The protonation process of Nisin

The protonation of Nisin is regulated by environmental pH, and its protonation state directly affects its charge properties, which in turn determines the strength of electrostatic interactions with bacterial cell membranes. Molecular dynamics (MD) simulations can precisely resolve the dynamic mechanisms and correlation laws of these two processes at the atomic level. The following are the specific simulation-related content and core conclusions:

Molecular Dynamics Simulation of Nisin's Protonation Process

Simulation Basis and Parameter Settings

Prior to simulation, the structural basis of Nisin must be clarified: as a lantibiotic containing 34 amino acid residues, it includes special residues such as dehydroalanine and dehydrobutyrine. For simulation, tools like PDB2PQR or PROPKA are commonly used to add hydrogen atoms and predict protonation sites under different pH conditions. Force fields such as CHARMM 36m or AMBER are preferred, with the solvent system using the TIP3P water model. Additionally, 0.15 mol/L NaCl is added to maintain physiological ionic strength, and the simulation temperature is set to 310 K to mimic the physiological environment. For example, the team at IISER Kolkata generated topological parameters for special residues using the Amber Leap program in their simulations, ensuring the accuracy of protonation state calculations.

Simulation Results and Laws of Protonation

Simulations clearly capture the regulatory effect of pH on Nisin protonation. In low-pH environments, basic groups such as amino groups in Nisin molecules are prone to protonation, resulting in a strong overall positive charge. As pH increases, protonation sites gradually deprotonate, reducing positive charge density. This aligns with experimental observations: Nisin solubility reaches 57 mg/ml at pH 2 but only 0.25 mg/ml at pH 8–12, as protonation enhances its hydrophilicity in polar solvents. Furthermore, simulations of the nisin@COFs system studied by the Nankai University team revealed that imine bonds in COFs also protonate and carry positive charges under low pH, generating electrostatic repulsion with protonated Nisin—confirming that Nisin's protonation state influences its intermolecular interactions.

Molecular Dynamics Simulation of Nisin's Membrane Adsorption

Simulation System Construction

Simulations typically use model membranes containing anionic phospholipids such as phosphatidylglycerol (PG), as bacterial cell membranes are rich in anionic lipids, which are key targets for Nisin adsorption. In system construction, Nisin with a defined protonation state is placed at the membrane-water interface. Equilibrium simulations are performed using engines like GROMACS: multiple cycles of equilibrium with gradually reduced constraints (50–100 ps per cycle, 5–6 cycles total) eliminate system stress, followed by production simulations of over 30 nanoseconds to record interaction trajectories between Nisin and the membrane.

Dynamic Mechanism of Adsorption

Initial Adsorption Stage: Simulations confirm that the C-terminal of Nisin is the core region for initial adsorption. Under low pH, protonated Nisin carries a positive charge, forming strong electrostatic attraction with phosphate groups of anionic phospholipids on the membrane—this is the primary driving force for adsorption. Simulations show that adsorption is significantly enhanced when anionic lipids account for over 50%–60% of the membrane composition.

Conformational Changes Post-Adsorption: Simulations reveal that Nisin's amide I band structure alters after adsorption, with an increased proportion of β-turns. This conformational adjustment facilitates deeper insertion of the N-terminal into the membrane. For example, PubMed studies observed that the N-terminal embeds more deeply into the lipid layer than the C-terminal, while the C-terminal remains anchored to the membrane surface—this differential adsorption mode lays the foundation for subsequent pore formation.

Influence of Lipids: Simulations with membranes of varying phospholipid compositions show that phosphatidylglycerol interacts most strongly with Nisin, causing membrane surface curvature. This aligns with the "wedge model" and is considered a preparatory step for pore formation.

Simulation of Synergistic Regulation Between Protonation and Membrane Adsorption

A core value of MD simulations is revealing the cross-regulatory relationship between protonation and membrane adsorption. Under low pH, Nisin exhibits high protonation and strong positive charge, leading to intense electrostatic attraction with anionic membranes—resulting in rapid adsorption and stable binding, as observed in simulations where Nisin rapidly accumulates and persists on the membrane surface. In contrast, under high pH, deprotonated Nisin has weakened positive charge, reducing electrostatic interactions with the membrane; adsorption efficiency drops significantly, and some Nisin molecules even detach from the membrane surface. Additionally, simulations show that protonation state indirectly regulates adsorption capacity by influencing Nisin's conformation: insufficient protonation prevents the formation of adsorption-favorable spatial conformations, leaving Nisin unable to bind stably even when near the membrane.