Single-molecule assays for investigating protein misfolding and aggregation

Protein misfolding and aggregation are relevant to many fields. Recently, their investigation has experienced a revival as a central topic in the research of numerous human diseases, including Parkinson's and Alzheimer's. Much has been learned from ensemble biochemical approaches, but the inherently heterogeneous nature of the underlying processes has obscured many important details. Single-molecule techniques offer unique capabilities to study heterogeneous systems, while providing high temporal and structural resolution to characterize them. In this Perspective, we give an overview of the single-molecule assays that have been applied to protein misfolding and aggregation, which are mainly based on fluorescence and force spectroscopy. We describe some of the technical challenges involved in studying aggregation at the single-molecule level and discuss what has been learned about aggregation mechanisms from the different approaches.