Molecular Dynamics Simulation to Uncover the Mechanisms of Protein Instability During Freezing

Abstract Freezing is a common process applied in the pharmaceutical industry to store and transport biotherapeutics. Herewith, multi-scale molecular dynamics simulations of Lactate dehydrogenase (LDH) protein in phosphate buffer with/without ice formation performed to uncover the still poorly understood mechanisms and molecular details of protein destabilization upon freezing. Both fast and slow ice growing conditions were simulated at 243 K from one or two-side of the simulation box, respectively. The rate of ice formation at all-atom simulations was crucial to LDH stability, as faster freezing rates resulted in enhanced structural stability maintained by a higher number of intramolecular hydrogen bonds, less flexible protein's residues, lower solvent accessibility and greater structural compactness. Further, protein aggregation investigated by coarse-grained simulations was verified to be initiated by extended protein structures and retained by electrostatic interactions of the salt bridges between charged residues and hydrogen bonds between polar residues of the protein. Lastly, the study of free energy of dissociation through steered molecular dynamics simulation revealed LDH was destabilized by the solvation of the hydrophobic core and the loss of hydrophobic interactions. For the first time, experimentally validated molecular simulations revealed the detailed mechanisms of LDH destabilization upon ice formation and cryoconcentration of solutes.