Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Multiple Substitutions of Methionine 129 in Human Prion Protein Reveal Its Importance in the Amyloid Fibrillation Pathway

“…Surewicz and co-workers (44) have reported that M129V and WT have the same fibril formation kinetics when subjected to mildly denaturing conditions. In that study, the WT and M129V variants did not fibrillate under native conditions when the agitation was slow, in accordance with our previous report on stagnant incubation of WT HuPrP(90 -231) (35). Nevertheless, studies of the D178N mutation in context with either WT or M129V showed faster fibrillation rates both regarding a shorter lag phase and faster growth rate when in context with Met in position 129 (44).…”

“…The ensembles of individual mutant trajectories of identical samples revealed that the protein showed a rather variable lag phase, indicating that PrP fibrillation kinetics is a process with a stochastic component, which appeared to be mutation-dependent. This notion is consistent with previous data showing an initial aggregation reaction preceding fibril formation, which renders a kinetic trap from which fibrils protrude (35). This is the background for the variations noted in the fibrillation trajectories.…”

“…Buffer F (50 mM Na 2 HPO 4 (pH 7.4), 100 mM NaCl, 50 mM KCl) was used for running buffer. Purity of obtained proteins was confirmed by SDS-PAGE (35). All described experiments with PrP were conducted in a biosafety P3** facility.…”

“…Amyloid Fibril Formation of HuPrP 129 Mutants-Previously, we have described in detail that vigorous shaking of natively folded PrP in 2-ml cryotubes induced protein aggregation and amyloidogenesis in physiological buffer at 37°C (35). Aggregates produced according to this protocol were collected after 7 h of agitation, stained with Congo red, and placed on glass slides.…”

“…4a, note the different y axis, and Table 3). The growth phase of the fibril formation trajectories was defined as the slope of the ThT fluorescence following the end of the lag phase to half the normalized maximum ThT intensity for each trajectory (35). In the amyloid fibril formation reactions, the highest mean growth rates were obtained for WT and M129A.…”