# 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
bridges with Na\({}^{+}\), K\({}^{+}\), Mg\({}^{2+}\), and Ca\({}^{2+}\) represent a negligible part of the interactions (<1%) compared to other types of molecular contacts, including direct ionic interactions between the phospholipid layer and the protein. In the DPPC/HSA system, ionic bridges are formed only between the phosphate group and the carboxyl group in GLU in the presence of Ca\({}^{2+}\) ions, with an average distance between the atoms and the ion of 3.19 \(\pm\) 0.14 A. In the DPPE/HSA system, ionic bridges are formed by both GLU and ASP, characterized by distances of 3.33 \(\pm\) 0.35 A for GLU and 3.41 \(\pm\) 0.25 A for ASP. The higher binding energy of HSA to the DPPE membrane, compared to DPPC, demonstrates greater mutual affinity of the components. This is attributed to structural differences in the phospholipid headgroup. As can be inferred from Figure 6, DPPE forms more direct ionic contacts (about 4-5 times) with HSA compared to DPPC. This is due to the difference in headgroup size and, consequently, the difference in positive charge density on groups capable of forming such contacts with HSA (-N(Me)\({}_{3}\) for DPPC and -NH\({}_{3}\) for DPPE) [88, 89]. Additionally, the steric hindrances caused by the methyl groups in -N(Me)\({}_{3}\) may also play a role in influencing the stability of HSA associates formed by these lipids. Considering the effect of mono- and divalent cations on the individual complexes, significant differences are observed, regardless of whether DPPC or DPPE membranes are considered. The percentages of ionic interactions formed by particular residues and HSA domains are presented in Table S1 (Supplementary Materials). The highest percentage of ionic interactions in the case of DPPC (approximately half of all ionic interactions in the complex) is formed by LYS (48%, 45%, 47%, and 52% for the complexes with Ca\({}^{2+}\), Mg\({}^{2+}\), K\({}^{+}\), and Na\({}^{+}\), respectively). About 20% of the ionic interactions are formed by ASP or GLU. The contributions of ASP contacts are 20% (Ca\({}^{2+}\)), 21% (Mg\({}^{2+}\)), 20% (K\({}^{+}\)), and 16% (Na\({}^{+}\)), while for GLU contacts they are 25% (Ca\({}^{2+}\)), 24% (Mg\({}^{2+}\)), 24% (K\({}^{+}\)), and 26% (Na\({}^{+}\)). Noteworthy, a low number of ionic interactions (2-6%) was observed in the case of ARG and HIS. In the case of DPPE complexes with HSA, GLU forms the highest number of ionic contacts: 46% (Ca\({}^{2+}\)), 45% (Mg\({}^{2+}\)), K\({}^{+}\) (42%), and Na\({}^{+}\) (46%). Similarly, as in the case of DPPC, ARG and HIS form only a small number of interactions (1-5%). As presented in Figure 1, HSA bound to the DPPC and DPPE bilayers by domain I and II. However, in the case of DPPE, interactions with domain III also occur (Figure 1C, Tables S1 and S2, Supplementary Materials). The experimental studies of [PERSON] et al. [90] showed that the adsorption of DPPC affects the structural features of all domains in HSA. This study, however, does not indicate which parts of albumin interact with a DPPC membrane. It is worth noting that although albumin is negatively charged as a whole molecule under physiological conditions, its polyelectrolyte nature allows it to  
Figure 6: Bar plot showing the average number and standard deviation of direct ionic interactions for all cases studied. The symbols PC and PE represent the phospholipid membranes formed by DPPC and DPPE, respectively. contain regions with positive charges. Hence, it can interact with both cationic and anionic surfactants [91]. According to MD simulation results reported by [PERSON] et al. [92], subdomain IA poses the highest negative charge density, while IB and IIIA are positively charged. In particular, the IIIA domain was found to be the most positively charged [92], which makes interaction with the positively charged amino groups of the phospholipid less favorable.