Structure and intermolecular dynamics of aggregates populated during amyloid fibril formation studied by...

The aggregation of proteins into amyloid fibrils is a complex and fascinating process associated with debilitating clinical disorders such as Alzheimer's and Parkinson's diseases. The process of aggregation involves a series of steps during which many intermediate aggregation states are populated. Recent evidence points to these intermediate states as the toxic moieties primarily responsible for cell damage or cell death, which are critical steps in the origin and progression of these disorders. To understand the molecular basis of these diseases, it is crucial to investigate and define the details of the aggregation process, and to achieve this objective, researchers need the tools to characterize the structure and kinetics of interconversion of the various species present during amyloid fibril formation. Hydrogen-deuterium (HD) exchange experiments are based on solvent accessibilities and provide one means by which this kind of information may be acquired. In this Account, we describe research based on HD exchange processes that is directed toward better understanding the dynamics and structural reorganizations involved in the formation of amyloid fibrils. Amide hydrogens that normally undergo rapid exchange with solvent hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent. The rates of exchange can be monitored by replacing some hydrogens with deuterons. When peptide and protein molecules assemble into amyloid fibrils, the fibrils contain a core region based on repetitive arrays of beta-sheets oriented parallel to the fibril axis. HD experiments have been applied extensively to map such structures in different amyloid fibril systems. By an extension of this approach, we have observed that HD exchange can be governed by a mechanism through which molecules making up the fibrils are continuously dissolving and reforming, revealing that amyloid fibrils are not static but dynamic structures. Under such circumstances, the kinetic parameters that define this "recycling" behavior can be determined, and they contain information that could be of significant value in the design of therapeutic strategies directed against amyloid-related diseases. More recently, to gain insights into the variety of intermediates that are thought to be involved in the aggregation process, we have applied a kinetic pulse labeling HD experiment that is able to characterize such species even if they are only transiently populated. Using this approach, we have been able to obtain structural insights into the different aggregates populated during the process of amyloid fibril formation as well as kinetic and mechanistic information on the structural reorganizations that take place during aggregation. HD exchange experiments, when carefully designed, constitute powerful tools for mapping the core structures of amyloid fibrils, for investigating the recycling of fibril components, and for characterizing the various types of structural reorganization that occur during aggregation. Such information is invaluable for understanding and addressing the molecular origins of the increasingly common and highly debilitating diseases associated with protein misfolding and aggregation.