Interaction of the noncovalent molecular adapter, β-cyclodextrin, with the staphylococcal α-hemolysin pore

Cyclodextrins act as noncovalent molecular adapters when lodged in the lumen of the alpha-hemolysin (alpha HL) pore. The adapters act as binding sites for channel blockers, thereby offering a basis for the detection of a variety of organic molecules with alpha HL as a biosensor element. To further such studies, it is important to find conditions under which the dwell rime of cyclodextrins in the lumen of the pore is extended. Here, we use single-channel recording to explore the pH- and voltage-dependence of the interaction of beta-cyclodextrin (beta CD) with alpha HL.beta CD can access its binding site only from the trans entrance of pores inserted from the cis side of a bilayer. Analysis of the binding kinetics shows that there is a single binding site for beta CD, with an apparent equilibrium dissociation constant that varies by >100-fold under the conditions explored. The dissociation rate constant for the neutral beta CD molecule varies with pH and voltage, a result that is incompatible with two states of the alpha HL pore, one of high and the other of low affinity. Rather, the data suggest that the actual equilibrium dissociation constant for the alpha HL.beta CD complex varies continuously with the transmembrane potential.