, or 14-subunit oligomers create a mixture of nanopores with different internal dimensions.…”

Biological Nanopore Approach for Single‐Molecule Protein Sequencing

“…During the last decade, numerous efforts with biological nanopores have been invested into peptide and protein sensing, including conformation/structure characterization, [51–56] variant recognition, [24, 26, 27, 57–63] interaction investigation, [64–69] and protein kinase exploration [70–74] . However, despite all the present achievements in proteomic analysis, there is still a long way toward SMPS.…”

Biological Nanopore Approach for Single‐Molecule Protein Sequencing

“…[49] Another work elucidated that translocations of positively charged peptides can be facilitated by engineering electrostatic binding sites of the nanopore sensing interface. [50] During the last decade,n umerous efforts with biological nanopores have been invested into peptide and protein sensing,i ncluding conformation/structure characterization, [51][52][53][54][55][56] variant recognition, [24,26,27,[57][58][59][60][61][62][63] interaction investigation, [64][65][66][67][68][69] and protein kinase exploration. [70][71][72][73][74] However, despite all the present achievements in proteomic analysis, there is still along way toward SMPS.This minireview focuses on recent progress in biological nanopore approach for SMPS, and attempts to provide acomprehensive overview of current experimental efforts and strategies for overcoming major hurdles of NPS.F inally,w ec lose it with our insights and perspectives on future developments in the field of NPS.…”