Single molecule studies of protein binding and aggregation

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The information of detailed molecular mechanisms of IDP binding is contained in binding pathways (binding transition paths) that are supposed to be heterogeneous as observed in molecular dynamics (MD) simulations. To probe and analyze diverse binding pathways, we have developed and used three-color FRET. By attaching two dyes to an IDP and the third dye to a binding partner, it is possible to detect conformational changes of the IDP and the interaction with the binding partner at the same time. Recently, we have developed fast three-color FRET in collaboration with Dr. Irina V. Gopich at LCP, which determines binding kinetics and all three FRET efficiencies between fluorophores. We have applied this method to the investigation of diffusion-limited binding of an IDP, the N-terminal transactivation domain (TAD) of p53, and one of its binding partners, the nuclear coactivator binding domain (NCBD) of CBP. We have demonstrated that binding pathways are highly heterogeneous. About half of the transitions follow a path involving strong non-native electrostatic interactions, resulting in a long transition time of several hundred microseconds. The remaining half follow more diverse paths characterized by weaker electrostatic interactions and much shorter transition path times. The chain flexibility and non-native interactions make diverse binding pathways possible, allowing disordered proteins to bind faster than folded proteins, whereas nonnative interactions slow the folding rate in protein folding. We are investigating binding of TAD with other binding partners with different binding kinetics to obtain a more general mechanistic picture of binding of this IDP. We have also developed various single-molecule fluorescence techniques including single-molecule FRET and fluorescence lifetime imaging and quantitative analysis methods based on deep-learning for studying oligomerization and aggregation of amyloid-beta (A-beta) that is associated with Alzheimers disease. We found that the concentration of oligomers, including dimers, is extremely low. Aggregation to form fibrils is highly heterogeneous in terms of the number of strands in a fibril, the elongation speed, and conformation of fibrils. This heterogeneity explains diverse results of experimental studies of amyloid-beta.