Single molecule studies of protein binding and aggregation

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Protein aggregation is implicated as the cause of pathology in various diseases such as Alzheimer’s and Parkinson’s disease. It is a complex process involving many different components to form amyloid fibrils. Polymorphism in amyloid fibril structure implies highly diverse aggregation pathways involving a variety of oligomeric intermediates, which are thought to be the primary cause of disease. The heterogeneity of the process makes its characterization extremely difficult. To investigate the heterogeneity of the aggregation process and the resultant fibril polymorphism of amyloid beta 42 peptide (Ab42), associated with Alzheimer’s disease, we employ Förster resonance energy transfer (FRET) imaging with pulsed interleaved excitation (PIE) and use a mixture of Alexa 488-labeled (donor), Alexa 594-labeled (acceptor), and unlabeled Ab42 peptide monomers. FRET images report on the relative positions of donor- and acceptor-labeled species within the fibrils. Fibrils with unique FRET efficiencies will have unique arrangements of monomers within the fibril. Our study demonstrates the influence of initial concentration of Ab42 monomers on the ultimate structure of the Ab42 fibrils. We find that lower initial monomer concentration promotes the assembly of fibril polymorphs with two markedly different FRET efficiencies. The pathways to forming each of these fibril polymorphs have distinct kinetics. Higher initial monomer concentration primarily promotes a quick-to-assemble high FRET species similar to the fastest forming species at lower concentration. The distinct heterogeneity in the fibril formation pathways depending on the monomer concentration highlights the importance of understanding heterogeneity in the context of biologically relevant aggregation environment. In collaboration with Dr. Irina Gopich in LCP, we have continued to develop quantitative analysis methods to accurately characterize diffusion-based single-molecule fluorescence experimental data. These methods rigorously treat molecular diffusion to avoid fluorescence burst selection bias resulting from the different brightness and diffusivity of molecular species. We have extended the previous method, which was applicable to a multi-component static system, to systems with state exchange in two- and three-color settings. The two-color study was published in The Journal of Physical Chemistry B and the three-color study was published in Biophysical Journal.