Effects of Arginine on Hierarchical Protein Aggregation

Arginine is widely used in protein formulations to suppress protein aggregation and prolong the lifetime of the native, functional state which undergoes different stresses during storage, shipping and handling. However, arginine's differential effects on protein aggregation pathways remain unclear. Here, we investigated the effects of arginine on heat- and acid-induced aggregation of immunoglobulin G and heat-induced aggregation of several model proteins. Arginine suppressed the formation of insoluble aggregates but had limited impact on monomer recovery, particularly on immunoglobulin G. Instead, arginine promoted the formation of soluble aggregates, suggesting a possibility that unfolded proteins possess heterogeneous surface properties, leading to formation of soluble and insoluble aggregates that are modulated by arginine. To explain these kinetic behaviors, we propose a hierarchical aggregation model, in which arginine preferentially modulates weaker intermolecular interactions to inhibit the growth of the soluble aggregates into large insoluble aggregates. Moreover, soluble aggregates were observed in a range of proteins with varying isoelectric points and molecular weights. These results highlight a previously unrecognized aspect of soluble protein aggregation. Understanding the pathway and mechanism of soluble aggregate formation may shed light into physiologically-relevant soluble assemblies, e.g., Alzheimer's disease-associated oligomers and microtubule-derived double rings in contrast to the soluble amorphous aggregates under current study.