Investigation of Gramicidin A Incorporation into Phospholipid Vesicle Bilayers Using Optical-Trapping Confocal Raman Microscopy

Pore-forming peptides are a pharmacologically relevant class of membrane-active molecules capable of self-assembling in phospholipid bilayers to form transmembrane ion channels that induce uncontrolled ion flux, disrupting cellular homeostasis. Developing mechanistic insight into how these molecules perturb lipid-bilayer structure is critical for understanding their biological activity and for rational design of next-generation antimicrobials. In this work, optical-trapping confocal Raman microscopy was employed to investigate the structural impact of gramicidin A (gA) incorporation into individual 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipid vesicle bilayers as a function of peptide concentration in the vesicle membrane. Raman spectra acquired from individual, optically trapped vesicles confirmed gA incorporation through observation of peptide-specific tryptophan vibrational markers. Concentration-dependent spectra collected from 0 to 20 mol% gA vesicles revealed systematic disordering of DMPC acyl chains, observed through changes in the C-C stretching, C-H twisting, and C-H bending regions, consistent with bilayer deformation driven by hydrophobic mismatch between the gA channel and surrounding lipid chains. Self-modeling curve resolution analysis of the concentration-dependent spectra identified two spectral components: an ordered bilayer of unperturbed DMPC chains, and a gA-perturbed disordered bilayer. The amplitude of the two components vary linearly with gA concentration but with opposite sign. This result is consistent with each additional gA channel generating a localized region of disordered boundary lipids whose population grows in proportion with peptide concentration. Lipids not within the local region perturbed by the presence of gA remain ordered and decrease in proportion to their diminishing population until they disappear when the gA concentration reaches ∼20 mol%, indicating that 7-8 lipids surrounding each gA channel are impacted by hydrophobic mismatch. These results establish optical-trapping confocal Raman microscopy as an effective method for quantitative, single-vesicle investigation of peptide-membrane interactions, and highlight the power of model-free spectral analysis for unconstrained resolution of the peptide impact on membrane structure.