Lq. Gu et al., Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores, J GEN PHYSL, 118(5), 2001, pp. 481-493
Noncovalent molecular adapters, such as cyclodextrins, act as binding sites
for channel blockers when lodged in the lumen of the alpha -hemolysin (alp
ha HL) pore, thereby offering a basis for the detection of a variety of org
anic molecules with alpha HL as a sensor element. beta -Cyclodextrin (beta
GD) resides in the wild-type alpha HL pore for several hundred microseconds
. The residence time can be extended to several milliseconds by the manipul
ation of pH and transmembrane potential. here, we describe mutant homohepta
meric alpha HL pores that are capable of accommodating beta CD 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 beta barrel, and fall into two classes. Members of the tight-
binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind beta CD si
milar to 10(4)-fold snore avidly than the remaining alpha HL pores, includi
ng WT-alpha HL. The lower K-d values of these mutants are dominated by redu
ced values of k(off). The major effect of the mutations is most likely a re
modeling of the binding site for PCD in the vicinity of position 113. In ad
dition, there is a smaller voltage-sensitive component of the binding, whic
h is also affected by the residue at 113 and may result from transport of t
he neutral PCD molecule by electroosmotic flow. The mutant pores for which
the dwell time of beta CD is prolonged can serve as improved components for
stochastic sensors.