The Role of Lys147 in the Interaction between MPSA-Gold Nanoparticles and the α-Hemolysin Nanopore

Abstract

Single channel recordings were used to determine the effect of direct electrostatic interactions between sulfonate-coated gold nanoparticles and the constriction of the Staphylococcus aureus α-hemolysin protein channel on the ionic current amplitude. We provide evidence that Lys147 of α-hemolysin can interact with the sulfonate groups at the nanoparticle surface, and these interactions can reversibly block 100% of the residual ionic current. Lys147 is normally involved in a salt bridge with Glu111. The capture of a nanoparticle leads to a partial current block at neutral pH values, but protonation of Glu111 at pH 2.8 results in a full current block when the nanoparticle is captured. At pH 2.8, we suggest that Lys147 is free to engage in electrostatic interactions with sulfonates at the nanoparticle surface. To verify our results, we engineered a mutation in the α-hemolysin protein, where Glu111 is substituted by Ala (E111A), thus removing Glu111-Lys147 interactions and facilitating Lys147-sulfonate electrostatic interactions. This mutation leads to a 100% current block at pH 2.8 and a 92% block at pH 8.0, showing that electrostatic interactions are formed between the nanopore and the nanoparticle surface. Besides demonstrating the effect of electrostatic interactions on cross channel ionic current, this work offers a novel approach to controlling open and closed states of the α-hemolysin nanopore as a function of external gears.

Ligand-Induced Structural Changes of Thiolate-Capped Gold Nanoclusters Observed with Resistive-Pulse Nanopore Sensing

“…We find that all three gold nanoclusters can be trapped in the sensing region of a single αHL channel for extended periods and this enables the observation of discrete changes in the current that we associate with ligand rearrangement on the particle. Similar multistep transitions have been reported previously for sulfonate-capped ligands (MPSA), but those fluctuations were assumed to result from Brownian diffusion or binding between the nanocluster ligands and residues in the constriction region of the αHL pore. , Here we show that the multistate fluctuations seen for our particles most likely result from intraparticle rearrangements. We report several experiments and develop a simplified geometric model to support this claim.…”

Biological nanopores elucidate the differences between isomers of mercaptobenzoic-capped gold clusters

“…This is mostly because ligand-pore interactions have been shown to be limited for wild-type αHL in near neutral pH conditions. 28,30 In any case, our results represent a significant development in the area of cluster characterization and provide a unique tool for observing ligand-induced cluster dynamics.…”

“…In particular, it is worth noting that the salt bridges formed between the glutamic acid and lysine residues in the constriction region of the pore limit the possibility of cluster-pore interactions. 28 Our work showed that the pore is capable of monitoring previously unknown discrete transitions between physical states whose properties were dependent on ligand mass and type. We suggested that these transitions result from reorganization of the ligands on the surface of the cluster through either lateral diffusion along the surface or through ligand folding.…”

Nanopore back titration analysis of dipicolinic acid

“…One challenge to develop nanopore sensors is to slow down the translocation of target analytes in the nanopore so that analyte events could be captured by the current recording technique (usually, ∼100 μs event residence time is required). In addition to taking advantage of the experimental conditions to regulate molecular and ionic transport , two major approaches have been utilized in the nanopore sensor design: one involves the structural modification of the nanopore interior to construct binding sites , while the other relies on a host or probe molecule to form host‐guest or probe‐analyte complexes . Although a wide variety of substances have been detected, nanopore detection of many small molecules, especially anions, by these two approaches is not successful.…”