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Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores.

Gu LQ, Cheley S, Bayley H - J. Gen. Physiol. (2001)

Bottom Line: 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 betaCD in the vicinity of position 113.The mutant pores for which the dwell time of betaCD is prolonged can serve as improved components for stochastic sensors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, TX 77843, USA.

ABSTRACT
Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the alpha-hemolysin (alphaHL) pore, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a sensor element. beta-Cyclodextrin (betaCD) resides in the wild-type alphaHL 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 alphaHL pores that are capable of accommodating betaCD 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 betaCD approximately 10(4)-fold more avidly than the remaining alphaHL pores, including WT-alphaHL. 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 betaCD 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 betaCD molecule by electroosmotic flow. The mutant pores for which the dwell time of betaCD is prolonged can serve as improved components for stochastic sensors.

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Relationships of the voltage dependence of kinetic constants for the interaction of αHL and βCD, and the charge selectivity of the pore. (A) Plot of β1/K versus α, where β1/K = log[(1/Kd+40)/(1/Kd−40)] = log(Kd−40/Kd+40) for the mutants in Table . β1/K > 0 reflects a stronger affinity at +40 mV than −40 mV, and β1/K < 0, the opposite. α = log(PK+/PCl−), a measure of the charge selectivity of each mutant. Where α > 0, a pore is cation selective, and where α < 0, a pore is anion selective. A line was fitted to the data by linear regression. The correlation coefficient is R2 = 0.80. (B) Plot of βkon versus α, where βkon = log(kon+40/kon-40). R2 = 0.79. (C) Plot of βkoff versus α, where βkoff = log(koff+40/koff-40). R2 = 0.77.
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Figure 7: Relationships of the voltage dependence of kinetic constants for the interaction of αHL and βCD, and the charge selectivity of the pore. (A) Plot of β1/K versus α, where β1/K = log[(1/Kd+40)/(1/Kd−40)] = log(Kd−40/Kd+40) for the mutants in Table . β1/K > 0 reflects a stronger affinity at +40 mV than −40 mV, and β1/K < 0, the opposite. α = log(PK+/PCl−), a measure of the charge selectivity of each mutant. Where α > 0, a pore is cation selective, and where α < 0, a pore is anion selective. A line was fitted to the data by linear regression. The correlation coefficient is R2 = 0.80. (B) Plot of βkon versus α, where βkon = log(kon+40/kon-40). R2 = 0.79. (C) Plot of βkoff versus α, where βkoff = log(koff+40/koff-40). R2 = 0.77.

Mentions: These data were quantified by using β1/K = log (Kd−40/Kd+40). β1/K > 0 reflects a stronger affinity for βCD at +40 mV than at −40 mV, and β1/K < 0, reflects the opposite. Similarly, α = log (PK+/PCl−) was used as a measure of the charge selectivity of each mutant. Where α > 0, a pore is cation selective, and where α < 0, a pore is anion selective (Table ). When α and β1/K were displayed on a scatter plot, they were seen to be correlated (Fig. 7 A). This means that βCD binds to a cation-selective pore more strongly at positive potentials than at negative potentials, whereas βCD binds to an anion-selective pore more strongly at negative potentials than at positive. Although the effect of mutagenesis on the affinity for βCD was largely reflected in koff, the smaller effect of voltage was manifested in both kon and koff (Fig. 7b and Fig. c).


Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores.

Gu LQ, Cheley S, Bayley H - J. Gen. Physiol. (2001)

Relationships of the voltage dependence of kinetic constants for the interaction of αHL and βCD, and the charge selectivity of the pore. (A) Plot of β1/K versus α, where β1/K = log[(1/Kd+40)/(1/Kd−40)] = log(Kd−40/Kd+40) for the mutants in Table . β1/K > 0 reflects a stronger affinity at +40 mV than −40 mV, and β1/K < 0, the opposite. α = log(PK+/PCl−), a measure of the charge selectivity of each mutant. Where α > 0, a pore is cation selective, and where α < 0, a pore is anion selective. A line was fitted to the data by linear regression. The correlation coefficient is R2 = 0.80. (B) Plot of βkon versus α, where βkon = log(kon+40/kon-40). R2 = 0.79. (C) Plot of βkoff versus α, where βkoff = log(koff+40/koff-40). R2 = 0.77.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233842&req=5

Figure 7: Relationships of the voltage dependence of kinetic constants for the interaction of αHL and βCD, and the charge selectivity of the pore. (A) Plot of β1/K versus α, where β1/K = log[(1/Kd+40)/(1/Kd−40)] = log(Kd−40/Kd+40) for the mutants in Table . β1/K > 0 reflects a stronger affinity at +40 mV than −40 mV, and β1/K < 0, the opposite. α = log(PK+/PCl−), a measure of the charge selectivity of each mutant. Where α > 0, a pore is cation selective, and where α < 0, a pore is anion selective. A line was fitted to the data by linear regression. The correlation coefficient is R2 = 0.80. (B) Plot of βkon versus α, where βkon = log(kon+40/kon-40). R2 = 0.79. (C) Plot of βkoff versus α, where βkoff = log(koff+40/koff-40). R2 = 0.77.
Mentions: These data were quantified by using β1/K = log (Kd−40/Kd+40). β1/K > 0 reflects a stronger affinity for βCD at +40 mV than at −40 mV, and β1/K < 0, reflects the opposite. Similarly, α = log (PK+/PCl−) was used as a measure of the charge selectivity of each mutant. Where α > 0, a pore is cation selective, and where α < 0, a pore is anion selective (Table ). When α and β1/K were displayed on a scatter plot, they were seen to be correlated (Fig. 7 A). This means that βCD binds to a cation-selective pore more strongly at positive potentials than at negative potentials, whereas βCD binds to an anion-selective pore more strongly at negative potentials than at positive. Although the effect of mutagenesis on the affinity for βCD was largely reflected in koff, the smaller effect of voltage was manifested in both kon and koff (Fig. 7b and Fig. c).

Bottom Line: 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 betaCD in the vicinity of position 113.The mutant pores for which the dwell time of betaCD is prolonged can serve as improved components for stochastic sensors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, TX 77843, USA.

ABSTRACT
Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the alpha-hemolysin (alphaHL) pore, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a sensor element. beta-Cyclodextrin (betaCD) resides in the wild-type alphaHL 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 alphaHL pores that are capable of accommodating betaCD 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 betaCD approximately 10(4)-fold more avidly than the remaining alphaHL pores, including WT-alphaHL. 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 betaCD 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 betaCD molecule by electroosmotic flow. The mutant pores for which the dwell time of betaCD is prolonged can serve as improved components for stochastic sensors.

Show MeSH
Related in: MedlinePlus