How Do Gyrating Beads Accelerate Amyloid Fibrillization?

Abstract

The chemical and physical mechanisms by which gyrating beads accelerate amyloid fibrillization in microtiter plate assays are unclear. Identifying these mechanisms will help optimize high-throughput screening assays for molecules and mutations that modulate aggregation and might explain why different research groups report different rates of aggregation for identical proteins. This article investigates how the rate of superoxide dismutase-1 (SOD1) fibrillization is affected by 12 different beads with a wide range of hydrophobicity, mass, stiffness, and topology but identical diameter. All assays were performed on D90A apo-SOD1, which is a stable and wild-type-like variant of SOD1. The most significant and uniform correlation between any material property of each bead and that bead's effect on SOD1 fibrillization rate was with regard to bead mass. A linear correlation existed between bead mass and rate of fibril elongation (R = 0.7): heavier beads produced faster rates and shorter fibrils. Nucleation rates (lag time) also correlated with bead mass, but only for non-polymeric beads (i.e., glass, ceramic, metallic). The effect of bead mass on fibrillization correlated (R = 0.96) with variations in buoyant forces and contact forces (between bead and microplate well), and was not an artifact of residual momentum during intermittent gyration. Hydrophobic effects were observed, but only for polymeric beads: lag times correlated negatively with contact angle of water and degree of protein adhesion (surface adhesion and hydrophobic effects were negligible for non-polymeric beads). These results demonstrate that contact forces (alone) explain kinetic variation among non-polymeric beads, whereas surface hydrophobicity and contact forces explain kinetic variation among polymeric beads. This study also establishes conditions for high-throughput amyloid assays of SOD1 that enable the control over fibril morphologies and produce eightfold faster lag times and fourfold less stochasticity than in previous studies.

Structure and Aggregation Mechanisms in Amyloids

“…[176,[182][183][184][185][186][187][188][189] The use of beads during agitation accelerates the aggregation process by enhancing cavitation. [190] Solid-liquid interface Solid-liquid interfaces may facilitate monomer encounters and initial monomer to monomer association and later further aggregation. In vitro, interaction with glass, silicone, graphite, polypropylene, Teflon, mica, gold, etc.…”

Acetylation of Aβ40 Alters Aggregation in the Presence and Absence of Lipid Membranes

“…The resulting solutions contained 20 μM Aβ 40 and the addition of 0.16, 0.32, 0.50, 1.00, and 1.50 mM NHSA Additionally, a 3 mm borosilicate bead was introduced to facilitate homogeneous mixing across wells. 122 The assay plate was sealed with plate tape and incubated at 37 °C for 24 h on a SpectraMax M2Multi-Mode plate reader (Molecular Devices, Sunnyvale, CA). Fluorescence intensities were measured in 5 min intervals at excitation and emission wavelengths of 440 and 484 nm, respectively, with shaking in between reads.…”

Bottom-Up Fabrication of Protein Nanowires via Controlled Self-Assembly of Recombinant Geobacter Pilins

“…The particle size controls the curvature and surface area of the substrate and the contact effects that modulate the rate of nucleation. Surface hydrophobicity impacts the effectiveness of seed particles in fibril formation (33) and could be controlled with the type of alkane coating (34) or with the use of nanoparticle materials with higher water contact angle than silica (33). Hydrophobe-bound silica nanoparticles also permitted their separation from the fibers after acetone precipitation of the proteins at the end of the assembly reaction.…”