Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Abstract

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼104-fold more avidly than the remaining αHL pores, including WT-αHL. The lower Kd values of these mutants are dominated by reduced values of koff. The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors.