Stochastic study of the effect of ionic strength on noncovalent interactions in protein pores

Abstract

Salt plays a critical role in the physiological activities of cells. We show that ionic strength significantly affects the kinetics of noncovalent interactions in protein channels, as observed in stochastic studies of the transfer of various analytes through pores of wild-type and mutant alpha-hemolysin proteins. As the ionic strength increased, the association rate constant of electrostatic interactions was accelerated, whereas those of both hydrophobic and aromatic interactions were retarded. Dramatic decreases in the dissociation rate constants, and thus increases in the overall reaction formation constants, were observed for all noncovalent interactions studied. The results suggest that with the increase of salt concentration, the streaming potentials for all the protein pores decrease, whereas the preferential selectivities of the pores for either cations or anions drop. Furthermore, results also show that the salt effect on the rate of association of analytes to a pore differs significantly depending on the nature of the noncovalent interactions occurring in the protein channel. In addition to providing new insights into the nature of analyte-protein pore interactions, the salt-dependence of noncovalent interactions in protein pores observed provides a useful means to greatly enhance the sensitivity of the nanopore, which may find useful application in stochastic sensing.

FIGURE 1

Single-channel recordings showing the effect of ionic strength on Y-P-F-F binding to a (M113F)₇ pore. The experiments were carried out at +50 mV (cis at ground) under a series of symmetrical buffer conditions with concentrations of NaCl ranging from 1 to 5 M, and 10 mM Tris · HCl (pH 7.5). (A) 1 M NaCl; (B) 2 M NaCl; (C) 3 M NaCl; (D) 4 M NaCl; and (E) 5 M NaCl.

FIGURE 2

Dependence of the open channel currents of protein pores and the kinetic constants of the noncovalent interactions on the salt concentration. (A) Open channel currents; (B) association rate constants k_on; (C) dissociation rate constants k_off; and (D) reaction formation constants K_f.

FIGURE 3

Effect of NaCl concentration on the (A) reversal potentials, (B) charge selectivities, and (C) streaming potentials of αHL pores, showing that with the increase of salt concentration, the streaming potentials for all the protein pores decrease, whereas the preferential selectivities of the pores for either cations or anions drop. The obtained values of streaming potentials for all the three protein pores were very similar at each salt concentration.

FIGURE 4

Single-channel recordings showing the application of salt effect for the differentiation of organophosphates HAE and APA in a (WT)₇ pore using βCD as an adaptor. (Left) Traces recorded at +40 mV. (Right) Traces recorded at −40 mV. (A) HAE in 1 M NaCl; (B) APA in 1 M NaCl; (C) HAE in 5 M NaCl; and (D) APA in 5 M NaCl. The experiments were carried out at ±40 mV (cis at ground) under symmetrical buffer conditions with the concentration of NaCl at 1 M (or 5 M), and 10 mM Tris · HCl (pH 7.5). αHL protein was added in the cis compartment of the chamber, whereas 40 μM βCD and 80 μM HAE (or APA) were added in the trans compartment of the chamber. The dashed lines 0, 1, and 2 indicate the current levels for open channel, βCD · αHL, and HAE · βCD · αHL (or APA · βCD · αHL), respectively.

FIGURE 5

Simultaneous detection of a mixture of two structure-related peptides in a (M113F)₇ pore. (Left) Current traces. (Right) Event histograms. The experiments were carried out at +40 mV (cis at ground) under symmetrical buffer conditions with 1 M (or 4 M) NaCl, and 10 mM Tris · HCl (pH 7.5). αHL protein was added in the cis compartment, whereas 1 μM Y-P-F-F or 1 μM Y-F-F was added in the trans compartment of the chamber.