Measurements of Ion Binding to Lipid-Hosted Ionophores by Affinity Chromatography

Abstract

The binding affinity between antibiotic ionophores and alkali ions within supported lipid bilayers was evaluated using affinity chromatography. We used zonal elution and frontal analysis methods in nanovolume liquid chromatography to characterize the binding selectivity of the carrier and channel ionophores valinomycin and gramicidin A within different phosphatidylcholine bilayers. Distinct binding sensitivity to the lipid phase, both in affinity and selectivity, is observed for valinomycin, whereas gramicidin is less sensitive to changes in a membrane environment, behavior that is consistent with ion binding occurring within the interior of an established channel. There is good agreement between the chromatographic retention and the reported binding selectivity measured by other techniques. Surface potential near the binding site affects ion retention and the apparent association binding constants, but not the binding selectivity or enthalpy measurements. A model accounting for the surface potential contributions of retained ions during frontal analyses yields values close to intrinsic binding constants for gramicidin A (K A for K+ between 70 and 120 M–1) using reasonable estimates of the initial potential that is postulated to arise from the underlying silica.

Ionophore properties of valinomycin in the model bilayer lipid membrane 1. Selectivity towards a cation

“…Valinomycin consists of twelve alternating amino acids, whose structure is shown in Scheme 1. Valinomycin has high cation selectivity by forming a complex with alkali metal cations, such as K + , Rb + , and Cs + , but not Na + or Li + [5]. The valinomycin-K + complex forms a bracelet conformation, in which K + stays in the center of the valinomycin ring cavity and binds to six ester C=O groups [6].…”

How Valinomycin Ionophores Enter and Transport K+ across Model Lipid Bilayer Membranes

“…This ion-transporting property is primarily due to the formation of a metal ion–peptide complex. Valinomycin forms stable complexes with K + , Rb + , and Cs + . The X-ray diffraction data of the K + –valinomycin complex revealed that the cation resides in the central cavity of the valinomycin ring and is coordinated to all six ester carbonyls. − The size of the cavity is suitable to accommodate K + ions but too large for Na + or Li + ions.…”

Ion-Pairing Mechanism for the Valinomycin-Mediated Transport of Potassium Ions across Phospholipid Bilayers

“…Valinomycin is a cyclic neutral peptide with 12 alternating amino acids, which has ionophore properties in biological cell membranes and artificial lipid bilayers. , Valinomycin is selective for alkali cations by forming a complex with K + , Rb + , and Cs + but not with Na + or Li + . , XRD data indicates that the valinomycin–K + complex adopts a bracelet conformation with a hydrophobic exterior and a polar cavity in the center of the valinomycin peptide where K + is coordinated with six ester CO bonds . In contrast, free (noncomplex) valinomycin assumes a propeller conformation forming hydrogen bonds with the surrounding solvent molecules …”