A Mechanistic Model for Amorphous Protein Aggregation of Immunoglobulin-like Domains

Abstract

Protein aggregation is associated with many debilitating diseases including Alzheimer’s, Parkinson’s, and light-chain amyloidosis (AL). Additionally, such aggregation is a major problem in an industrial setting where antibody therapeutics often require high local concentrations of protein domains to be stable for substantial periods of time. However, despite a plethora of research in this field, dating back over 50 years, there is still no consensus on the mechanistic basis for protein aggregation. Here we use experimental data to derive a mechanistic model that well describes the aggregation of Titin I27, an immunoglobulin-like domain. Importantly, we find that models that are suitable for nucleated fibril formation do not fit our aggregation data. Instead, we show that aggregation proceeds via the addition of activated dimers, and that the rate of aggregation is dependent on the surface area of the aggregate. Moreover, we suggest that the “lag time” seen in these studies is not the time needed for a nucleation event to occur, but rather it is the time taken for the concentration of activated dimers to cross a particular solubility limit. These findings are reminiscent of the Finke–Watzky aggregation mechanism, originally based on nanocluster formation and suggest that amorphous aggregation processes may require mechanistic schemes that are substantially different from those of linear fibril formation.

Free energy landscapes for initiation and branching of protein aggregation

“…The mutant dimer and trimer constructs maintain marginal stability. Our result for the wild-type construct is consistent with the aggregation experiments on I27 under denaturing conditions [28% Trifluoroethanol (TFE)] done by Wright et al (12) and Borgia et al (14), in which the number of monomers involved in the critical step for initial aggregation was inferred to be 2.…”

“…The tetramer appears to be a critical size for the mutant construct insofar as the relative population of the misfolded state increases abruptly when going from I27 Ordered vs. Amorphous Aggregation. Borgia et al (14) note that no fibers were observed during the aggregation of I27. Instead, the aggregate that does form remains amorphous over several months, and the kinetic data can be fitted well using a model wherein the rate of aggregation is proportional to the surface area of the aggregate.…”

“…In aggregation experiments, the structural and thermodynamic details of the transiently populated species that seed aggregation are hidden beneath a cascade of events. Aggregation experiments that monitor the concentration of macroscopic aggregates in bulk (12,14) typically observe a lag phase and then a fast growth phase after aggregation has been seeded. The size of the critical initiating species can be roughly inferred by repeating these types of experiments using constructs with a variable number of domains, but the wide range of time and spatial scales involved makes these inferences nontrivial.…”

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

“…The best studied of these conditions include amyotrophic lateral sclerosis (superoxide dismutase), Parkinson's disease (α-synuclein), serpinopathies (α1-antitrypsin), cancers with P53 and P21 tumor suppressor defects, and lens cataract (crystallins) (1)(2)(3)(4)(5). Some of these aggregates contain the well-known amyloid structure, and others do not; even in cases where amyloid is the final aggregated state, oligomers, prefibrillar species, and amorphous aggregates are often closely linked to pathology (6,7). Structures and interactions of specific locally unfolded or misfolded intermediate states are critically important in the mechanisms of non-amyloid aggregation (1).…”

Mechanism of initiation of aggregation of p53 revealed by Φ-value analysis

“…Generally, however, especially at physiological temperature and pH, p53 forms amorphous aggregates, which still contain the β-structural elements responsible for ThT binding and other properties (28,29). Amorphous aggregates are very commonly found, but there are few systematic studies of their mechanism of formation (30). We are analyzing the mechanism of amorphous aggregation of p53 in its own right, as well as to aid in the design of inhibitors (31).…”