Abstract
Presenilin (PS1 or PS2) functions as the catalytic subunit of γ-secretase, which produces the toxic amyloid beta peptides in Alzheimer’s disease (AD). The dependence of folding and structural stability of PSs on the lipophilic environment and mutation were investigated by far UV CD spectroscopy. The secondary structure content and stability of PS2 depended on the lipophilic environment. PS2 undergoes a temperature-dependent structural transition from α-helical to β-structure at 331 K. The restructured protein formed structures which tested positive in spectroscopic amyloid fibrils assays. The AD mutant PS1L266F, PS1L424V and PS1ΔE9 displayed reduced stability which supports a proposed ‘loss of function’ mechanism of AD based on protein instability. The exon 9 coded sequence in the inhibitory loop of the zymogen was found to be required for the modulation of the thermal stability of PS1 by the lipophilic environment.
Highlights
For proteolytic activity PS zymogens undergo activation by auto-cleavage in the α-helical region of the third cytoplasmic loop between TM6 and TM7
For fos-choline detergent with chain length of 12 (FC12), four apparent un-separated peaks were seen on stepwise decreased to obtain finally (SEC), whereas for FC14 and FC16 only two major peaks were observed
Samples with increased homogeneity for biophysical characterization were obtained by a second SEC20 from the pooled fractions (Fig. 1)
Summary
Effect of detergent on the purification and oligomerization of presenilins. We purified PS2 using fos-choline detergent with chain length of 12 (FC12), 14 (FC14) and 16 (FC16) to investigate the effect of detergent chain length on the purification yield and biophysical property of the PS2-detergent complex. Used to compare the stability of the protein-detergent complexes[25,26] because the kinetics of aggregation did not disturb the unfolding (see Supplementary Figure S6): Similar Tm values were obtained with different heating rates. The purified protein-detergent complexes were subjected to far and near UV CD and fluorescence spectroscopy to study the effect of mutation on the secondary and tertiary structure as well as the thermal stability of PS1 (Fig. 8). Thermal unfolding of PS1 AD mutants showed varied melting curves (Fig. 8c) with lower Tm values (see Supplementary Figure S9) than the wild type protein This is in accordance with the reduced stability predicted for point mutations based on the structure of wild type PS1. PS1 E9 shows differences in the near UV CD region at ~275 nm and at 292, which is in agreement with the differences observed for the intrinsic fluorescence (Fig. 8b)
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