Tunable Energetic Stabilization Governs Self- and Mixed-Aggregation of M？bius and Bracelet Cyclotides

The environmental persistence and off-target toxicity of conventional synthetic pesticides continue to motivate the development of sustainable, biologically derived alternatives. Cyclotides—plant-derived, backbone-cyclized peptides stabilized by a cystine-knot motif—are promising candidates due to their exceptional chemical stability and intrinsic insecticidal and antimicrobial activities. However, the molecular determinants governing cyclotide aggregation, which directly affect formulation stability and bioavailability, remain insufficiently understood. Here, atomistic molecular dynamics simulations in explicit solvent were used to investigate self-aggregation and mixed-composition assembly of representative M？bius and Bracelet cyclotides across multiple concentrations. Time-resolved analyses of total potential, van der Waals (vdW), and Coulombic energies were employed as quantitative descriptors of stability. Aggregation occurred rapidly in all systems, with energies converging to stable equilibrium plateaus. Although statistically significant differences in equilibrium energies were observed as a function of peptide composition and concentration (p < 0.05), overall energetic trends were conserved. Distinct morphological differences emerged, with homogeneous systems forming compact clusters and heterogeneous mixtures producing more dispersed assemblies. Energetic decomposition revealed that stabilization is dominated by dispersion-driven packing interactions, while screened electrostatics provide secondary, composition-dependent modulation. Together, these findings define a conserved mechanistic framework with tunable energetic stabilization for cyclotide aggregation in aqueous environments, supporting flexible formulation strategies and advancing the development of cyclotide-based biopesticides as sustainable agrochemical alternatives.