# 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  
The types of molecular contacts discussed in this study are presented in Figure 3. As shown, the examined systems are primarily stabilized by hydrogen bonds and ionic interactions formed by the hydrophilic heads of phospholipids and specific residues in HSA. Figure 2: The effect of HSA adsorption on mass profiles of DPPC (**A**) and DPPE (**B**) phospholipid bilayers. The z-parameter denotes the distance from the membrane center. Smoothed plots were obtained using a moving average. The mass density values of head group nitrogens were scaled by a factor of 20 due to their low magnitude. Figure 3: Examples of key membrane–HSA interactions are DPPC/HSA (**A,B**) and DPPE/HSA (**C,D**). Pink solid lines indicate ionic interactions. Dotted pink—H-bonds. Atoms of lipids are presented in ball-like models and colored in the following fashion: turquoise—carbon, red—oxygen, blue—nitrogen, and yellow—phosphorus. The calcium cation is represented by a pink ball. Large yellow balls indicate magnesium ions. Solid lines with additional side groups in a stick-like model represent the HSA molecule. Its color corresponds to the domains depicted in Figure 1. The panels highlight interactions with specific residues in HSA: Panel A: GLU 321; Panel B: LYS 317, LYS 313, HIS 367, and ASP 365; Panel C: HIS 9, LYS 12, and GLU 16; Panel D: GLU 321, ASP 314, and LYS 317. Given the polyelectrolyte nature of proteins, the distribution of electrostatic charge within the molecule, dictated by pH, determines the type and number of intermolecular interactions they form. It is well known that albumin, which is characterized by an isoelectric point of c.a. 5, is negatively charged under physiological pH conditions (pH \(\approx\) 7) [73; 74]. As expected, the hydrophilic head groups of the phospholipid membranes interact with albumin through the ionic interactions formed by positively charged ammonium groups and negatively charged GLU and ASP residues (Figure 3). The other important interactions are hydrogen bonds formed by HIS and LYS residues, with the phosphate group playing the accepting role. It is of significant importance to examine the energetic aspects, which reveal substantial differences in the mutual affinity of membranes and HSA. In Figure 4, the distribution of binding energy values determined for the complexes in the presence of Na\({}^{+}\), K\({}^{+}\), Ca\({}^{2+}\), and Mg\({}^{2+}\) cations is presented. The confirmation that stable optimal structures were obtained is provided by the plot of root mean square deviation (RMSD) versus time (Figure 5). Additional trajectories are presented in Figures S2-S5 (Supplementary Materials). The trajectory analysis is useful for determining the equilibrium state, as indicated by a plateau. The attainment of a stable conformation is clearly observed both for the entire structure, as evidenced by considering the binding energy as a function of time (Supplementary Figure S1), and for individual binding sites (Supplementary Figure S2). In the latter case, however, broader thermal fluctuations and generally lower binding energy values are observed for DPPC compared to DPPE. For structures that have reached equilibrium, the binding energy values further suggest a higher affinity of HSA for the membrane composed of DPPE (Figure 4). The average binding energy values for DPPC in complexes containing Na\({}^{+}\), K\({}^{+}\), Ca\({}^{2+}\), and Mg\({}^{2+}\) are 700, 600, 1300, and 1100 kJ/mol, while for DPPE, these values are 1800, 1400, 1800, and 1200 kJ/mol, respectively. Apart from energetic characteristics, changes in structural parameters during MD simulations serve as an indicator of system stability, as demonstrated in previous studies on protein-phospholipid bilayer interactions [75; 76; 77]. Minor variations in structural parameters suggest the formation of stable and energetically favorable interactions.