Cross-linking and SDS-PAGE analysis. Aβ peptides were covalently cross-linked using the technique of cross-linking of unmodified proteins (PICUP), as described 31 . Briefly, 1 μL of 40 mM ammonium persulfate (APS) and 1 μL of 2 mM Tris(2,2′- bipyridyl)dichlororuthenium(II)) were added to 18 μL of 40 μM Aβ. The mixture was irradiated for 1s with visible light (Dolan-Jenner Industries Fiber-Lite Model 170-D, Boxborough, MA) and the reaction was quenched immediately with 1 μL of 1M dithiothreitol in H2O. Tricine SDS Sample Buffer (2X) (Invitrogen, Carlsbad, CA) was then added to the solution. Five µL of each cross-linked Aβ was analyzed by SDS-PAGE using 10–20% Tricine gels (1.0 mm × 12 well) and subsequently silver stained following the Silver Xpress Silver Staining Protocol (Invitrogen). Gels were dried for one hour using Novex DryEase Mini cellophane in Novex drying frames and then scanned with a Canon CanoScan 9950F flatbed scanner at 300 dpi or greater. Densitometry was performed using ImageJ 1.50d (http://imagej.nih.gov/ij), which we used to determine the normalized intensity of each band ; where Ii is the intensity of band i and is the sum of all band intensities. Three independent experiments were performed with each peptide.
Thioflavin T fluorescence. Aβ42 and its variants were initially dissolved in ~1 mL of 4 °C hexaflouro-2-propanol (HFIP), sonicated 5 min to completely dissolve the Aβ, incubated for 30 min incubation at RT, and aliquoted into a 1.5 mL low retention microcentrifuge tube (FisherBrand). The HFIP was completely evaporated overnight in a chemical fume hood. The following day, the tubes were rotary evaporated for 1 h in a SpeedVac concentrator (Thermo Scientific, Savant SPD121P), which ensured complete removal of the HFIP. The peptides within the films then were prepared for use as described above for peptide lyophilizates.
Immediately after sample preparation, 100 μL aliquots of each of the peptides (40 μM Aβ40, 20 μM Aβ42) were mixed with 100 μL of 120 μM ThT in 10 mM sodium phosphate, pH 7.4 in wells of a Thermo Scientific™ Nunc™ Microwell™ 96-Well optical-bottom plate. The plate was sealed with SealPlate film (Excel Scientific) incubated at 37°C with shaking at 160 rpm. ThT fluorescence then was measured using a Hitachi F4500 fluorometer (Hitachi Instruments Inc., Rye, NH) with excitation and emission filters set to 450 nm and 480 nm, respectively. Slit-widths for excitation and emission were 5 and 10 nm, respectively. Three independent experiments were performed for each peptide. The time at which half-maximal ThT intensity (t1/2) was observed was determined by calculating the difference between the final and initial fluorescence levels, visually locating that value on a smooth fit of the graph of the time-dependence of fluorescence intensity, and then identifying the corresponding time.
Circular dichroism spectroscopy (CD) . Peptides were prepared as described above for the ThT experiments. Spectra were acquired periodically during incubation of the peptides at 37°C, without agitation, in 0.1 cm path-length quartz cuvettes (Hellma, Forest Hills, NY). Spectra were acquired using a Jasco Model J-810 spectropolarimeter (Jasco corporation, Japan) over a wavelength range of ≈190–260 nm. Parameters for measurements were: standard sensitivity, data pitch=0.2 nm, scanning speed=100 nm/min, and bandwidth=1 nm. 10 spectra were taken per sample. Three independent experiments were performed with each peptide.
Transmission electron microscopy (TEM). Aliquots of 8 µL volume were removed periodically from assembly reactions being monitored by CD. Each aliquot was applied to 400 mesh carbon-coated formvar EM grids (Electron Microscopy Sciences, Hatfield, PA). Each grid was incubated for 2 min at RT. The liquid was then wicked off by gently bringing the tip of 55 mm diameter, #2 filter paper (Whatman) to the edge of the grid. Subsequently 8 μL of 1% (w/v) uranyl acetate in MilliQ water was applied to the grid and immediately wicked off. A JEOL 1200 EX transmission electron microscope was used to visualize the sample morphologies.