Aggregation behavior of the amyloid model peptide NACore

Abstract

The aggregation of the 11 residue long NACore peptide segment of α-synuclein (68-GAVVTGVTAVA-78) has been investigated using a combination of cryogenic transmission electron microscopy (cryo-TEM), small- and wide-angle X-ray scattering, and spectroscopy techniques. The aqueous peptide solubility is pH dependent, and aggregation was triggered by a pH quench from pH 11.3 to approximately pH 8 or 6, where the average peptide net charge is weakly negative (pH 8), or essentially zero (pH 6). Cryo-TEM shows the presence of long and stiff fibrillar aggregates at both pH, that are built up from β-sheets, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. The fibrils are crystalline, with a wide angle X-ray diffraction pattern that is consistent with a previously determined crystal structure of NACore. Of particular note is the cryo-TEM observation of small globular shaped aggregates, of the order of a few nanometers in size, adsorbed onto the surface of already formed fibrils at pH 6. The fibrillation kinetics is slow, and occurs on the time scale of days. Similarly slow kinetics is observed at both pH, but slightly slower at pH 6, even though the peptide solubility is here expected to be lower. The observation of the small globular shaped aggregates, together with the associated kinetics, could be highly relevant in relation to mechanisms of secondary nucleation and oligomer formation in amyloid systems.

NACore Amyloid Formation in the Presence of Phospholipids

“…Here, the scattered intensity I(q) is plotted against the scattering vector q. These X-ray scattering patterns are in close agreement with previously reported data for NACore peptide fibrils formed under similar conditions, showing a characteristic power law scattering pattern at low q-values, and several Bragg peaks at higher q-values (Pallbo et al, 2019). The two main peaks occurring at q = 13.6 nm −1 and 7.6 nm −1 , are associated with the β-strand separation of 0.46 nm and the β-sheet lamination distance of 0.82 nm (PDB ID: 4RIL) (Rodriguez et al, 2015).…”

“…We have previously reported that pH can be used to control the fibrillation of NACore (Pallbo et al, 2019). At high pH, the peptide has a net negative charge and a higher aqueous solubility than at lower pH closer to its isoelectric point (∼ pH 5.5).…”

“…Vesicles composed of either POPC:POPS 8:2 or POPC only were prepared, and peptide fibrillation was induced by a change in pH. After 4 h of quiescent incubation at room temperature, a small part of the sample was taken out, vitrified, and observed with a JEM-2200FS (JEOL) transmission electron microscope with an accelerating voltage of 200 kV and an in-column energy filter for zero-loss imaging (Omega Filters, India), as has previously been described (Pallbo et al, 2019). Images were recorded digitally using a TVIPS TemCam-F416 digital camera.…”

“…To further investigate these differences, we performed CD measurement on peptide fibrillated in the presence and absence of POPC:POPS or POPC vesicles at various time points of incubation ( Figure 6). The CD measurements give information about the secondary structure of the peptide and can be used to monitor the peptide fibrillation process (Kelly et al, 2005;Pallbo et al, 2019). Initially, the peptide has a disordered structure as judged from the CD spectra.…”

“…In this report we have investigated amyloid formation by the amyloid model peptide NACore and how the formed aggregates are affected by the presence of phospholipids, from a structural perspective on nanometer length-scales. NACore is an eleven amino acid residue peptide from the so-called non-amyloid-β component (NAC) region of the Parkinson's disease associated protein α-synuclein (68-GAVVTGVTAVA-78) (Bodles et al, 2001;Rodriguez et al, 2015;Pallbo et al, 2019). This peptide has previously shown cytotoxic effects on cells in vitro (Bodles et al, 2001;Rodriguez et al, 2015).…”