Using FCS to Study Protein Disorder and Aggregation

Abstract

Fluorescence correlation spectroscopy (FCS) is powerful approach for studying the dynamic properties of fluorescently labeled biomolecules including diffusion, binding, and conformational dynamics. We have applied FCS to the study of membrane binding, dynamics, and aggregation of two intrinsically disordered proteins. The first, α-synuclein, is a small, neuronal proteins whose aggregation is implicated in Parkinson’s disease. Its native functions are poorly understood, but are thought to involve binding to cellular membranes. Here we have measured the free energy of binding of α-Synuclein to lipid membranes using FCS. Our results show that the binding energy can be strongly altered by changes to the bilayer curvature and composition, suggesting that even modest changes to lipid components in vivo may be able to exert significant effects on α-Synuclein function. Furthermore, modifications to the C-terminus of α-Synuclein, which does not directly interact with the lipid bilayer but exerts control of the aggregation of the protein, is also able to modulate binding interactions. Measurements of intrachain dynamics indicate that modifications alter the flexibility of the C-terminus as well as the conformational ensemble populated by the protein. The second protein, tau, is a microtubule binding protein that is found in aggregated paired-helical filaments in Alzheimer’s and other diseases. We have characterized the lipid-induced aggregation of tau by FCS. We find that aggregation occurs when the amount of protein bound to the lipid bilayer exceeds a critical surface density. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein as well as by increasing the local protein concentration, resulting in rapid aggregation. Our work highlights the versatility of FCS in studying this important class of proteins.