In our previous work, we simulated the behaviour of CCBu with a pure DPPC lipid bilayer. More specifically, the free energy profiles (FEP) for CCBu in both Eand Z-configuration were calculated and it was found that only the E-conformer presented an absolute minimum inside the membrane and close to the lipid polar head groups. On the contrary, the Zisomer only presented a metastable state when internalized in the membrane, and a free energy barrier needed to be overcome to allow the interaction with the polar heads. Coherently with those results during the equilibrium MD simulations only spontaneous penetration of the E-isomer was observed. Conversely, in the present case (see SI) we observe spontaneous penetration of both isomers inside the mixed membrane and their positioning in the vicinity of the polar head region. Furthermore, meta-eABF was used to calculate the FEP for one CCBu molecule in both Eand Z-configurations penetrating inside a complex membrane composed of DPPC, DOPC and cholesterol and it was observed that both isomers can enter the mixed lipid bilayer unimpeded (Figure ). Furthermore, both isomers present a global free energy minimum at the interface between polar heads and lipid tails, as can also be appreciated by the snapshots reported in Figure . Interestingly, a free energy gain of about 6 kcal/mol can be appreciated for both isomers, compared to the situation in which CCBu is in the water bulk, and at the global minimum CCBu is interacting with the polar groups of the DPPC and DOPC lipids. This situation represents a first very crucial difference experienced by the more complex membrane under study here. The easiness of penetration of CCBu may be ascribed to the membrane higher fluidity and flexibility, which most notably enables even the sterically bulkier Z-isomer to penetrate the membrane, while the more rigid DPPC-only membrane made penetration of Z-CCBu impossible and energetically unfavorable due to the larger energetic barrier. We have also identified the main interactions stabilizing CCBu inside the lipid membrane. Our findings reveal that hydrogen bonds (HBs) between CCBu and the lipid polar heads play a crucial role the most important role. Specifically, we found that HBs involving either the hydroxyl group of the phenyl ring and the carbonyl oxygen of the pyron ring (C=O) are the most important occurrences and takes place with both DPPC and DOPC polar moieities, as shown in Figure . Interestingly, by analysing the MD trajectories we evidenced that distances shorter than 3 Å between CCBU and the polar heads occurred for 6% of the whole simulation span while distances shorter than 4 Å were populated for 59% of the simulation span. Furthermore, we also observed for 58% of the MDs trajectory the establishment of HBs (with a distance of 3 Å) between CCBu and water molecules (Figure ). Furthermore, we also evidenced that weak hydrophobic interactions between the hydrophobic tail of DPPC and DOPC lipids and the butyl chain of CCBu contribute to the overall stability of the membrane aggregate. We also considered the effects of CCBu orientation inside the lipid membrane. To accomplish this, we constructed two extreme configurations obtained after full equilibration of CCBu in E-conformation and situated within the lipid bilayer. In the first system, CCBu is positioned perpendicular to the lipid's hydrophobic chains, i.e. the membrane axis, while in the second system, it is oriented parallel to them. To investigate the effects of these orientations, we conducted MD simulations for a total of 100 ns for both systems. We observed that for the first system CCBu remains in a perpendicular orientation for the whole duration of the simulation, the average angle between the membrane Oz axis and the CCBu main axis stabilizing at 120°. Conversely, in the second system CCBu rapidly reorients to a more perpendicular arrangement after 50 ns. Thus, it appears that slightly perpendicular orientation is the most favourable arrangement, at least when considering only one ligand. This result is not unexpected, since this conformation allows to maximize at the same time HBs with the polar head and hydrophobic interactions with the lateral chains. Having assessed that both isomers are indeed penetrating inside the lipid membrane, we then turn to consider the impact of various concentrations of Eand Z-conformers on the membrane structural properties. Because of the more complex composition of the membrane, we computed the average area per lipid separately for each lipid in the system and the results are presented in Figure . Once again, we may observe a striking difference with the situation observed for the pure DPPC membrane, which was showing the coexistence of three different phases, characterized by different ordered and disordered arrangement of CCBu. As reported in Figure , in the case of the mixed membrane studied here we may observe an almost perfectly linear increase of the area per lipid as a function of the concentration, whatever the isomer considered. Interestingly, the same global behavior is maintained for the two amphiphilic lipids, i.e. DPPC and DOPC, while cholesterol presents a more intricate behavior as shown in SI. Interestingly, the constant increase of the area per lipid leads to a grow of area per lipid of about 4% for DPPC and 7% for DOPC for 50 CCBu molecules. Unsurprisingly, we also observe a globally stronger perturbation induced by the bulkier and more compact Zisomer, indeed the average area per lipid after the forced E/Z isomerization and ensuing equilibration are systematically higher than in the case of the E-isomer. Although, the perturbation appears relatively small, the observed differences are statistically significant, and thus indicative of an effective perturbation. Nevertheless, it is also important to note that the difference appears slightly lower than the one observed for concentrations compatible with the ordered phases in the case of the pure membrane. This difference can also be ascribed to the higher flexibility of the mixed membrane, which is thus, more efficient in accommodating the structural perturbations brought by the isomerization. Yet, as we already observed, it appears that the inclusion of bulkier peripheral substituents could be beneficial to maximize the membrane perturbation. Similar results can also be appreciated for the evolution of membrane interdigitation and the deuterium order parameters which are shown in SI. The evolution of the membrane structural properties with the concentration shows no discontinuity and thus points to the absence of specific phase transitions and concentration dependent specific arrangements. This can also be pictorially appreciated by the representative snapshots reported in Figure for the Eand Z-isomer, respectively. Indeed, a globally disordered arrangement is always and consistently observed, without the propensity of the CCBu moieties to orient parallelly to each other as it was observed for the pure membrane system. Interestingly, the same disordered pattern is observed for the planar Eand the more globular Z-isomer, coherently with the linear behavior of the area per lipid evolution as reported in Figure . Furthermore, and even at slightly high concentration we may notice the absence of clustering of CCBu suggesting that the hydrophobic interactions developed at the interface with the lipid tails are actually preventing aggregation, which is instead highly favorable in aqueous environment. Next, we analysed the orientation of CCBu within the lipid bilayer with respect to the membrane axis. We observed that CCBu in the E-conformation orient at angles of 50° and 130° relative to the Oz axis, i.e. the membrane axis, whatever the concentration (Figure ). This observation is also in agreement with the results previously discussed and obtained for a single ligand embedded in the membrane. Upon photoisomerization, we observed that the Z-CCBu retains the same global behavior, with however a slightly more perpendicular orientation the angle peaking, at 60°. Interestingly, for more isomers, the distributions are quite large and the orientation spans a wide allowed region. Once again, this observation is coherent with the more fluid nature of the mixed membrane, as compared to the simple model.