Molecular insights into the surface-catalyzed secondary nucleation of amyloid-β40 (Aβ40) by the peptide fragment Aβ16–22

Combined experiment and simulation reveals a structural mechanism of surface-catalyzed nucleation in Aβ amyloid formation.


General materials and methods for organic synthesis
Non-aqueous reactions were carried out in washed and oven-dried glassware. Solvents and reagents were used as received from major suppliers without prior purification unless stated. Anhydrous tetrahydrofuran (THF), ethanol (EtOH), acetonitrile (MeCN), dichloromethane (DCM) and diethyl ether (Et 2 O) were obtained from the in-house solvent purification system from Innovative Technology Inc. PureSolv®. Anhydrous dimethyl formamide (DMF), methanol (MeOH) and chloroform (CHCl 3 ) were obtained from major chemical suppliers equipped with a SureSeal™ (or equivalent). For reactions under non-anhydrous conditions, the solvents used were of HPLC quality and provided by Fisher or Sigma-Aldrich. Water in aqueous solutions and used for quenching was deionized, and water used for buffers and HPLC was ultra-pure 18 MΩ from an ELGA Purelab system. 1 H NMR spectra were recorded on Bruker DPX 300 (300 MHz) or Avance 500 (500 MHz) spectrometers and referenced to either residual non-deuterated solvent peaks or tetramethylsilane. 1 H spectra are reported as follows: δ H (spectrometer frequency, solvent): ppm to two decimal places (number of protons, multiplicity, J coupling constant in hertz, assignment). Chemical shifts are quoted in ppm with signal splitting recorded as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quin.) multiplet (m), broad (br) and apparent (app.). Coupling constants, J, are measured to the nearest 0.1 Hz.

Synthesis of N-Fmoc-protected TFMD-Phe
The synthesis of TFMD-Phe was carried out according to the literature procedure originally developed by Fishwick and co-workers and improved upon by Smith and co-workers. 5,6 A change in protecting group has been made from the original Smith and co-workers synthesis. All synthetic products have been analysed by at least 1 H NMR to confirm the identity and purity of each compound. Principally, 1 H NMR, FTIR and LC-MS analysis were used. All data has been consistent with those previously reported within the group and in the literature. 6,7 Scheme S1. Synthesis of TFMD-Phe.
After cleavage from the resin using TFA:TIPS:H 2 O (98:1:1, 3 mL, 2 hr) the crude product was precipitated using ice cold Et 2 O (50 mL) and the supernatant removed. The crude peptides identity was confirmed by LC-MS prior to HPLC purification.

General materials and methods for HPLC purification
Peptides were purified by preparative scale HPLC using an X-bridge C18 preparative column (reversed phase) on an increasing gradient of MeCN to H 2 O. Crude peptides were dissolved in DMSO and injected into the column in 300 μL aquilots. The solvent gradient (5 -95% MeCN in H 2 O + 0.1% formic acid) was increased linearly over a 15 min run time at a flow rate of 10 mL/min -1 . The fixed-wavelength detector was set to scan the eluent at either 220, 254 or 270 nm with peak-based collection for 30 s after the diode was triggered. Fractions were analyzed by LC-MS and fractions containing the desired peptide were pooled, concentrated under reduced pressure and lyophilized. The purity of the peptide samples was determined by the School of Chemistry HPLC service. Identity was confirmed by mass using either the LC-MS or HRMS.

General materials and methods for recombinant peptide synthesis
Commercial E. coli strain BL21 (DE3) cells (Agilent) were transformed with a pETSAC plasmid containing the sequences for Aβ 40 (a gift from Profs. Sara Linse and Dominic Walsh) , including an N-terminal methionine residue that has no effect on the fibrillation of Aβ 40 or the morphology of fibrils formed.(44) Cultures were grown in LB media and Aβ 40 purified as described previously.(45) Final protein concentrations were estimated from UV absorption in 6 M guanidinium hydrochloride at 280 nm using an extinction coefficient of 1490 M -1 cm -1 , and averaged 4 mg pure peptide/L culture. HRMS was used to confirm the identity of the final product (expected molecular weight 4459.21 Da). The size exclusion chromatography trace from the second purification step (in 50 mM ammonium bicarbonate) is also shown. Fraction 11 and 12 were pooled and lyophilised prior to use.

Fig. S2. SEC trace of Aβ 40 indicates that there is a single peak, and ESI-IMS-MS
indicates that in the gas phase Aβ 40 is largely monomeric. The observed m/z ratio is stated above each peak.