The Influence of Lipid Bilayer Physicochemical Properties on Gramicidin A Conformer Preferences.

The conformational preferences adopted by gramicidin A (GA) dimers inserted into phospholipid bilayers are reported as a function of the bilayer cholesterol content, temperature, and incubation time. Through use of vesicle capture-freeze drying methodology, GA dimers were captured in lipid bilayers and the conformational preferences of the complex were analyzed using ion mobility-mass spectrometry. Perturbations that affect the physicochemical interactions in the lipid bilayer such as cholesterol incorporation, temperature, and incubation time directly alter the conformer preferences of the complex. Regardless of bilayer cholesterol concentration, the antiparallel double helix (ADH) conformation was observed to be most abundant for GA dimers in bilayers composed of lipids with 12 to 22 carbon acyl chains. Incorporation of cholesterol into lipid bilayers yields increased bilayer thickness and rigidity, and an increased abundance of parallel double helix (PDH) and single-stranded head-to-head (SSHH) dimers were observed. Bilayers prepared using 1,2-dilauroyl-sn-glycero-3-phosphocholine, a lipid with 12 carbon acyl chains, yielded a nascent conformer that decreased in abundance as a function of bilayer cholesterol content. High resolution ion mobility-mass spectrometry data revealed two peaks in the ADH region suggesting that ADH populations are composed of two distinct conformers. The conformer preferences of GA dimers from 1,2-distearoyl-sn-glycero-3-phosphocholine bilayers were significantly different for samples incubated at 4°C vs. 60°C; increased cholesterol content yielded more PDH and SSHH at 60°C. The addition of cholesterol as well as incubating samples of 1,2-distearoyl-sn-glycero-3-phosphocholine at 60°C for 24-72 h yielded an increase in PDH and SSHH abundance.