Thermodynamic and structural characterization of the cyclic peptide G4CP2 binding to DOPC liposomes: an integrated experimental/computational study

The interaction of the cyclic peptide G4CP2 (βA-RYFFDMWY) with zwitterionic DOPC liposomes was investigated as a minimal membrane model by combining spectroscopy, calorimetry, and enhanced-sampling molecular dynamics. G4CP2 was originally reported as a functional inhibitor of the transcription factor GAL4 and was selected here to provide a physicochemical, membrane-focused characterization of this biologically validated cyclic scaffold. UV-Vis titrations showed hypochromic effects upon lipid addition, while steady-state fluorescence quenching (including synchronous fluorescence) confirmed membrane association without large spectral shifts, suggesting predominantly interfacial binding environments for the Trp reporter. Circular dichroism indicated no major change in spectral shape, with a progressive increase in signal magnitude with lipid concentration consistent with an increasing membrane-associated fraction. Isothermal titration calorimetry revealed micromolar binding with an endothermic enthalpy compensated by a strongly favorable entropy contribution, indicating an entropy-driven association regime. DLS and TEM showed no major vesicle disruption under the tested conditions. To provide a molecular interpretation, reweighted free-energy surfaces were reconstructed from OPES simulations along peptide insertion depth and tilt, and the resulting ensemble was projected onto hydration, aromatic insertion, headgroup contacts, and conformational descriptors. The simulations indicated a broad low-free-energy basin consistent with multiple interconverting membrane-associated microstates and supported an ensemble shifted toward deeper interfacial association characterized by stronger dehydration and aromatic engagement. Overall, the combined experimental-computational data provide a coherent thermodynamic and structural picture of how an aromatic-rich cyclic peptide associates with a zwitterionic bilayer through heterogeneous interfacial states.