Abstract
The data here consists of time-dependent experimental parameters from chemical and biophysical methods used to characterize Aβ monomeric reactants as well as soluble oligomer and amyloid fibril products from a slow (3–4 week) assembly reaction under biologically-relevant solvent conditions. The data of this reaction are both of a qualitative and quantitative nature, including gel images from chemical cross-linking and Western blots, fractional solubility, thioflavin T binding, size exclusion chromatograms, transmission electron microscopy images, circular dichroism spectra, and fluorescence resonance energy transfer efficiencies of donor–acceptor pair labels in the Aβ chain. This data enables future efforts to produce the initial monomer and eventual soluble oligomer and amyloid fibril states by providing reference benchmarks of these states pertaining to physical properties (solubility), ligand-binding (thioflavin T binding), mesoscopic structure (electron microscopy, size exclusion chromatography, cross-linking products, SDS and native gels) and molecular structure (circular dichroism, FRET donor-acceptor distance).Aβ1-40 soluble oligomers are produced that are suitable for biophysical studies requiring sufficient transient stability to exist in their “native” conformation in biological phosphate-saline buffers for extended periods of time. The production involves an initial preparation of highly monomeric Aβ in a phosphate saline buffer that transitions to fibrils and oligomers through time incubation alone, without added detergents or non-aqueous chemicals. This criteria ensures that the only difference between initial monomeric Aβ reactant and subsequent Aβ oligomer products is their degree of peptide assembly. A number of chemical and biophysical methods were used to characterize the monomeric reactants and soluble oligomer and amyloid fibril products, including chemical cross-linking, Western blots, fraction solubility, thioflvain T binding, size exclusion chromatography, transmission electron micrscopy, circular dichroism spectroscopy, and fluorescence resonance energy transfer.
Highlights
The data here consists of time-dependent experimental parameters from chemical and biophysical methods used to characterize Aβ monomeric reactants as well as soluble oligomer and amyloid fibril products from a slow (3–4 week) assembly reaction under biologicallyrelevant solvent conditions
The production involves an initial preparation of highly monomeric Aβ in a phosphate saline buffer that transitions to fibrils and oligomers through time incubation alone, without added detergents or non-aqueous chemicals
This criteria ensures that the only difference between initial monomeric Aβ reactant and subsequent Aβ oligomer products is their degree of peptide assembly
Summary
Size-exclusion chromatography (SEC) was performed with a Sephacryl S-200HR column (GE Life Sciences) with a Pharmacia peristaltic pump and Gilson Fraction collector. Absorbance spectra were acquired on a Cary 100 spectrophotometer (Cary). Steady-state fluorescence measurements were acquired on a PTI Quanta Master Steady-State fluorimeter (Photon Techonologies Inc). Time-resolved fluorescence measurements were acquired on a PTI EasyLife equipped with a 295 nm LED excitation source and 395 cut-on emission filter. CD measurements were acquired on a Chirascan spectropolarimeter (Applied Photophysics). Electron microscopy was performed using a Morgagni 268 Transmission Electron Microscope (TEM) equipped with a Hamamatsu digital camera. A subtracted spectra of B-C shows an estimated spectrum of insoluble Aβ on Day 42 (D, ————————). (d) FRET efficiency E between the aminobenzamide donor at residue 35 and nitrotyrosine acceptor at position 10 of FRET –labeled Aβ peptides in soluble states (MAβ and nOAβ) over the course of 365 days. The FRET-labeled Aβ was 2 μM (4%) of the total 50 μM Aβ sample, a dilution sufficient to observe only intramolecular FRET and no intermolecular FRET
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