Kinetics of a Three‐Step Reaction Observed at the Single‐Molecule Level

Luchian, Tudor; Shin, Seong‐Ho; Bayley, Hagan

doi:10.1002/ange.200250666

Cited by 12 publications

(8 citation statements)

References 30 publications

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“…We have developed an approach through which the covalent chemistry of individual molecules can be monitored in an aqueous environment inside a "nanoreactor", that is, the transmembrane protein pore formed by a-hemolysin. [6][7][8][9] By monitoring an ionic current driven through the pore by a transmembrane potential, subtle changes in the structures of individual reactants tethered within the lumen can be detected. We now use this approach to follow both the formation of a chiral center at As III (through the making of an As À S bond) and the inversion taking place at that center.…”

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Formation of a Chiral Center and Pyrimidal Inversion at the Single‐Molecule Level

et al. 2007

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The recent development of single-molecule techniques has been largely targeted at solving biological problems; [1][2][3][4][5] for example, protein folding has been examined by means of force spectroscopy, and enzyme kinetics have been investigated by single-molecule fluorescence. These approaches provide insight into individual trajectories that might be obscured in ensemble measurements. In contrast, bondforming and bond-breaking reactions of small molecules in solution have rarely been observed at the single-molecule level. We have developed an approach through which the covalent chemistry of individual molecules can be monitored in an aqueous environment inside a "nanoreactor", that is, the transmembrane protein pore formed by a-hemolysin.[6-9] By monitoring an ionic current driven through the pore by a transmembrane potential, subtle changes in the structures of individual reactants tethered within the lumen can be detected. We now use this approach to follow both the formation of a chiral center at As III (through the making of an As À S bond) and the inversion taking place at that center. The nanoreactor also serves to isolate the relatively short-lived As III adduct and thereby prevent additional reaction steps that would complicate the chemistry. AsÀS bond making and breaking are important reactions in pharmacology, toxicology, [10][11][12][13] and experimental cell biology. [14,15] We earlier reported reversible covalent As À S bond formation between the side chain of a cysteine residue at position 117 in the a-hemolysin pore and 4-sulfophenylarsonous acid (Figure 1 a).[9] Herein, we examine the same reaction at a different position within the nanoreactor (residue 137) and observed two distinct conductance levels, which corresponded to the formation of two As À S adducts at the wall of the protein (see Figure 1 b). Re-examination of the earlier data from the reaction at position 117 also revealed two current levels, but they were very closely spaced. The unitary conductance of P 137-SH -a heteromeric pore with just one of the seven subunits containing a cysteine residue at position 137-in 2 m KCl at À50 mV is (1.71 AE 0.04) nS (this is Figure 1. Two adducts are formed when 4-sulfophenylarsonous acid reacts with the side chain of Cys-137 in the P 137-SH pore. a) Structure of 4-sulfophenylarsonous acid and schematic of the P 137-SH pore. In aqueous solution, the dehydrated form, that is, 4-sulfophenylarsane oxide, may exist in equilibrium with 4-sulfophenylarsonous acid. The P 137-SH pore consists of six cysteine-free wild-type subunits (green) and one mutated subunit (blue) in which Gly 137 is substituted by Cys. The sulfur atom of the Cys residue is shown in yellow, the carboxy oxygen of residue 137 in red. b) Single-channel recording at À50 mV and 23 8C with a buffer containing 2 m KCl, 80 mm 3-(4-morpholinyl)-1-propanesulfonic acid (MOPS), 100 mm ethylenediaminetetraacetate (EDTA) (pH 8.4) in both chambers. 4-Sulfophenylarsonous acid (100 mm) was in the trans chamber. The current levels corresponding ...

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Formation of a Chiral Center and Pyrimidal Inversion at the Single‐Molecule Level

et al. 2007

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“…In this work, we explore the use of ion channels derived from gA to detect chemicals that display particular functional properties (such as the ability to facilitate specific chemical transformations). Bayley and co‐workers have previously demonstrated that wild‐type and genetically modified α‐hemolysin (a 232.4 kDa protein7) can be used to monitor the conversion of a light‐driven reaction by recording distinct conductances of intermediates throughout the course of the reaction 3c. Here, we demonstrate that it is possible to monitor the synthetic conversion of reactive chemical groups on molecules attached to gA in the presence of external chemical reagents in solution to afford identifiable and isolatable products with measurably different single‐channel conductance.…”

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Monitoring Chemical Reactions by Using Ion‐Channel‐Forming Peptides

Blake

Mayer

et al. 2006

ChemBioChem

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This paper presents a method for monitoring chemical reactions on individual molecules. We exploit the functional properties of an ion-channel-forming peptide to follow the conversion of chemical groups on molecules attached near the opening of these semisynthetic nanopores. It is known that the conductance properties of derivatives of gramicidin A (gA) can be measurably different.[1] Here, we take advantage of singlechannel conductance measurements to monitor in situ the multistep conversion of a tert-butyloxycarbonyl-protected (Boc-protected) amine to the free amine and the subsequent diazotization/hydrodediazoniation of the amine to an alcohol functionality on molecules attached to gA. These model reactions show that gA provides a simple nanoscale platform for sensing external chemical reagents with high sensitivity and selectivity.Gramicidin A is a natural ion-channel-forming peptide (molecular weight 1.9 kDa, secreted from the bacterium Bacillus brevis) that incorporates into lipid bilayers and facilitates the transmembrane flux of monovalent cations upon reversible dimerization in bilayers (Figure 1 A).[2] Although several artificial ion channels based on genetically engineered proteins, [3] peptides, [4] or oligomers of organic molecules [5] have been explored for their potential use as chemo-and biosensors, [6] most of these sensors are designed to sense particular structural properties of chemical or biochemical species (such as the ability of a ligand to interact with a binding pocket). In this work, we explore the use of ion channels derived from gA to detect chemicals that display particular functional properties (such as the ability to facilitate specific chemical transformations). Bayley and co-workers have previously demonstrated that wild-type and genetically modified a-hemolysin (a 232.4 kDa protein [7] ) can be used to monitor the conversion of a light-driven reaction by recording distinct conductances of intermediates throughout the course of the reaction.[3c] Here, we demonstrate that it is possible to monitor the synthetic conversion of reactive chemical groups on molecules attached to gA in the presence of external chemical reagents in solution to afford identifiable and isolatable products with measurably different single-channel conductance. This research extends the pioneering work on a-hemolysin to a synthetically accessible platform-gA-that is readily available in high purity and in useful quantities.We synthesized and characterized derivatives of gA carrying N-Boc-protected glycine (1), glycine (2), and glycolic acid (3) moieties attached to the C terminus of commercially available gA (Figure 1 B). The Supporting Information summarizes the details of the syntheses. We measured the single-channel current of 1-3 by incorporating them into planar lipid bilayers. Figure 1 C shows representative current-versus-time traces of 1-3 under an applied potential of 100 mV in buffered solution (1 M KCl, 0.01 M HEPES buffer, pH 7.4). These traces show that the conductance of ions through deriva...

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“…Durch die geschickte Messung der Zeitabhängigkeit des Ionenstromflusses durch einzelne Proteinporen, z. B. α‐Hämolysin ( α HL), konnte die Gruppe um Bayley in Echtzeit dem Fluss irreversibler (und einfacher reversibler) Transformationen folgen, die an einem einzelnen Molekül stattfinden, das kovalent an die dafür entwickelten Proteinporenwände gebunden ist …”

Section: Sonntag 5 Maiunclassified