Single molecule studies of protein binding and aggregation

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

Protein aggregation is implicated as the cause of pathology in various diseases such as Alzheimers and Parkinsons disease. Polymorphism in the structure of fibrils formed by aggregation suggests the existence of many different assembly pathways and therefore a heterogeneous ensemble of soluble oligomers. Characterization of this heterogeneity is the key to understanding the aggregation mechanism and toxicity of specific oligomers, but in practice it is extremely difficult to probe individual aggregation pathways in a mixture. We have 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 (Abeta) that is associated with Alzheimers disease. The individual fibril analysis allows for processing a large amount of fibril data, which has led to quantitative description of the fibril heterogeneity in terms of the number of strands in a fibril, the elongation speed, and conformation of fibrils. This heterogeneity explains diverse experimental results involving amyloid-. We also found a new nucleation mechanism, which we call heterogeneous secondary nucleation, from the observation that fibrils can grow from oligomers or protofibrils with different structures. This mechanism describes possible nucleation and aggregation pathways of Abeta at very low concentration in vivo. We carried out a single-molecule FRET experiment to detect stable Abeta oligomers that appear during the aggregation process. However, quantitative analysis was difficult because of their extremely low concentration, structural heterogeneity, and a broad range of oligomer size. We have developed a molecular diffusion analysis method of diffusion-based single molecule FRET data in collaboration with Dr. Irina V. Gopich at LCP. This method can accurately determine the brightness, diffusion time, FRET efficiency, and population of oligomer species in solution. We have applied this method to the oligomer formation of 42-residue Abeta and a mixture of 40- and 42-residue Abeta. In collaboration with Dr. Jinwei Zhangs group in LMB, we have studied conformational dynamics and interactions of noncoding RNAs using two- and three-color single-molecule FRET spectroscopy. We found that mRNA riboswitch has a broad conformational distribution, all of which can bind tRNA in an induced fit manner. Using a maximum likelihood analysis of single-molecule fluorescence trajectories, we could extract complex kinetic model parameters.