Journal
JOURNAL OF GENERAL PHYSIOLOGY
Volume 118, Issue 5, Pages 481-493Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.118.5.481
Keywords
alpha-toxin; molecular adapter; mutagenesis; pore; stochastic sensing
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Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the alpha -hemolysin (alpha HL) pore, thereby offering a basis for the detection of a variety of organic 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 manipulation of pH and transmembrane potential. here, we describe mutant homoheptameric 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 similar to 10(4)-fold snore avidly than the remaining alpha HL pores, including WT-alpha HL. The lower K-d values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for PCD 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 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.
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