Discrimination among Protein Variants Using an Unfoldase-Coupled Nanopore

Nivala, Jeff; Mulroney, Logan; Li, Gabriel; Schreiber, Jacob; Akeson, Mark

doi:10.1021/nn5049987

Cited by 112 publications

(124 citation statements)

References 67 publications

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“…Activity has begun in this area of nanopore proteomics [71,72], and there is a pressing need for improved means to translocate polypeptide chains through pores. A long-term goal is to characterize proteins from single cells.…”

Section: Applications Of Engineered Poresmentioning

confidence: 99%

Engineered transmembrane pores

Ayub

Bayley

2016

Current Opinion in Chemical Biology

139

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Today, hundreds of researchers are working on nanopores, making an impact in both basic science and biotechnology. Proteins remain the most versatile sources of nanopores, based on our ability to engineer them with sub-nanometer precision. Recent work aimed at the construction and discovery of novel pores has included unnatural amino acid mutagenesis and the application of selection techniques. The diversity of structures has now been increased through the development of helix-based pores as well as the better-known β barrels. New developments also include truncated pores, which pierce bilayers through lipid rearrangement, and hybrid pores, which do away with bilayers altogether. Pore dimers, which span two lipid bilayers, have been constructed and pores based on DNA nanostructures are gaining in importance. While nanopore DNA sequencing has received enthusiastic attention, protein pores have a wider range of potential applications, requiring specifications that will require engineering efforts to continue for years to come.

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Section: Applications Of Engineered Poresmentioning

confidence: 99%

Engineered transmembrane pores

Ayub

Bayley

2016

Current Opinion in Chemical Biology

139

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“…Although several studies utilizing peptide and protein translocation through nanopores, have been published over the last several years [47–49,54–58], direct method for validating peptide translocations are lacking. A recent publication tried to solve the problem by conjugating a single-stranded DNA to unfolded protein and then amplify the DNA using PCR after translocation[69].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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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.

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“…3). 12 Analysis of the current showed that its temporal sequence was repeatable and able to be associated with pre-unfolding and translocation events of the four domains of the protein.…”

Section: Protein Folding Studied With Unfoldase-coupled Nanoporesmentioning

confidence: 97%

Research highlights: nanopore protein detection and analysis

2015

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In this article we highlight recent work using nanopores to detect and study proteins. Nanopores are excellent single molecule sensors, capable of rapidly characterizing small molecules with relatively modest instrumentation requirements. Although the vast majority of recent effort and attention surrounding nanopores has centered on detection and sequencing of nucleic acids, proteins represent a more difficult and diverse analyte population, with a wide range of sizes, structures, charges, among other characteristics. Nanopores can be used to detect the presence of proteins of interest as well as to study their enzymatic activity, binding to ligands, and secondary structure. We highlight new work describing detection of specific protein species in solution by coupling them to a strand of carrier DNA that is used to electrophoretically transport the proteins through conical glass nanopores. Additionally, we spotlight another approach for nanopore detection of protein and other analytes through detection of their binding to aptamers-measurements which were quantitative to pM concentrations. Finally, we highlight studies in which protein secondary structure and folding energetics were studied through the use of an unfoldase enzyme coupled to a protein nanopore, a technique capable of detecting the effects of single amino acid mutations on the stability of the folded protein.

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Discrimination among Protein Variants Using an Unfoldase-Coupled Nanopore

Cited by 112 publications

References 67 publications

Engineered transmembrane pores

Engineered transmembrane pores

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

Research highlights: nanopore protein detection and analysis

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