Conductance and Ion Selectivity of a Mesoscopic Protein Nanopore Probed with Cysteine Scanning Mutagenesis

Abstract

Nanometer-scale proteinaceous pores are the basis of ion and macromolecular transport in cells and organelles. Recent studies suggest that ion channels and synthetic nanopores may prove useful in biotechnological applications. To better understand the structure-function relationship of nanopores, we are studying the ion-conducting properties of channels formed by wild-type and genetically engineered versions of Staphylococcus aureus alpha-hemolysin (alphaHL) reconstituted into planar lipid bilayer membranes. Specifically, we measured the ion selectivities and current-voltage relationships of channels formed with 24 different alphaHL point cysteine mutants before and after derivatizing the cysteines with positively and negatively charged sulfhydryl-specific reagents. Novel negative charges convert the selectivity of the channel from weakly anionic to strongly cationic, and new positive charges increase the anionic selectivity. However, the extent of these changes depends on the channel radius at the position of the novel charge (predominantly affects ion selectivity) or on the location of these charges along the longitudinal axis of the channel (mainly alters the conductance-voltage curve). The results suggest that the net charge of the pore wall is responsible for cation-anion selectivity of the alphaHL channel and that the charge at the pore entrances is the main factor that determines the shape of the conductance-voltage curves.

Theory for polymer analysis using nanopore-based single-molecule mass spectrometry

“…1C). Electroosmotic flow does not cause the latter effect because the slightly anion selective nature of the αHL causes a net solvent flow through the pore that is opposite that of the applied electric field (25,(28)(29)(30). This would lead to a decrease in the PEG capture rate with increasing electric field.…”

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

“…A change in the charges on these side chains would markedly alter the barriers to ion transport through the pore. 73 Therefore, all PA 63 channels were reconstituted into the membrane at pH c|t 7.2|7.2 as a reference point for channel performance.…”

Energy Conversion in Protocells with Natural Nanoconductors

“…Synthetic counterparts of ion channels can be synthesized by protein engineering, such as α-hemolysin and its mutants [63,71]. Artificial ion channels may also be fabricated by precisely tailoring the radii of SiO 2 nanopores through organic group functionalization [93,94].…”

“…The wildtype α-HL is normally weakly anion selective [69,70]; it can also be made cation selective by protein engineering with or without further chemical modification [63,71]; for instance, 2-sulfonatoethyl methanethiosulfonate-(MTSES-) treated G133C mutant has been reported with cation selectivity of 170 (g K /g Cl ) [63].…”

“…[63]. The total voltage output (E t ) is shown with the contribution from ion transport through the channels (E r ) and contributions from the electrodes (E c ).…”