Aggregation Intermediates

The formation of insoluble aggregates due to protein misfolding is a phenomenon associated with various human diseases, inclusion body formation, and in vitro aggregation. In vivo, this folding problem frequently arises with heterologous proteins overexpressed in E. coli, which form inclusion bodies, or amorphous aggregates within the cell. The analogous aggregation problem occurs in vitro via a similar association mechanism. Non-native multimerization subsequently leads irreversibly to the formation of large aggregates or inclusion bodies. A frequently employed strategy for minimizing in vitro aggregation is to refold protein under dilute conditions. The drawback of this practice is that it dilutes the final product, and the strategy does not work for oligomeric proteins that cannot assemble efficiently under dilute conditions. Therefore, further study of the aggregation reaction is essential in designing effective refolding strategies.

To study the polymerization process, nondenaturing polyacrylamide gel electrophoresis (PAGE) has been used to trap a sequential ladder of multimeric intermediates in the in vitro aggregation of P22 tailspike polypeptide chains. The aggregation intermediates aggregation intermediates microscopy gel polymerized by noncovalent association of a monomeric folding intermediate rather than aggregation of nativelike trimeric species. These multimers were sensitive to SDS and protease unlike the thermostable native trimer. Using a modified nondenaturing Western blot procedure, aggregation intermediates were determined to have nonnative epitopes in common with productive folding intermediates. The aggregate epitopes were found on folding and aggregation intermediates but not on denatured or native tailspike protein. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, and this structural information has implications on the aggregation mechanism.

Although the understanding of the mechanism of protein folding and aggregation is somewhat limited, the ability to influence the kinetic competition between productive folding and aggregation is a requirement for improving the recovery of active protein expressed in a heterologous host cell. In certain polymerization processes, such as crystallization and amyloid fibril formation, the initial nucleation complex forms slowly, but rapid polymerization can occur once the aggregate reaches a certain critical size. Biological systems that display nucleation-growth kinetics include the polymerization of sickle cell hemoglobin, amyloid fibril formation, actin polymerization, bacterial flagella assembly, and collagen refolding. Many biological self assembly occur via a sequential polymerization mechanism, in which monomeric subunits add sequentially to the growing fibril. Two-dimensional PAGE data show that aggregation occurs by multimeric polymerization, in which two multimers of any size can associate to form a larger aggregate. This mechanism allows the generation of large aggregates even after the pool of monomers has been depleted. The aggregation intermediates are not in rapid equilibrium with each other, and multimerization is essentially an irreversible reaction.

There is evidence that the aggregation process is specific, involving certain structural moieties such as helix-helix docking or cross-ß-sheet formation. Inclusion bodies formed within cells are relatively pure and do not contain a significant fraction of other cellular proteins. In addition, mixed aggregation studies of the P22 coat and tailspike proteins suggest that the tailspike does not form heterogeneous aggregates with the P22 coat protein. This methodology of isolating early multimers along the aggregation pathway has been shown to be applicable to other proteins, such as the P22 coat protein and carbonic anhydrase II, and may potentially be used to monitor inclusion body formation or detect amyloid fibil intermediates associated with Alzheimers disease.