# Molecular Insights into the Interactions Between Human Serum Albumin and Phospholipid Membranes
## 2 Methods  
In this study, all-atom molecular dynamics (MD) simulations employing the NPT ensemble were conducted utilizing the AMBER14 force field [48] to investigate the intermolecular interactions between human serum albumin (HSA) and bilayers composed of either 1,2-dipalmitoyl-sn-glycero-3-phosphorylicholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE). The HSA structure (PDB code: 1e78) was obtained from the Protein Data Bank [49] and subjected to structural refinement procedures. Specifically, missing hydrogen atoms were added using the YASARA software (2021) [50], followed by homology modeling to incorporate absent amino acids based on the PDB structure and secondary structure prediction protocols. The resulting HSA model considered for analysis exhibited a molecular mass of 66.5 kDa.  
The DPPC and DPPE bilayers, each comprising 700 lipids, were constructed and initially equilibrated utilizing the CHARMM-GUI software platform ([[http://www.charmm-gui.org](http://www.charmm-gui.org)]([http://www.charmm-gui.org](http://www.charmm-gui.org)), accessed on 1 March 2023) [51; 52; 53]. Subsequently, a 10-nanosecond (ns) equilibration period was employed to mitigate potential atomic clashes within the membrane. Following equilibration, the HSA molecule was introduced adjacent to the bilayer surface without perturbing its structural integrity. Ten unique orientations of HSA were randomly positioned approximately 0.5 nm from the bilayer surface to explore potential binding sites.  
Initial MD simulations spanning 100 ns have been performed to select the most stable binding sites. DPPC showed more specific HSA domain binding affinity, resulting in protein desorption from the surface (except for those orientations involving mostly domain II). DPPE membrane formed stable complexes with HSA regardless of the orientation. After this initial period, binding was retained in only five DPPC systems. Therefore, further analyses and computations included only the five most stable structures for both DPPE/HSA and DPPC/HSA systems. For this reason, the simulations for selected structures were repeated and extended to 200 ns. Periodic boundary conditions were enforced to simulate an \"infinite\" bilayer, and the TIP3P water model [54; 55] was employed.  
All simulations were conducted under physiological conditions (temperature: 310 K, pH: 7.0, P = 1 Atm, NPT procedure [50; 56]) employing a 2 fs time step. Two scenarios were investigated: (i) HSA at the bilayer interfaced with an aqueous solution of NaCl and (ii) HSA at the bilayer interfaced with a CaCl\({}_{2}\) aqueous solution. Additionally, simulations with KCl and MgCl\({}_{2}\) solutions were also performed. Each simulation box encompassed water, a single HSA molecule, a DPPC/PE bilayer, 240 cations for bivalent ions, and around 480 cations for monovalent ions, along with an appropriate number of anions to maintain electroneutrality. The default ion parameterization in YASARA was applied throughout the study.  
The equilibrium state of the system was achieved after approximately 40 ns, following which simulations were continued for an additional 200 ns to gather data. To maintain constant pressure and temperature throughout the simulations, we utilized a modified [PERSON] barostat and thermostat [57], each with a relaxation time of 1 fs.  
Analysis of the MD simulations involved the evaluation of binding free energies, the formation of hydrogen bonds, and ionic interactions. These interactions were elucidated through contact maps illustrating the frequency of specific molecular pairings. Furthermore, the distribution of water molecules surrounding the HSA-bilayer complexes, as well as the distribution of calcium and sodium ions, were analyzed to provide insights into the solvation environment and potential ion-mediated interactions.