Investigating Daptomycin–Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs

Abstract

Daptomycin is a cyclic lipopeptide antibiotic that targets the lipid membrane of Gram-positive bacteria. Membrane fluidity and charge can affect daptomycin activity, but its mechanisms are poorly understood because it is challenging to study daptomycin interactions within lipid bilayers. Here, we combined native mass spectrometry (MS) and fast photochemical oxidation of peptides (FPOP) to study daptomycin−membrane interactions with different lipid bilayer nanodiscs. Native MS suggests that daptomycin incorporates randomly and does not prefer any specific oligomeric states when integrated into bilayers. FPOP reveals significant protection in most bilayer environments. Combining the native MS and FPOP results, we observed that stronger membrane interactions are formed with more rigid membranes, and pore formation may occur in more fluid membranes to expose daptomycin to FPOP oxidation. Electrophysiology measurements further supported the observation of polydisperse pore complexes from the MS data. Together, these results demonstrate the complementarity of native MS, FPOP, and membrane conductance experiments to shed light on how antibiotic peptides interact with and within lipid membranes.

Antimicrobial Peptides and Their Assemblies

“…The biophysics of peptide-membrane interaction was recently investigated using an interesting approach combining mass spectrometry (MS) and fast photochemical oxidation of peptides (FPOP). The daptomycin-membrane interactions with different lipid bilayer nanodiscs were studied; MS suggested that daptomycin incorporated randomly and did not prefer any specific oligomeric states when integrated into bilayers, whereas FPOP revealed significant protection in most bilayer environments; the stronger membrane interactions occurred with more rigid membranes, and pore formation took place in more fluid membranes exposing daptomycin to FPOP oxidation; membrane conductance supported the observation of polydisperse pore complexes depicted from the MS analysis [80].…”

Decoding the molecular interplay in the central dogma: An overview of mass spectrometry‐based methods to investigate protein‐metabolite interactions

“…Application of native MS of proteinlipid complexes following either gas-phase ejection or solution-phase detergent extraction showed a selective interaction of membrane proteins with lipids [78]. Whereas the combination of native MS with photochemical oxidation of peptides enabled the identification of not preferential daptomycin interaction with membrane lipids [79]. A key advancement in the field of native MS has been the introduction of single ion detection methods that enable charge detection (CD) MS [39,80,81].…”

Precursor Reagent Hydrophobicity Affects Membrane Protein Footprinting

“…Currently, there are two major ways of using reactive species to footprint MPs. , The most popular footprinting reagent is the hydroxyl radical generated by the photolysis of hydrogen peroxide (commonly known as fast photochemical oxidation of proteins (FPOP)), radiolysis of water, or other methods. , This type of reaction modifies a range of protein residues on a short time scale. The hydroxyl radical, − other FPOP reagents, − or other footprinting methods , are suitable probes for water-soluble proteins and extra-membrane domains of MPs. For the TMDs, however, there is little modification − , because the hydrophobic core resists the penetration of reagents.…”