Aggregation kinetics of native-state cataract-forming proteins

We have investigated the aggregation kinetics of Human γD-crystallin (HγD) protein, which is responsible for cataracts in humans, under physiological conditions, using light scattering. We have monitored the aggregation kinetics by following two kinds of heating pathways, namely gradient heating and direct heating. The gradient heating involved many intermediate temperatures and at each intermediate temperature, the system was annealed for a certain long time. We find the aggregation kinetics to significantly depend on the heating pathway. In both heating pathways, the final temperature was below the protein's melting temperature. Unlike the typical thermally induced aggregation involving protein unfolding, the aggregation observed here occurred in the native state, resulting in amorphous aggregates rather than amyloid fibers. The critical temperature to trigger aggregation was lower with direct heating compared to gradient heating, and direct heating resulted in a shorter nucleation time, indicating that rapid heating provides sufficient energy to overcome kinetic barriers more effectively. Remarkably, gradient heating delays the aggregation kinetics by presumably allowing distractive pathways from the optimal pathway to the final aggregates. Furthermore, presence of additional sodium chloride significantly accelerated the aggregation process, exhibiting the important role of electrostatic interactions among the protein molecules in the aggregation kinetics. These findings provide new insights into native-state aggregation, with potential implications for understanding cataractogenesis and developing preventive measures for protein aggregation-related diseases.