Amyloidogenic peptides and proteins have a property to convert from their native functional states into fibrillar amyloid aggregates. This property is associated with neurodegenerative disorders, such as Alzheimer's or Parkinson's diseases, as well as prionopathies [1]. It has been observed that the environment conditions in which amyloid aggregation takes place have an important effect on fibril polymorphism. Moreover, amyloid aggregates have recently been shown to have the property to adopt more than one different conformation under the same environmental conditions [2]. One example of such environmental conditions, whose effect on amyloid structural variability is not fully understood, is agitation.
In this work, we examined the effect of three different agitation conditions on the aggregation kinetics of mouse prion protein fragment (MoPrP 89-230) and analyzed the secondary structure of the resulting fibrils.
Protein MoPrP 89-230 samples were incubated under three agitation conditions (200, 400 and 600 RPM) at 37°C, under denaturing conditions (2 M guanidinium hydrochloride, 50 mM sodium phosphate, pH 6.0). The kinetics of aggregation were determined by recording the fluorescence intensity of the amyloidophilic dye thioflavin-T (ThT). The secondary structure of fibrils was determined by analyzing each sample's FTIR spectra.
The results suggest that the intensity of agitation has a minimal influence on primary nuclei formation and that the rate of elongation does to scale with the level of agitation. In all three cases, a diverse collection of secondary structures (at least three structure types) were observed, with the highest variability detected under 200 RPM agitation conditions.