Stochastic Sensing of Nanomolar Inositol 1,4,5-Trisphosphate with an Engineered Pore

Cheley, Stephen; Gu, Li Qun; Bayley, Hagan

doi:10.1016/s1074-5521(02)00172-2

Cited by 138 publications

(157 citation statements)

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“…Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (∼10 7 to 10 8 M -1 ·s -1 ), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.…”

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confidence: 75%

“…1E). Current-voltage relationships for the open states of (αHL-D127N) 7 and αHL-D127N-PLM show modest changes in rectification with respect to (αHL-WT) 7 , presumably because of alterations in charge distribution near the trans mouth arising from the modifications (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…The versatility and specificity of stochastic sensing have been enhanced by introducing sensing elements (analyte binding sites) into the αHL pore by protein engineering, chemical modification, or the use of adapter molecules (1,4,5). Through such approaches, the stochastic detection of ions (1,6), small molecules (1,7,8), reactive molecules (9,10), and proteinligand interactions has been achieved (1,(11)(12)(13)(14). The stochastic detection of proteins has also been achieved with functionalized solid-state nanopores (15).…”

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confidence: 99%

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Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate

Harrington

Cheley

Alexander

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (∼10 7 to 10 8 M -1 ·s -1 ), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.

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confidence: 75%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate

Harrington

Cheley

Alexander

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The mutants were made by using the WT or RL2 genes as templates. The RL2 gene encodes 4 amino acid replacements in the barrel domain (Val-124 3 Leu, Gly-130 3 Ser, Asn-139 3 Gln, Ile-142 3 Leu) and one at the cis entrance of the pore (Lys-8 3 Ala) (42). The properties of the mutants differed significantly from the WT pore with respect to the open-pore current and the frequency of occurrence of DNA translocation events ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

Maglia

Rincón-Restrepo²,

Mikhailova³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the ␣-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at ؉120 mV by Ϸ10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event⅐s ؊1 ⅐ M ؊1 . Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification.DNA sequencing ͉ electroosmosis ͉ nanopore ͉ protein engineering ͉ single-molecule detection P ores with diameters of a few to hundreds of nanometers, ''nanopores,'' are being developed for a wide variety of analytical applications (1-5). Nanopores can be fabricated by using particle beams and etchants to treat various substrates, including silicon nitride (3, 6) and plastics, for instance poly(ethylene terephthalate) (4). Alternatively, protein pores such as ␣-hemolysin (␣HL) can be used (1, 5). In both cases, to be detected, an analyte must travel into the pore by electrodiffusion, but few systematic attempts have been made to optimize this process. In the present work, we show how the manipulation of charge on the internal surface of the ␣HL pore can be used to improve the rate of capture of an important analyte, DNA.The ␣HL protein nanopore is advantageous in sensing applications because it can be engineered with subnanometer precision with reference to the high resolution crystal structure (7). Further, the ␣HL pore is far more stable than commonly held, operating as normal at temperatures approaching 100°C (8). In stochastic sensing, a binding site for a family of analytes is formed within the pore by site-directed mutagenesis or targeted chemical modification (1). The concentration of an analyte is estimated from the number of binding events per unit time to a single pore. An analyte is identified through the signature provided by the amplitude and mean duration of the individual binding events. In this way, a great variety of analytes have been examined including: cations, anions, organic molecules, and various polymers (1). For example, all 4 DNA bases can be detected as nucleoside monophosphates by an ␣HL pore equipped with an aminocyclodextrin adapter (9). In addition, individual covalent chemical reactions occurring within the lumen of the pore can be observed (5), offering a basis for the detection of reactive molecules (10).Polymers can also be analyzed from the characteristics of transit events through the ␣HL pore (11-15). Studies of nucleic acids have been especially intensive, following the demonstration of the transit of single str...

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“…The efficacy of non-canonical sites is demonstrated by the intriguing finding that providing a ring of arginines in the lumen of the α-hemolysin pore confers upon it the ability to bind IP3 with low nanomolar affinity, but not another negatively charged second messenger, cAMP (Cheley et al, 2002).…”

Section: Where Is the Molecular Selectivity Filter?mentioning

confidence: 99%

Connexin channel permeability to cytoplasmic molecules

Harris¹

2007

Progress in Biophysics and Molecular Biology

412

346

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Connexin channels are known to be permeable to a variety of cytoplasmic molecules. The first observation of second messenger junctional permeability, made ∼30 years ago, sparked broad interest in gap junction channels as mediators of intercellular molecular signaling. Since then, much has been learned about the diversity of connexin channels with regard to isoform diversity, tissue and developmental distribution, modes of channel regulation, assembly and expression, biochemical modification and permeability, all of which appear to be dynamically regulated. This information has expanded the potential roles of connexin channels in development, physiology and disease, and made their elucidation much more complex -30 years ago such an orchestra of junctional dynamics was unanticipated. Only recently, however, have investigators been able to directly address, in this more complex framework, the key issue: What specific biological molecules, second messengers and others, are able to permeate the various types of connexin channels, and how well? An important related issue, given the ever-growing list of connexin-related pathologies, is how these permeabilities are altered by disease-causing connexin mutations. Together, many studies show that a variety of cytoplasmic molecules can permeate the different types of connexin channels. A few studies reveal differences in permeation by different molecules through a particular type of connexin channel, and differences in permeation by a particular molecule through different types of connexin channels. This article describes and evaluates the various methods used to obtain these data, presents an annotated compilation of the results, and discusses the findings in the context of what can be inferred about mechanism of selectivity and potential relevance to signaling. The data strongly suggest that highly specific interactions take place between connexin pores and specific biological molecular permeants, and that those interactions determine which cytoplasmic molecules can permeate and how well. At this time, the nature of those interactions is unclear. One hopes that with more detailed permeability and structural information, the specific molecular mechanisms of the selectivity can be elucidated.

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Stochastic Sensing of Nanomolar Inositol 1,4,5-Trisphosphate with an Engineered Pore

Cited by 138 publications

References 44 publications

Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate

Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

Connexin channel permeability to cytoplasmic molecules

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