2021 Annual Meeting

Molecular Dynamics Simulations to Investigate Stability of Lysozyme Under Different Freezing Rates

Freezing processes are integral in drug storage and preservation, especially in the protein biotherapeutics that have complex structures which must remain unperturbed in order to ensure stability. Protein freezing results in a lessened reaction rate and decreased risk of microbial growth. An increase in hydrogen bonds after freezing inhibits flexibility and increases structural compactness. Consequences associated with freezing, such as solute rejection along the ice-water interface, induces negative impacts on stability and activity. Molecular dynamics (MD) simulations examine this phenomena in the Lysozyme protein, desired for its antibiotic and immune boosting properties . The use of MD simulations allow for the discovery of underlying causes of protein instability during freezing.

All-atom MD simulations reveal the interactions causing instability. Necessary softwares to perform simulations and analyze data include GROMACS, VMD, Pymol, and Gnuplot. Two Lysozyme proteins (PDB:1AKI ) are randomly put into a simulation box (6.4nm x 6.8nm x 13 nm) with 10mM sodium phosphate buffer: pH = 7. Three “non-isothermal” models show a temperature change from 270K to 247K. One is slow freezing represented by cooling 5-6K per 490 ns. Medium and rapid freezing are represented as cooling 7-8K and 11-12K respectively over 490 ns. An ice seeding method propagates the formation of a freezing environment. Equilibration is achieved through application of 1000 conjugate-gradient minimization cycles and a 500 ps MD simulation in the NPT assembly. Temperature and pressure control is achieved using a Langevin thermostat and Nose-Hoover barostat. The equilibrated system is subjected to another MD simulation using an NVT ensemble. Simulations are subject to periodic boundary conditions, and compiled on a supercomputer cluster called HPC.

Ice seeding populates the protein buffer system to simulate freezing conditions. The system is equilibrated by an analysis of the convergence of potential energy. Root-mean-square-deviation (RMSD) evaluates the structural similarity between conformations. There are large fluctuations in early stages of freezing due to the proximity of Lysozyme to the ice-water interface, but remain stable later in the freezing process. Root-mean-square-fluctuation (RMSF) identifies the most flexible residues in Lysozyme by analyzing H-bond formation between the protein and solvent molecules as well as intramolecularly. Analyses correlate variables that affect stability in Lysozyme. These include the effects of temperature, solute concentration, and ionic strength on a number of factors dictating stability such as unfolding, aggregation, degradation, and molecular interactions. This simulation and analysis improves understanding of these correlations in Lysozyme.