Computational protocol: Preclinical Validation of the Heparin-Reactive Peptide p5+14 as a Molecular Imaging Agent for Visceral Amyloidosis

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Protocol publication

[…] Synthetic rVλ6Wil fibrils formed by agitation of a 1 mg/mL solution [] were coated onto wells of NUNC Maxisorb plates (8 µg/mL, 50 µL) and the plates incubated overnight at 37 °C. The dried wells were washed ×1 with assay buffer (PBS containing 0.05% Tween 20) blocked with 200 µL per well of 1% BSA (w/v) in PBS at 37 °C for 1 h, followed by addition of biotinylated p5+14 diluted over the range 1 × 10−10–1 × 10−6 M. To determine background, triplicate wells lacking amyloid fibrils were used that contained only the peptide. After a 1-h incubation at 37 °C, the wells were washed ×2 and a 1/1000 dilution of europium/streptavidin (Perkin Elmer) added for 1 h before being developed with Perkin Elmer enhancement solution. Bound europium was measured by using a Victor 3 Wallac microplate reader (Perkin Elmer). The data were analyzed using SigmaPlot (SPSS: An IBM Company, Armonk, NY, USA) [...] For the docking studies, the ZDOCK server was used to generate 100 theoretical models of the interaction of p5+14 with amyloid fibrils composed of Aβ(17–42) (PDB # 2BEG; []) ZDOCK searches all possible binding modes in the translational and rotational space between the two proteins and evaluates each pose using an energy-based scoring function. The lowest energy p5+14 and Aβ(17–42) fibril complex was first solvated in a TIP3P water box, and then subjected to 20 ns of molecular dynamics (MD) simulation using NAMD 2.9 [] with the CHARMM C36 force field []. In the simulation, the van der Waals interaction was smoothly turned off between 8.5–10 Å using a switching function. Long-range electrostatic interactions were treated using the Particle-Mesh Ewald (PME) method [] with a 1.0 Å grid spacing. The time step for integration was 1 fs. Langevin dynamics was used to maintain a constant temperature at 310 K, while the Nosé-Hoover Langevin-piston algorithm was used to maintain a constant pressure at 1 bar.The molecular mechanics-Poisson Boltzmann/surface area (MM-PB/SA) method [,] was used to compute the binding free energy of the peptide with the Aβ(17–42) fibril. The total binding energy ΔGbind was defined as ΔGbind = Gcomplex − Greceptor − Gligand. Each free energy term consisted of the gas phase MM energy (ΔEgas), the solvation free energy (ΔGsol), and the vibrational entropy contributions (TΔS). ΔGsol was estimated from the PB theory and solvent accessible surface area (SASA) calculations which yielded ΔGpolar and ΔGnonpolar. In the PB, energies were evaluated at 0.1 M, and 1.0 M NaCl concentrations to interrogate the effect of ionic strength on the electrostatic interactions between p5+14 and the Aβ fibril. A surface tension coefficient (γ) of 0.0072 kcal/(mol·Å2) was used to calculate the nonpolar solvation free energy contribution. Due to its prohibitive computational cost and the inherent difficulty in determining accurate absolute entropy for large protein-peptide complex systems, the vibrational entropy contribution was not included in our calculation. Egas and ΔGsol were computed for 1000 snapshots extracted evenly from the last 2 ns of the MD trajectory. […]

Pipeline specifications

Software tools SigmaPlot, SPSS, ZDOCK, NAMD
Applications Miscellaneous, Protein interaction analysis
Organisms Mus musculus, Homo sapiens
Diseases Mastocytosis, Systemic