De novo coiled-coil peptides as scaffolds for disrupting protein–protein interactions† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc02643b

Homo- and hetero-dimeric coiled coils as scaffolds for the presentation of α-helical protein-binding motifs.


Synthesis of wt NOXA-B
Fmoc-protected amino acids were purchased from Novabiochem (Merck), or Sigma-Aldrich. The peptide was prepared using a microwave-assisted automated peptide synthesiser (Liberty Blue; CEM) on Rink amide MBHA resin (0.057 mmol) using standard Oxyma/DIC chemistry with systematically repeated steps of coupling and deprotection (20% piperidine in DMF) interspaced with washing (note: The Arg6 residue was double-coupled). Once assembly was complete, the resin-attached peptide was N-terminally acetylated (acetic anhydride (10 eq.), DIPEA (10 eq.) in DMF for 2 h) before being washed and dried. The peptide was then cleaved from the resin with simultaneous removal of side-chain protection by treatment with a cocktail of trifluoroacetic acid/H2O/triisopropylsilane (95:2.5:2.5 v/v, 3 × 2 mL) for 2 h at room temperature. Resin was removed by filtration, and the peptide precipitated by the addition of ice-cold Et2O (25 mL) and centrifuged. Supernatant was removed, and the peptide pellet dried under a stream of nitrogen, before being dissolved in H2O and freeze-dried.
Crude wt NOXA-B was purified by HPLC in two stages. Firstly, using a linear gradient at 12 mL/min of 5-50% acetonitrile in water (each containing 0.1% TFA) across a RediSep ® Rf gold C18 reverse-phase column (Teledyne Isco); and secondly, by MD-HPLC using a Jupiter Proteo (Phenomenex) preparative column employing a linear gradient of acetonitrile in water (each containing 0.1% formic acid).

Analytical Ultracentrifugation
Analytical ultracentrifugation (AUC) was performed at 20 °C in a Beckman Proteomelab XL-A or Beckman Proteomelab XL-I analytical ultracentrifuge using an An-60 Ti rotor and 2-channel centrepieces. Sedimentation equilibrium experiments were made up in PBS (137 mM NaCl, 2.7 mM KCl, 8.2 mM Na2HPO4 and 1.8 mM KH2PO4) at 50 µM peptide concentration for homomeric assemblies and 50 µM peptide concentration of both peptides for heteromeric assemblies and to 120 µL. The reference channel was loaded with 130 µL of PBS solution. Equilibrium distributions were measured twice per speed, in 4 krpm increments, and with rotor speeds from 40 to 60 krpm. Data were fitted to single ideal species models using Ultrascan II (http:/www.ultrascan.uthscsa.edu). A better fit for CC-Di_E2 data was found using monomer-dimer equilibrium with a fixed monomer mass. 95% confidence limits were obtained by Monte Carlo analysis of the fits. The partial specific volume ( ̅ ) for each of the peptides and the buffer density were calculated using Ultrascan II.

MCL-1/FITC-BID Direct Titration
Titration of MCL-1 into FITC-BID was performed in a 96-well plate in Tris Buffer (50 mM Tris, 150 mM NaCl, pH 7.4) + 0.01% Triton-X-100 with the concentration of MCL-1 starting at 3.75 µM, diluted over 12 points in a 1/2 regime with [FITC-BID] fixed at 25 nM. Plates were read after 1 hour and 21 hours incubation. Assays were performed in triplicate (both test wells and blank control wells).

BCL-xL /FITC-BID Direct Titration
Titration of BCL-xL into FITC-BID was performed in a 384-well plate in Tris Buffer + 0.01% Triton-X-100 + 0.02 mg/mL BSA with the concentration of BCL-xL starting from 5 µM, diluted over 16 points in a 1/2 regime with [FITC-BID] fixed at 25 nM. Plates were read after 1 hour and 21 hours incubation. Assays were performed in triplicate (both test wells and blank wells).