Protein-protein interactions and protein cluster formation from scattering experiments and coarse-grained molecular models

Measuring and predicting colloidal protein-protein interactions (PPI) in solution is important for applications ranging from protein phase behavior to aggregation of biopharmaceuticals. However, the nature of these interactions is poorly understood since it is affected by several factors such as temperature and protein sequence. Typically, PPI are characterized via the second viral coefficient (B22), as determined from Rayleigh light scattering (LS). However, discrepancies and confusion have arisen regarding recent interpretations of B22 from classical treatment of LS data, as well as questions about refinement of coarse-grained (CG) models of PPI against experimental results such as B22. Here, an alternative treatment of Rayleigh scattering in multi-component systems is provided based on Kirkwood-Buff solution theory, demonstrating clearly that the correct B22 value can be obtained directly from LS. The model is compared against existing approximations for simulated LS data, as well as experimental scattering data for acidic solutions of alpha-chymotrypsinogen A (aCgn) from low to high protein concentrations. The results illustrating this approach provides quantitatively accurate values for protein interactions vs. protein concentration from low to high concentrations without assumptions on the nature of interactions and/or the thermodynamics of the system. Additionally, a CG model is developed to study the role of colloidal protein interactions on the stability and aggregation propensity of proteins. The novel CG model considers proteins as folded rigid bodies that interact through van der Waals, hydrophobic, and screened Coulomb forces between individual amino acids, which allow one to evaluate the thermodynamic stability of proteins at different interacting conditions. The CG model was applied to investigate the dimerization human gamma-D-Crystallin (gDCrys). The results suggest that the thermodynamic equilibrium between monomeric and dimeric gDCrys is balanced by the formation of weakly bound dimers, and changes in the stability of this species may be a cornerstone for controlling protein cluster formation and aggregation. Furthermore, the results indicate that at experimentally relevant conditions, entropic contributions are predominant in the free-energy of protein cluster formation and colloidal protein interactions, arguing against interpretations that treat B22 primarily from energetic considerations alone.