The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

The development of sensing systems for the measurement of small molecules is an active area of research. A sensor based approach for the measurement of metabolites can potentially provide the simplicity and portability required for widespread use. Rapid detection and quantitation of small-molecule metabolites can potentially emerge as an effective way to link the metabolite profile to the disease state. Surface plasmon resonance (SPR) combined with molecular recognition elements to deliver high specificity is a sensing platform that has been widely applied for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. The work described here combines a periplasmic binding protein for recognition with target modified gold nanoparticles (AuNPs) in a competitive assay format for folic acid (FA) detection. Specifically, a SPR imaging substrate containing immobilized folate binding protein (FBP) is used to measure the adsorption of FA conjugated AuNPs. The immobilization of the FBP and the binding of the FA conjugated AuNPs are characterized and optimized. It is shown that free FA in solution can be quantitatively measured by competition for the surface binding sites with the functionalized AuNPs. We demonstrate that the dynamic range can be lowered from micromolar to nanomolar by simply decreasing the concentration of FA conjugated AuNPs, thus lowering the limit of detection to 2.9 nM. This type of competitive assay can be applied to a range of small molecules, which paves the way for future multiplexed analysis of metabolites using SPR.