# Molecular Insights into the Interactions Between Human Serum Albumin and Phospholipid Membranes
## 3 Results and Discussion
### The Affinity of HSA for DPPC and DPPE in the Presence of Mono- and Divalent Cations
As illustrated in Figure 5, the RMSD values of DPPC complexes with HSA are lower than those observed for DPPE complexes. It is noteworthy that, in simulations of DPPC/HSA in the presence of K\({}^{+}\) ions, two structures failed to reach equilibrium, resulting in complex dissociation. The destabilizing influence of K\({}^{+}\) is clearly reflected in the RMSD vs. time profile (Figure 5). Figure 4: Bar plot showing the average free binding energy of HSA to the bilayer and its standard deviation for all cases studied. The symbols PC and PE represent the phospholipid membranes formed by DPPC and DPPE, respectively. As previous studies have shown [38], Na\({}^{+}\), Mg\({}^{2+}\), and Ca\({}^{2+}\) impact the stability of complexes involving HSA. Notably, Ca\({}^{2+}\) ions were found to be responsible for linking various peptides with phospholipid bilayers [79; 80; 81]. In contrast, Na\({}^{+}\) and K\({}^{+}\) ions are known to interact only slightly with albumin compared to Ca\({}^{2+}\) and Mg\({}^{2+}\)[82; 83]. The binding of cations to phospholipids increases the positive charge of the bilayer, thereby promoting interactions with negatively charged albumin. Considering the atomic radius and ionic charge, the hydration ability of the studied ions increases as follows: K\({}^{+}\)\(<\) Na\({}^{+}\)\(<\) Ca\({}^{2+}\)\(<\) Mg\({}^{2+}\), which aligns with experimental observations [84]. Ion hydration appears to be a competing effect against binding with phospholipids. On the other hand, the binding of ions of high positive charge (Mg\({}^{2+}\), Ca\({}^{2+}\)) to the membrane is expected to promote stronger interactions with the negatively charged protein. However, as shown in Figure 4, this expectation is not met in the case of DPPE, as the presence of Mg\({}^{2+}\) ions results in the weakest binding of HSA by the phospholipid bilayer. In the case of DPPC, an advantage of divalent ions in stabilizing the complex over monovalent ions can be observed, as the highest affinity for HSA was found in systems containing Ca\({}^{2+}\) and Mg\({}^{2+}\) ions. Notably, experimental data on the interactions of soybean lecithin with albumin support this observation [46]. PC phospholipids, including DPPC, are the main components of lecithins, contributing to their functional properties [85]. These phospholipids have been found to form efficient interactions with albumin [46; 86; 87]. The results of infrared spectroscopic measurements conducted by [PERSON] et al. [46] have demonstrated that bovine serum albumin binds more effectively to hydrogenated soybean lecithin films in the presence of CaCl\({}_{2}\) compared to NaCl. On the other hand, the distributions of E\({}_{\text{bind}}\) in the case of DPPE complexes containing Na\({}^{+}\) and Ca\({}^{2+}\) cations are very similar. Further, both cations increase the stability of the complex when compared to K\({}^{+}\) and Mg\({}^{2+}\), respectively. For DPPC, complexes containing the K\({}^{+}\) ion exhibit the lowest stability. As previously mentioned, we have observed that two of these complexes disintegrate during the simulation period. Indeed, both experimental and theoretical studies conducted by other researchers [43; 44; 45; 47] suggest a low affinity of K\({}^{+}\) for DPPC compared to other monovalent and divalent metal cations. The stability of HSA-phospholipid complexes in the presence of mono- and divalent cations is primarily driven by ionic contacts between the phospholipid membrane and HSA, along with hydrogen bonds. However, the influence of ions on HSA affinity for the membrane appears to be more indirect than direct. It might seem that the differences in complex stability arise from the binding of components via ions. Nevertheless, ionic  
Figure 5: Root mean square deviation (RMSD) between the Cartesian atom coordinates in the two selections of HSA evolution in time. PC and PE symbols denote the phospholipid membranes formed by DPPC and DPPE, respectively.