Dynamical Observations of Brownian Motions on Single Protein Molecules Using Diffracted Electron Tracking (DET)

Abstract

Recent evidence suggests that soluble amyloid-b (Ab) oligomers as small as dimers may be linked to the progression of Alzheimer's disease. We have used single-pair FRET measurements to investigate heterogeneity in surfacetethered dimers of Ab40, probing for preferred structures. Dimers are prepared by combining monomers singly labeled with donor and acceptor dyes; dimers prepared in solution (prior to surface-tethering) and on the functionalized surface have been examined. Donor and acceptor fluorescence are separated onto two detectors, such that co-localized spots in two-color images are indicative of at least two associated peptides. Dimers are verified based on the observation of single-step photobleaching in each detection channel; larger oligomers are excluded from analysis. By measuring donor and acceptor fluorescence as a function of time, we have determined time-dependent FRET efficiencies for dozens of individual dimers, permitting insight into inter-dye distances and dimer structures. These results are further complemented by comparison to published structures of simulated Ab40 dimers. Together, experiment and simulation may reveal a subset of preferred structures for Ab40 dimers.