Molecular Mechanism of Amino Acids for Stabilizing Protein Dispersions.

Abstract

In living cells, numerous biomolecules coexist in highly crowded environments while maintaining their biological functions. Under such conditions, biomolecules are constantly exposed to the risk of aggregation, leading to the loss of their biological function. To mitigate these risks, cells employ various strategies, such as the accumulation of small molecules in the cytosol. For example, free amino acids (AAs) are well-known to inhibit protein association and stabilize protein dispersions. A recent experimental study using a simple theoretical model suggested that the adsorption of AAs onto protein surfaces induces repulsive interactions between proteins. However, the molecular mechanism by which the adsorption of AAs inhibits protein association remains unclear. In this study, we conducted molecular dynamics simulations to investigate the molecular mechanisms by which AAs inhibit protein association. As a model system, insulin was simulated in the presence of a 1 M proline solution using both monomeric and dimeric models. For the monomeric system, the adsorption behavior of proline on the protein surface was characterized, revealing that proline molecules preferentially and uniformly adsorbed onto the protein surface. For the dimeric system, we examined how proline adsorption influences protein association by calculating the potential of mean force (PMF) and the osmotic second virial coefficient. These analyses demonstrate that proline induces net repulsive interactions among proteins, which are consistent with experimental observations. Furthermore, the number of proline molecules at the protein-protein interface reached a maximum around the intermediate state in the PMF prior to protein association. This accumulation of interfacial proline molecules increases the effective free energy required for further protein association. In conclusion, this study provides a direct molecular-level mechanism by which the adsorption of AAs stabilizes protein dispersions.