Effect of charge, topology and orientation of the electric field on the interaction of peptides with the α‐hemolysin pore

Christensen, Carol M.; Baran, Christian; Krasniqi, Besnik; Stefureac, Radu I.; Nokhrin, Sergiy; Lee, Jeremy S.

doi:10.1002/psc.1393

Cited by 23 publications

(39 citation statements)

References 42 publications

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“…4A) reveals two peaks; a bumping peak at -30 pA and a larger peak at about -80 pA. As has been discussed elsewhere, for many proteins it is difficult to distinguish between intercalation and translocation because the blockade time of these events is little changed by changes in the voltage. 21 By comparison, the such as SOD, implicated in ALS, it might be more appropriate to use solid state pores. 14,15 These can be fabricated in silicon nitride membranes with diameters in the range of 5-20 nm, allowing the translocation of large proteins or aggregates.…”

Section: Protein Misfoldingmentioning

confidence: 99%

Nanopore analysis

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Christensen

et al. 2012

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Section: Protein Misfoldingmentioning

confidence: 99%

Nanopore analysis

Madampage

Tavassoly

Christensen

et al. 2012

Prion

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“…The effect of voltage on the translocation parameters of peptides and proteins has been studied previously by our group and others [2], [3], [5], [6], [13], [36], [37]. It's been suggested that the voltage effect on the interaction of a molecule with the pore can be used to provide indirect evidence of protein or peptide translocation through the pore.…”

Section: Resultsmentioning

confidence: 99%

“…However, in order to differentiate between translocation and intercalation events a detailed voltage study must be carried out. For an elecrophoretically driven translocation, duration time will be inversely proportional to the applied voltage [11], [35], [36], [37]. In contrast, for an intercalation event the opposite is expected [35], [36].…”

Section: Introductionmentioning

confidence: 99%

“…It has been reported that the interaction of molecules with the α-hemolysin pore causes three general types of events: bumping, translocation, and intercalation [35], [36]. Bumping events (i.e small blockade events) can be distinguished from the translocation and intercalation events (i.e large blockade events) on the basis of blockade amplitude.…”

Section: Introductionmentioning

confidence: 99%

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RNase A Does Not Translocate the Alpha-Hemolysin Pore

Krasniqi

Lee

2014

PLoS ONE

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The application of nanopore sensing utilizing the α-hemolysin pore to probe proteins at single-molecule resolution has expanded rapidly. In some studies protein translocation through the α-hemolysin has been reported. However, there is no direct evidence, as yet, that proteins can translocate the α-hemolysin pore. The biggest challenge to obtaining direct evidence is the lack of a highly sensitive assay to detect very low numbers of protein molecules. Furthermore, if an activity based assay is applied then the proteins translocating by unfolding should refold back to an active confirmation for the assay technique to work. To overcome these challenges we selected a model enzyme, ribonuclease A, that readily refolds to an active conformation even after unfolding it with denaturants. In addition we have developed a highly sensitive reverse transcription polymerase chain reaction based activity assay for ribonuclease A. Initially, ribonuclease A, a protein with a positive net charge and dimensions larger than the smallest diameter of the pore, was subjected to nanopore analysis under different experimental conditions. Surprisingly, although the protein was added to the cis chamber (grounded) and a positive potential was applied, the interaction of ribonuclease A with α-hemolysin pore induced small and large blockade events in the presence and the absence of a reducing and/or denaturing agent. Upon measuring the zeta potential, it was found that the protein undergoes a charge reversal under the experimental conditions used for nanopore sensing. From the investigation of the effect of voltage on the interaction of ribonuclease A with the α-hemolysin pore, it was impossible to conclude if the events observed were translocations. However, upon testing for ribonuclease A activity on the trans chamber it was found that ribonuclease A does not translocate the α-hemolysin pore.

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“…electrophoretically driven), the dwell time of a polymeric molecule in the nanopore decreases as the voltage increases. 48, 49 …”

Section: Resultsmentioning

confidence: 99%

Click Addition of a DNA Thread to the N-Termini of Peptides for Their Translocation through Solid-State Nanopores

et al. 2015

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Foremost among the challenges facing single molecule sequencing of proteins by nanopores is the lack of a universal method for driving proteins or peptides into nanopores. In contrast to nucleic acids, the backbones of which are uniformly negatively charged nucleotides, proteins carry positive, negative and neutral side chains that are randomly distributed. Recombinant proteins carrying a negatively charged oligonucleotide or polypeptide at the Ctermini can be translocated through a α-hemolysin (α-HL) nanopore, but the required genetic engineering limits the generality of these approaches. In this present study, we have developed a chemical approach for addition of a charged oligomer to peptides so that they can be translocated through nanopores. As an example, an oligonucleotide PolyT20 was tethered to peptides through first selectively functionalizing their N-termini with azide followed by a click reaction. The data show that the peptide-PolyT20 conjugates translocated through nanopores whereas the unmodified peptides did not. Surprisingly, the conjugates with their peptides tethered at the 5′-end of PolyT20 passed the nanopores more rapidly than the PolyT20 alone. The PolyT20 also yielded a wider distribution of blockade currents. The same broad distribution was found for a conjugate with its peptide tethered at the 3′-end of PolyT20, suggesting that the larger blockades (and longer translocation times) are associated with events in which the 5′-end of the PolyT20 enters the pore first.

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Effect of charge, topology and orientation of the electric field on the interaction of peptides with the α‐hemolysin pore

Cited by 23 publications

References 42 publications

Nanopore analysis

Nanopore analysis

RNase A Does Not Translocate the Alpha-Hemolysin Pore

Click Addition of a DNA Thread to the N-Termini of Peptides for Their Translocation through Solid-State Nanopores

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