Ion selectivity of alpha-hemolysin with beta-cyclodextrin adapter. II. Multi-ion effects studied with grand canonical Monte Carlo/Brownian dynamics simulations

Abstract

In a previous study of ion selectivity of alpha-hemolysin (alphaHL) in complex with beta-cyclodextrin (betaCD) adapter, we calculated the potential of mean force (PMF) and characterized the self-diffusion coefficients of isolated K(+) and Cl(-) ions using molecular dynamics simulations (Y. Luo et al., "Ion Selectivity of alpha-Hemolysin with beta-Cyclodextrin Adapter: I. Single Ion Potential of Mean Force and Diffusion Coefficient"). In the present effort, these results pertaining to single isolated ions in the wide aqueous pore are extended to take into account multi-ion effects. The grand canonical Monte Carlo/Brownian dynamics (GCMC/BD) algorithm is used to simulate ion currents through the wild-type alphaHL ion channel, as well as two engineered alphaHL mutants, with and without the cyclic oligosaccaride betaCD lodged in the lumen of the pore. The GCMC/BD current-voltage curves agree well with experimental results and show that betaCD increases the anion selectivity of alphaHL. Comparisons between multi-ion PMFs from GCMC/BD simulations and single-ion PMFs demonstrate that multi-ion effects and pore shape are crucial for explaining this behavior. It is concluded that the narrow betaCD adapter increases the anion selectivity of alphaHL because it reduces the pore radius locally, which decreases the ionic screening and the dielectric shielding of the strong electrostatic field induced by a nearby ring of positively charged alphaHL side chains.

Figure 1

GCMC/BD simulation system based on the crystal structure of (M113N)₇·βCD (Montoya and Gouaux, unpublished). Parts of the protein and the membrane are truncated to open a view into the channel lumen. The cut surface is grey. The protein surface is colored according to the electrostatic potential (blue is positive, red negative). The slab surrounding the lower part of the channel represents the low dielectric core of the membrane. The βCD molecule (magenta) is lodged at the narrowest part of the pore. Note, in (M113F)₇·βCD the orientation of the βCD ring is upside down. The simulation box (big black frame, 62Å×62Å×132Å in size) contains K⁺ (yellow) and Cl⁻ (green) ions, which are the only moving parts of the simulation system. Buffer regions (small black frames, 3Å thick) are located on both ends of the simulation box. Within these buffers a GCMC algorithm is creating and destroying ions to keep the chemical potential on the cis and the trans side of the membrane constant. The image was produced using the program UCSF Chimera. The electrostatic potential was computed with the adaptive Poisson-Boltzmann solver (APBS).

Figure 2

Single ion PMFs from BD simulations (solid lines) for (a) wt-αHL, (b) (M113F)₇, and (c) (M113F)₇·βCD extracted from BD simulations via Eq. (4). Also shown in dashed lines are the free energy profiles W(x = 0, y = 0,z) of a single K⁺ or Cl⁻ located along the channel axis calculated via Eq. (2). The BD PMFs are aligned with the corresponding dashed lines.

Figure 3

Single ion PMFs from BD simulations (solid lines) for (a) (M113N)₇, (b) (M113N)₇·βCD, and (c) (M113N)₇·βCD with all charges on the βCD atoms set to zero Free energy profiles of a single K⁺ or Cl⁻ located along the channel axis calculated via Eq. (2) are shown as dashed lines. See Figure 2 for further information.

Figure 4

I-V curves from GCMC/BD simulations with symmetric salt concentration (1.0 M KCl on cis and trans side). (a) wt-αHL (including experimental results measured at pH 5.0 and pH 7.5 using a symmetric salt concentration of 1 M NaCl10), (b) (M113F)₇, and (c) (M113F)₇·βCD (including experimental results for this channel without and with covalent linker between protein and βCD13).

Figure 5

I-V curves from GCMC/BD simulations with symmetric salt concentration (1.0 M KCl on cis and trans side). (a) (M113N)₇, (b) (M113N)₇·βCD (including experimental results for this channel without and with covalent linker between protein and βCD13), and (c) (M113N)₇·βCD with all charges on the βCD atoms set to zero.

Figure 6

Multi-ion PMFs from GCMC/BD simulations at zero current and zero membrane voltage with 1.0 M symmetric KCl concentration for (a) wt-αHL, (b) (M113F)₇, and (c) (M113F)₇·βCD.

Figure 7

Multi-ion PMFs from GCMC/BD simulations at zero current and zero membrane voltage with 1.0 M symmetric KCl concentration for (a) (M113N)₇, (b) (M113N)₇·βCD, and (c) (M113N)₇·βCD with all charges on the βCD atoms set to zero.

Figure 8

I-V curves from GCMC/BD simulations with asymmetric salt concentration. (a) 1.0 M KCl on cis side and 0.2 M KCl on trans side (location of cis and trans side is indicated in Figure 1). (b) 0.2 M KCl on cis side and 1.0 M KCl on trans side. The reversal potential of a simulation system is the voltage at the intersection of the corresponding I-V curve with the zero current axis.