The Effect of Arginine on the Phase Stability of Aqueous Hen Egg-White Lysozyme Solutions

Abstract

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the HEWL solutions. The computer simulation results indicate that arginine molecules tend to self-associate. If arginine residues are located on the protein surface, the free arginine molecules stay in their vicinity and prevent the way protein molecules “connect” through them to form clusters. The results are not sensitive to a particular force field and suggest a possible microscopic mechanism of the stabilizing role of arginine as an excipient.

The mechanism of self-association of human γ-D crystallin from molecular dynamics simulations

“…3 , free arginine does not self-assemble only in solution, but also binds on already bound free arginines on the surface of the protein, creating arginine clumps that act as inter-protein spacers. This phenomenon of arginine association has already been observed by others, and in the course of our research we also detected it in simulations of HEWL solutions [ 11 , 23 , 24 ].…”

“…To be consistent with previous studies on HEWL [ 11 ] we first investigated the influence of 0.3 M concentration of free arginine by adding 740 arginine molecules in the native γ -D crystallin solution.…”

“…Since our previous results show that protein-protein interactions in the case of γ -D crystallin are dominated by arginine residues [ 10 ], we first examined the stability of these solutions with added free arginine. The free arginine was previously found to interact with the arginine residues on protein surface, and in such way hinders the protein self-association process [ 11 ].…”

“…Systems containing 0.3 and 0.6 M free arginine, in addition to proteins, contained also 740 and 1480 arginine molecules, respectively. We also ran a simulation of pure aqueous 0.3 M arginine solution, which was prepared as described in [ 11 ]. The mutated γ -D crystalline were obtained interactively using Swiss-PdbViewer 4.1.0 [ 19 ], with which we managed to successfully mutate the surface of γ -D crystallin by replacing 21 positively charged arginine residues with 21 neutral glycine residues and in the second case with 21 positively charged lysine residues.…”

“…By performing an extensive analysis of intermolecular residue–residue interactions, we showed that arginine residues on the protein surface are of paramount importance in the initial stage of the aggregation of these proteins [ 10 ]. To further address the role of arginine in protein self-association we have additionally performed MD simulations of HEWL in the presence of free arginine which suggested that free arginine interacts with the arginine residues on the protein surface preventing further protein-protein interactions [ 11 ].…”