A supramolecular complex of palmitoylated Orai C-terminal peptide and cyclodextrin targets the plasma membrane to reduce store-operated calcium channel activity.

Palmitoylation is a widely employed strategy for anchoring peptides to lipid membranes, but it can also lead to uncontrolled self-assembly. The lipid moieties can promote clustering into heterodispersed aggregates that distribute unevenly, thereby reducing accessibility and inducing undesired uptake mechanisms that limit the functional performance of peptides. Controlling this lipid-driven aggregation remains a key challenge for membrane-active peptide systems. Here, we introduce a supramolecular entrapment strategy for controlling the assembly and membrane insertion of bioactive lipopeptides. We synthesize palmitoylated Orai3-derived peptides and use randomly methylated α-cyclodextrin (RAMEA) to complex the fatty acid chain through a host-guest inclusion mechanism, thereby effectively shielding its intermolecular hydrophobic interactions. In addition, we incorporate an octaethylene glycol (PEG8) spacer to improve peptide flexibility and access to its molecular target at the membrane interface. Biophysical characterization and molecular dynamics simulations show that the complex formation between the cyclodextrin and the lipopeptide yields an ultralow-sized molecular dispersion system with reduced hydrophobic exposure and aggregation propensity. In cell-based systems, this supramolecular organization enhances membrane localization and reduces endosomal internalization, while the PEG8 spacer further facilitates intracellular target engagement. This strategy was evaluated in the context of store-operated calcium (SOC) channel regulation, a well-characterized membrane-associated system that critically depends on precise spatial organization of interacting components, namely STIM1-Orai1 at the junctions between plasma and endoplasmic reticulum membranes. In this context, we show that this lipopeptide-cyclodextrin multiplex enables inhibition of SOC channel activity. Overall, this work demonstrates that our lipopeptide-RAMEA inclusion complexation strategy provides a general approach to control palmitoylation-driven lipopeptide aggregation and establishes a broadly applicable design principle for tuning the performance of membrane-associated peptide systems.