Aggregates of α-chymotrypsinogen anneal to access more stable states.

Non-native protein aggregates present a variety of problems in fundamental and applied biochemistry and biotechnology, from quality and safety issues in pharmaceutical development to their association with a number of chronic diseases. The aggregated, often amyloid, protein state is often considered to be more thermodynamically and kinetically stable than (partially) unfolded or folded monomers except under highly denaturing conditions. However, evolution of the structure and stability of aggregated states has received much less attention. Here it is shown that under mildly-denaturing conditions (elevated temperature or [urea]), where the native monomer (N) is slightly favored compared to the unfolded state (U), α-chymotrypsinogen A (aCgn) non-native aggregates undergo a structural relaxation or annealing process to reach even more stable states. The annealed aggregates are more resistant to dissociation than aggregates that do not undergo this relaxation process. Aggregates without annealing dissociate via linear chain depolymerization, and annealing is accelerated under conditions that promote slow dissociation (partially denaturing conditions). This is consistent with a free energy landscape with multiple barriers and local minima that allows for a kinetic competition between aggregate dissociation and structural relaxation to more stable aggregate states. This highlights added complexities for protein refolding or aggregate dissociation processes, and may explain why it is often difficult to completely recover monomeric protein from aggregates.