β-Hairpin Alignment Alters Oligomer Formation in Aβ-Derived Peptides

Amyloid-β (Aβ) forms heterogeneous oligomers, which are implicated in the pathogenesis of Alzheimer’s disease (AD). Many Aβ oligomers consist of β-hairpin building blocks—Aβ peptides in β-hairpin conformations. β-Hairpins of Aβ can adopt a variety of alignments, but the role that β-hairpin alignment plays in the formation and heterogeneity of Aβ oligomers is poorly understood. To explore the effect of β-hairpin alignment on the oligomerization of Aβ peptides, we designed and studied two model peptides with two different β-hairpin alignments. Peptides Aβm17–36 and Aβm17–35 mimic two different β-hairpins that Aβ can form, the Aβ17–36 and Aβ17–35 β-hairpins, respectively. These hairpins are similar in composition but differ in hairpin alignment, altering the facial arrangements of the side chains of the residues that they contain. X-ray crystallography and SDS-PAGE demonstrate that the difference in facial arrangement between these peptides leads to distinct oligomer formation. In the crystal state, Aβm17–36 forms triangular trimers that further assemble to form hexamers, while Aβm17–35 forms tetrameric β-barrels. In SDS-PAGE, Aβm17–36 assembles to form a ladder of oligomers, while Aβm17–35 either assembles to form a dimer or does not assemble at all. The differences in the behavior of Aβm17–36 and Aβm17–35 suggest β-hairpin alignment as a source of the observed heterogeneity of Aβ oligomers.

Materials and Methods 1

General information
All chemicals were used as received unless otherwise noted.All Fmoc-protected amino acids, coupling agents, triisopropylsilane (TIPS), and 2-chlorotrityl chloride resin was purchased from Chem-Impex.HPLC grad acetonitrile (MeCN) was purchased from Fischer Scientific.
Methylene chloride (DCM) was passed through alumina under nitrogen prior to use. was added immediately and the suspension was gently agitated for 12 h.The solution was then drained and a mixture of DCM/MeOH/N,N-diisopropylethylamine (DIPEA) (17:2:1, 6 mL) was added immediately.The mixture was gently agitated for 1 h to cap the unreacted 2-chlorotrityl chloride resin sites.The resin was then washed with dry DCM (2x) and dried by passing nitrogen through the vessel.This procedure typically yields 0.12-0.18mmol of loaded resin (0.4-0.6 mmol/g loading).
Peptide coupling.The Boc-Orn(Fmoc)-2-chlorotrityl resin generated from the previous step was transferred to a solid phase reaction vessel and washed with DMF (5x).The linear peptide was synthesized from the C-terminus to the N-terminus.Each coupling cycle consisted of i. Fmoc-deprotection with 20% (v/v) piperidine in DMF for 5 min, ii.washing with DMF (5x), iii. coupling of the amino acid (0.75 mmol, 5 equiv) in the presence of HCTU (0.675 mmol, 4.5 equiv) and 20% (v/v) 2,4,6-collidine in DMF for 20 min iv.washing with DMF (5x).Special coupling conditions were used for the isoleucine that followed the N-methyl-glycine in Aβm 17-36 and for the isoleucine that followed the N-methyl-isoleucine in Aβm 17-35 : The isoleucine was double coupled (0.75 mmol, 5 equiv.)and allowed to react for 1 h per coupling with HATU (5 equiv) and HOAt (5 equiv) in 20% (v/v) 2,4,6-collidine in DMF.After coupling of the last amino acid, the terminal Fmoc group was removed with 20% (v/v) piperidine in DMF (5 min).The resin was transferred from the reaction vessel to a Bio-Rad Poly-Prep chromatography column.
Cleavage of the peptide from the resin.The linear peptide was cleaved from the resin by agitating the resin for 45 min with a solution of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in DCM (1:4, 8 mL).The suspension was filtered and the filtrate was collected in a 250-mL roundbottomed flask.An additional 8 ml of the HFIP cleavage cocktail was added to the resin and agitated for 30 min.The solution was then filtered into the same flask.The combined filtrates were concentrated by rotary evaporation to give a white solid, which was cyclized without further purification.
Cyclization of the linear peptide.The crude protected linear peptide was dissolved in dry DMF (150 mL).HOBt (114 mg, 0.75 mmol, 5 equiv) and HBTU (317 mg, 0.75 mmol, 5 equiv) were added to the solution.DIPEA (0.33 mL, 1.8 mmol, 12 equiv) was added to the solution and the mixture was stirred under nitrogen for 48 h.The mixture was by rotary evaporation to afford the crude protected cyclic peptide.Cyclized peptides were further dried by vacuum pump.
Global deprotection and Ether Precipitations.The protected cyclic peptide was dissolved in TFA/triisopropylsilane (TIPS)/H 2 O (18:1:1, 20 mL) in a 250-mL round-bottomed flask equipped with a nitrogen-inlet adaptor.The solution was stirred for 1.5 h.The reaction mixture was then separated evenly into two 50 ml conical tubes.30 ml of ice-cold diethyl ether was added to each tube and left to sit on ice for 15 minutes.The 50-ml conical tubes were centrifuged (2000 x g) for 10 min to pellet the crude peptide.The supernatant was removed, and the pellets were redissolved in 5 ml of MeCN.The MeCN peptide solutions were combined in a 250 ml round-bottom flask concentrated by rotary evaporation to afford the crude cyclic peptide.The crude cyclic peptide was immediately subjected to purification by reverse-phase HPLC (RP-HPLC), as described below.
Reverse-phase HPLC purification.(v/v) acetic acid in deionized water) for 20 min.Next, the fixing solution was discarded and the gel was rocked in 50% (v/v) aqueous MeOH for 10 min.Next, the 50% methanol was discarded and the gel was rocked in deionized water for 10 min.Next, the water was discarded and the gel was rocked in 0.02% (w/v) sodium thiosulfate in deionized water for 1 min.The sodium thiosulfate was discarded and the gel was rinsed with deionized water for 1 min (2X).After the last rinse, the gel was submerged in chilled 0.1% (w/v) silver nitrate in deionized water and rocked at 4 °C for 20 min.Next, the silver nitrate solution was discarded and the gel was rinsed with deionized water for 1 min (2X).To develop the gel, the gel was incubated in developing solution (2% (w/v) sodium carbonate, 0.04% (w/v) formaldehyde until the desired intensity of staining was reached (~1-3 min).When the desired intensity of staining was reached, the development was stopped by discarding the developing solution and submerging the gel in 5% aqueous acetic acid.

Crystallization conditions for Aβm 17-36 and Aβm 17-35
Crystallization conditions for Aβm 17-36 were determined using a 4x6 matrix Hampton VDX 24-well plate.The conditions mimicked the optimization screen previously used for a homologue of Aβm 17-36 that contained ornithine in place of Met 35 (PDB 4NTR) which previously crystallized in HEPES with Jeffamine M-600. 4 The HEPES buffer pH was varied in each row in increments of 0.5 pH units (6.5, 7.0, 7.5, and 8.0) and the Jeffamine concentration in each column in increments of 2% (24%, 26%, 28%, 30%, 32%, 34%).The first well in the 4x6 matrix for Aβm 17-36 was prepared by combined 100 μL of 1 M HEPES buffer at pH 6.5, 480 μL of 50% v/v aqueous Jeffamine M-600 pH 7.0, and 420 μL of deionized water.The other wells were prepared in analogous fashion, by combining 100 μL of HEPES buffer of varying pH, Jeffamine in varying amounts, and deionized water for a total volume of 1 mL in each well.

Synthesis of Aβm 17-36 and Aβm 17-35
NANOpure Diamond water purification system.Analytical reverse-phase HPLC was performed on an Agilent 1260 instrument equipped with a Phenomonex Aeris PEPTIDE 2.6 μm XB-C18 150 mm x 4.6 mm column.Preparative reverse-phase HPLC was performed on a Rainin Dynamax instrument equipped with an Agilent Zorbax 300SB-C18 21.2 mm x 250 mm (7 μm) column.Peptides were first purified on a Biotage® Isolera™ One system equipped with a 25 g Biotage® Sfär Bio C18 -Duo 300 Å 20 μm column, before repurification on the Rainin Dynamax.UV detection (214 nm) was used for analytical and preparative HPLC.HPLC grade The peptide was dissolved in H 2 O and MeCN (4:1, 10 mL), and purified on a Biotage® Isolera™ One system equipped with a 25 g Biotage® Sfär Bio Aβm 17-36 and Aβm 17-35 was compared with a molecular weight protein ladder (SpectraTM Multicolor Low Range Protein Ladder, ThermoFisher Scientific, catalog #: 26628).Staining with silver nitrate was used to visualize Aβm 17-36 and Aβm 17-35 in the SDS- 218 -Duo 300 Å 20 μm column using a MeCN (15%-45%) gradient in H 2 O. Fractions were analyzed by MALDI-TOF and analytical HPLC.Fractions containing the desired peptide were combined in a 250 ml round-bottom flask and concentrated by rotary evaporation.The peptide was then redissolved in H 2 O and MeCN (4:1, 10 mL), filtered through a 0.2 μm syringe filter, and purified by RP-HPLC on a Rainin Dynamax instrument equipped with an Agilent Zorbax 300SB-C18 21.2 mm x 250 mm (7 μm) column (gradient elution with 20-45% MeCN in H 2 O over 70 min).Fractions were analyzed by MALDI-TOF and analytical HPLC.Pure fractions were concentrated by rotary evaporation and lyophilized.Typical syntheses yielded ~30 mg of the peptide as the TFA salt.SDS-PAGE and silver stainingSolutions of Aβm 17-36 and Aβm 17-35 were prepared gravimetrically by dissolving lyophilized peptide in the appropriate amount of 18 MΩ deionized water to achieve a 10 mg/ml stock.Stock solutions of all peptides were diluted with 18 MΩ deionized water to create 400 μM, 200 μM, and 100 μM sample solutions.1 μl of 6X SDS-PAGE sample loading buffer (G 2 Reagents for Tricine SDS-PAGE were prepared and used according to recipes and procedures detailed in the Mini-PROTEAN Precast Gels Instruction Manual and Application Guide (2011) from Bio-Rad Laboratories.2Thegel was run at a constant 100 V for approximately 2 hours.The migration of Three hanging-drops were prepared per borosilicate glass slide by combining a solution of peptide 2 or peptide 4 (10 mg/mL in deionized water) and the well solution in the following amounts: 1 μL:1 μL, 2 μL:1 μL, and 1 μL:2 μL.Crystallization conditions for Aβm 17-36 were further optimized using the same method.The HEPES buffer pH was varied in each row in Molecular Dynamics Morpheus, MemPlus/MemSys, PGA).Three 150 nL hanging drops that differed in the ratio of peptide to well solution were made per condition in each 96-well plate for a total of 1728 experiments.Hanging drops were made by combining an appropriate volume of Aβm 17-36 or Aβm 17-35 (10 mg/mL in deionized water) with an appropriate volume of well solution to create three 150 nL hanging drops with 1:1, 1:2, and 2:1 peptide:well solution.The hanging drops were made using a TTP LabTech Mosquito nanodisperse instrument.Crystals of