19 F solid-state NMR approaches to probe antimicrobial peptide interactions with membranes in whole cells.

To address the global problem of bacterial antibiotic resistance, antimicrobial peptides (AMPs) are considered promising therapeutic candidates due to their broad-spectrum and membrane-lytic activity. As preferential interactions with bacteria are crucial, it is equally important to investigate and understand their impact on eukaryotic cells. In this study, we employed ¹⁹ F solid-state nuclear magnetic resonance (ssNMR) as a novel approach to examine the interaction of AMPs with whole red blood cells (RBCs). We used RBC ghosts (devoid of hemoglobin) and developed a protocol to label their lipid membranes with palmitic acid (PA) monofluorinated at carbon positions 4, 8, or 14 on the acyl chain, allowing us to probe different locations in model and intact RBC ghost membranes. Our work revealed that changes in the ¹⁹ F chemical shift anisotropy, monitored through a CF bond order parameter (S _CF ), can provide insights into lipid bilayer dynamics. This information was also obtained using magic-angle spinning ¹⁹ F ssNMR spectra with and without ¹ H decoupling, by studying alterations in the second spectral moment (M ₂ ) as well as the ¹⁹ F isotropic chemical shift, linewidth, T ₁ , and T ₂ relaxation times. The appearance of an additional isotropic peak with a smaller chemical shift anisotropy, a narrower linewidth, and a shorter T _1, induced by the AMP caerin 1.1, supports the presence of high-curvature regions in RBCs indicative of pore formation, analogous to its antimicrobial mechanism. In summary, the straightforward incorporation of monofluorinated FAs and rapid signal acquisition offer promising avenues for the study of whole cells using ¹⁹ F ssNMR.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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