Measuring the Size and Spontaneous Fluctuations of Amyloid Aggregates with Fluorescence Correlation Spectroscopy.

Bacterial amyloids decorate the cell surface of many bacteria by forming functional amyloid fibers. These amyloids have structural and biochemical similarities with many disease-related amyloids in eukaryotes. Amyloid aggregation starts at the individual monomer level, and the end product is the amyloid fibril. The process of amyloid aggregation involves a continuous increase of the aggregate size, and therefore size is a critical parameter to measure in aggregation experiments. Also, our understanding of the aggregation process, and our ability to design interventions, can benefit from a measurement of the conformational dynamics of proteins undergoing aggregation. Fluorescence correlation spectroscopy (FCS) is perhaps the most sensitive and rapid technique available currently for this purpose. It can measure the average size and the size distribution of molecules and aggregates down to sub-nm length scales and can also measure fast nanosecond time-scale conformational dynamics, all in an equilibrium solution. FCS achieves this by measuring the fluorescence intensity fluctuations of freely diffusing protein molecules in an optically defined microscopic probe volume in a solution. Here, we present a set of instructions for effectively measuring the size and dynamics of amyloid aggregates with FCS.