Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores - PubMed (original) (raw)

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

L Q Gu et al. J Gen Physiol. 2001 Nov.

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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Figures

Scheme S1

Scheme S1

Figure 1

Figure 1

Representations of staphylococcal α-hemolysin (αHL) and β-cyclodextrin (βCD). (A) Sagittal section through the WT-αHL pore showing the location of Met-113 (dark green), Glu-111 (red), Lys-147 (blue), Asn-139 (yellow), and Leu-135 (green). (B) Structure of βCD. (C) Schematic of the WT-αHL pore showing βCD lodged in the lumen of the channel. The location is based on mutagenesis data (Gu et al. 1999, Gu et al. 2001), including that described here. (D) Sequence of the transmembrane β barrel of αHL-RL2. WT residues are shown in parentheses.

Figure 2

Figure 2

Representative current traces from single αHL pores showing the blockade of Met-113 mutants by βCD. All traces were recorded under symmetrical conditions in buffer containing 1 M NaCl, 10 mM sodium phosphate, pH 7.5; 40 μM βCD was added to the trans chamber. (left) Traces recorded at −40 mV; (right) traces recorded at +40 mV. The mutants shown are all derived from RL2 (see the first paragraph of

results

). The broken line indicates zero current. The mutants are ordered (top to bottom) according to increasing affinity for βCD.

Figure 4

Figure 4

Plots of kinetic constants for the Met-113 mutants versus the van der Waals volume (Creighton 1993) of the residue at position 113. (A) 1/K d; (B) kon; and (C) koff. (closed gray circle) −40 mV; (○) +40 mV. Some of the points are obscured, but the values can be found in Table .

Figure 3

Figure 3

Dwell times for the interaction between βCD and the Met-113 mutants. (A) Values of τoff, the dwell time of βCD in the pore, and τon, the inter-event interval at 40 μM trans βCD. (closed gray circle) −40 mV; (○) +40 mV. The mutants are ordered (left to right) according to increasing affinity for βCD. (B) Dependence of τoff and τon on the concentration of βCD (trans) for WT-αHL and selected mutants.

Figure 3

Figure 3

Dwell times for the interaction between βCD and the Met-113 mutants. (A) Values of τoff, the dwell time of βCD in the pore, and τon, the inter-event interval at 40 μM trans βCD. (closed gray circle) −40 mV; (○) +40 mV. The mutants are ordered (left to right) according to increasing affinity for βCD. (B) Dependence of τoff and τon on the concentration of βCD (trans) for WT-αHL and selected mutants.

Figure 5

Figure 5

Plots of single-channel conductance values for the Met-113 mutants with and without βCD bound versus the van der Waals volume of the residue at position 113. (A) Conductance values at −40 mV: (closed gray circle), αHL conductance; (○) αHL•βCD conductance. (B) Conductance values at +40 mV. (C) Ratio of conductance at +40 mV to conductance at −40 mV (g+40/g-40). (D) Conductance of the pore with βCD bound divided by the conductance of the pore itself. (closed gray square), −40 mV; (□) +40 mV.

Figure 6

Figure 6

Representative current traces from single αHL pores showing blockades by βCD. All traces were recorded under symmetrical conditions in buffer containing 1 M NaCl, 10 mM sodium phosphate, pH 7.5. βCD (40 μM) was present on the trans side of the membrane. (A) WT-αHL, (B) N139Q(WT), (C) L135N(RL2), (D) M113N(WT), (E) M113N/N139Q(WT), and (F) M113N/L135N(RL2). The broken line indicates zero current.

Figure 7

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/K d+40)/(1/K d−40)] = log(K d−40/K d+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.

Figure 7

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/K d+40)/(1/K d−40)] = log(K d−40/K d+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.

Figure 7

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/K d+40)/(1/K d−40)] = log(K d−40/K d+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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